Laurence Hurley

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• Ho, D., Kretzmann, J. A., Norret, M., Toshniwal, P., Veder, J. P., Jiang, H., Guagliardo, P., Munshi, A. M., Chawla, R., Evans, C. W., Clemons, T. D., Nguyen, M., Kretzmann, A. L., Blythe, A. J., Saunders, M., Archer, M., Fitzgerald, M., Keelan, J. A., Bond, C. S., , Kilburn, M. R., et al. (2018). Intracellular speciation of gold nanorods alters the conformational dynamics of genomic DNA. Nature nanotechnology, 13(12), 1148-1153.
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  Gold nanorods are one of the most widely explored inorganic materials in nanomedicine for diagnostics, therapeutics and sensing. It has been shown that gold nanorods are not cytotoxic and localize within cytoplasmic vesicles following endocytosis, with no nuclear localization, but other studies have reported alterations in gene expression profiles in cells following exposure to gold nanorods, via unknown mechanisms. In this work we describe a pathway that can contribute to this phenomenon. By mapping the intracellular chemical speciation process of gold nanorods, we show that the commonly used Au-thiol conjugation, which is important for maintaining the noble (inert) properties of gold nanostructures, is altered following endocytosis, resulting in the formation of Au(I)-thiolates that localize in the nucleus. Furthermore, we show that nuclear localization of the gold species perturbs the dynamic microenvironment within the nucleus and triggers alteration of gene expression in human cells. We demonstrate this using quantitative visualization of ubiquitous DNA G-quadruplex structures, which are sensitive to ionic imbalances, as an indicator of the formation of structural alterations in genomic DNA.
• Hurley, L., Amato, J., Madanayake, T. W., Iaccarino, N., Novellino, E., Randazzo, A., & Pagano, B. (2018). HMGB1 binds to the KRAS promoter G-quadruplex: A new player in oncogene transcriptional regulation?. Chem. Comm, 54, 9442-9445.
• Hurley, L., Onel, B., Carver, M., Agrawal, P., & Yang, D. (2018). The 3′-end region of the human PDGFR-β core promoter nuclease hypersensitive element forms a mixture of two unique end-insertion G-quadruplexes. Biochimica et Biophysica Acta, 1862, 846-854.
• Hurley, L., Weldon, C., Dacanay, J., Gokhale, V., Boddupally, P., Behm-Ansmant, I., Burley, G., Dominguez, C., & Eperon, I. (2018). Specific G-quadruplex ligands modulate the alternative splicing of Bcl-X. Nucleic Acids Research, 46, 886-896.
• Weldon, C., Dacanay, J. G., Gokhale, V., Boddupally, P. V., Behm-Ansmant, I., Burley, G. A., Branlant, C., Hurley, L. H., Dominguez, C., & Eperon, I. C. (2018). Specific G-quadruplex ligands modulate the alternative splicing of Bcl-X. Nucleic acids research, 46(2), 886-896.
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  Sequences with the potential to form RNA G-quadruplexes (G4s) are common in mammalian introns, especially in the proximity of the 5' splice site (5'SS). However, the difficulty of demonstrating that G4s form in pre-mRNA in functional conditions has meant that little is known about their effects or mechanisms of action. We have shown previously that two G4s form in Bcl-X pre-mRNA, one close to each of the two alternative 5'SS. If these G4s affect splicing but are in competition with other RNA structures or RNA binding proteins, then ligands that stabilize them would increase the proportion of Bcl-X pre-mRNA molecules in which either or both G4s had formed, shifting Bcl-X splicing. We show here that a restricted set of G4 ligands do affect splicing, that their activity and specificity are strongly dependent on their structures and that they act independently at the two splice sites. One of the ligands, the ellipticine GQC-05, antagonizes the major 5'SS that expresses the anti-apoptotic isoform of Bcl-X and activates the alternative 5'SS that expresses a pro-apoptotic isoform. We propose mechanisms that would account for these see-saw effects and suggest that these effects contribute to the ability of GQC-05 to induce apoptosis.
• Brown, R. V., Wang, T., Chappeta, V. R., Wu, G., Onel, B., Chawla, R., Quijada, H., Camp, S. M., Chiang, E. T., Lassiter, Q. R., Lee, C., Phanse, S., Turnidge, M. A., Zhao, P., Garcia, J. G., Gokhale, V., Yang, D., & Hurley, L. H. (2017). The Consequences of Overlapping G-Quadruplexes and i-Motifs in the Platelet-Derived Growth Factor Receptor β Core Promoter Nuclease Hypersensitive Element Can Explain the Unexpected Effects of Mutations and Provide Opportunities for Selective Targeting of Both Structures by Small Molecules To Downregulate Gene Expression. Journal of the American Chemical Society, 139(22), 7456-7475.
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  The platelet-derived growth factor receptor β (PDGFR-β) signaling pathway is a validated and important target for the treatment of certain malignant and nonmalignant pathologies. We previously identified a G-quadruplex-forming nuclease hypersensitive element (NHE) in the human PDGFR-β promoter that putatively forms four overlapping G-quadruplexes. Therefore, we further investigated the structures and biological roles of the G-quadruplexes and i-motifs in the PDGFR-β NHE with the ultimate goal of demonstrating an alternate and effective strategy for molecularly targeting the PDGFR-β pathway. Significantly, we show that the primary G-quadruplex receptor for repression of PDGFR-β is the 3'-end G-quadruplex, which has a GGA sequence at the 3'-end. Mutation studies using luciferase reporter plasmids highlight a novel set of G-quadruplex point mutations, some of which seem to provide conflicting results on effects on gene expression, prompting further investigation into the effect of these mutations on the i-motif-forming strand. Herein we characterize the formation of an equilibrium between at least two different i-motifs from the cytosine-rich (C-rich) sequence of the PDGFR-β NHE. The apparently conflicting mutation results can be rationalized if we take into account the single base point mutation made in a critical cytosine run in the PDGFR-β NHE that dramatically affects the equilibrium of i-motifs formed from this sequence. We identified a group of ellipticines that targets the G-quadruplexes in the PDGFR-β promoter, and from this series of compounds, we selected the ellipticine analog GSA1129, which selectively targets the 3'-end G-quadruplex, to shift the dynamic equilibrium in the full-length sequence to favor this structure. We also identified a benzothiophene-2-carboxamide (NSC309874) as a PDGFR-β i-motif-interactive compound. In vitro, GSA1129 and NSC309874 downregulate PDGFR-β promoter activity and transcript in the neuroblastoma cell line SK-N-SH at subcytotoxic cell concentrations. GSA1129 also inhibits PDGFR-β-driven cell proliferation and migration. With an established preclinical murine model of acute lung injury, we demonstrate that GSA1129 attenuates endotoxin-mediated acute lung inflammation. Our studies underscore the importance of considering the effects of point mutations on structure formation from the G- and C-rich sequences and provide further evidence for the involvement of both strands and associated structures in the control of gene expression.
• Kaiser, C. E., Van Ert, N. A., Agrawal, P., Chawla, R., Yang, D., & Hurley, L. H. (2017). Insight into the Complexity of the i-Motif and G-Quadruplex DNA Structures Formed in the KRAS Promoter and Subsequent Drug-Induced Gene Repression. Journal of the American Chemical Society, 139(25), 8522-8536.
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  Activating KRAS mutations frequently occur in pancreatic, colorectal, and lung adenocarcinomas. While many attempts have been made to target oncogenic KRAS, no clinically useful therapies currently exist. Most efforts to target KRAS have focused on inhibiting the mutant protein; a less explored approach involves targeting KRAS at the transcriptional level. The promoter element of the KRAS gene contains a GC-rich nuclease hypersensitive site with three potential DNA secondary structure-forming regions. These are referred to as the Near-, Mid-, and Far-regions, on the basis of their proximity to the transcription start site. As a result of transcription-induced negative superhelicity, these regions can open up to form unique DNA secondary structures: G-quadruplexes on the G-rich strand and i-motifs on the C-rich strand. While the G-quadruplexes have been well characterized, the i-motifs have not been investigated as thoroughly. Here we show that the i-motif that forms in the C-rich Mid-region is the most stable and exists in a dynamic equilibrium with a hybrid i-motif/hairpin species and an unfolded hairpin species. The transcription factor heterogeneous nuclear ribonucleoprotein K (hnRNP K) was found to bind selectively to the i-motif species and to positively modulate KRAS transcription. Additionally, we identified a benzophenanthridine alkaloid that dissipates the hairpin species and destabilizes the interaction of hnRNP K with the Mid-region i-motif. This same compound stabilizes the three existing KRAS G-quadruplexes. The combined effect of the compound on the Mid-region i-motif and the G-quadruplexes leads to downregulation of KRAS gene expression. This dual i-motif/G-quadruplex-interactive compound presents a new mechanism to modulate gene expression.
• Kendrick, S., Muranyi, A., Gokhale, V., Hurley, L. H., & Rimsza, L. M. (2017). Simultaneous Drug Targeting of the Promoter MYC G-Quadruplex and BCL2 i-Motif in Diffuse Large B-Cell Lymphoma Delays Tumor Growth. Journal of medicinal chemistry, 60(15), 6587-6597.
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  Secondary DNA structures are uniquely poised as therapeutic targets due to their molecular switch function in turning gene expression on or off and scaffold-like properties for protein and small molecule interaction. Strategies to alter gene transcription through these structures thus far involve targeting single DNA conformations. Here we investigate the feasibility of simultaneously targeting different secondary DNA structures to modulate two key oncogenes, cellular-myelocytomatosis (MYC) and B-cell lymphoma gene-2 (BCL2), in diffuse large B-cell lymphoma (DLBCL). Cotreatment with previously identified ellipticine and pregnanol derivatives that recognize the MYC G-quadruplex and BCL2 i-motif promoter DNA structures lowered mRNA levels and subsequently enhanced sensitivity to a standard chemotherapy drug, cyclophosphamide, in DLBCL cell lines. In vivo repression of MYC and BCL2 in combination with cyclophosphamide also significantly slowed tumor growth in DLBCL xenograft mice. Our findings demonstrate concurrent targeting of different DNA secondary structures offers an effective, precise, medicine-based approach to directly impede transcription and overcome aberrant pathways in aggressive malignancies.
• Liang, W. S., Hendricks, W., Kiefer, J., Schmidt, J., Sekar, S., Carpten, J., Craig, D. W., Adkins, J., Cuyugan, L., Manojlovic, Z., Halperin, R. F., Helland, A., Nasser, S., Legendre, C., Hurley, L. H., Sivaprakasam, K., Johnson, D. B., Crandall, H., Busam, K. J., , Zismann, V., et al. (2017). Integrated genomic analyses reveal frequent TERT aberrations in acral melanoma. Genome research, 27(4), 524-532.
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  Genomic analyses of cutaneous melanoma (CM) have yielded biological and therapeutic insights, but understanding of non-ultraviolet (UV)-derived CMs remains limited. Deeper analysis of acral lentiginous melanoma (ALM), a rare sun-shielded melanoma subtype associated with worse survival than CM, is needed to delineate non-UV oncogenic mechanisms. We thus performed comprehensive genomic and transcriptomic analysis of 34 ALM patients. Unlike CM, somatic alterations were dominated by structural variation and absence of UV-derived mutation signatures. Only 38% of patients demonstrated driver BRAF/NRAS/NF1 mutations. In contrast with CM, we observed PAK1 copy gains in 15% of patients, and somatic TERT translocations, copy gains, and missense and promoter mutations, or germline events, in 41% of patients. We further show that in vitro TERT inhibition has cytotoxic effects on primary ALM cells. These findings provide insight into the role of TERT in ALM tumorigenesis and reveal preliminary evidence that TERT inhibition represents a potential therapeutic strategy in ALM.
• Onel, B., Carver, M., Agrawal, P., Hurley, L. H., & Yang, D. (2017). The 3'-end region of the human PDGFR-β core promoter nuclease hypersensitive element forms a mixture of two unique end-insertion G-quadruplexes. Biochimica et biophysica acta, 1862(4), 846-854.
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  While the most stable G-quadruplex formed in the human PDGFR-β promoter nuclease hypersensitive element (NHE) is the 5'-mid G-quadruplex, the 3'-end sequence that contains a 3'-GGA run forms a less stable G-quadruplex. Recently, the 3'-end G-quadruplex was found to be a transcriptional repressor and can be selectively targeted by a small molecule for PDGFR-β downregulation.
• Hurley, L. H., Kang, H., Cui, Y., Yin, H., Scheid, A., Kong, D., & Mao, H. (2016). A pharmacological chaperone molecule induces cell death by restoring tertiary DNA structures in mutant hTERT promoters. Nature Medicine.
• Kang, H. J., Cui, Y., Yin, H., Scheid, A., Hendricks, W. P., Schmidt, J., Sekulic, A., Kong, D., Trent, J. M., Gokhale, V., Mao, H., & Hurley, L. H. (2016). A Pharmacological Chaperone Molecule Induces Cancer Cell Death by Restoring Tertiary DNA Structures in Mutant hTERT Promoters. Journal of the American Chemical Society.
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  Activation of human telomerase reverse transcriptase (hTERT) is necessary for limitless replication in tumorigenesis. Whereas hTERT is transcriptionally silenced in normal cells, most tumor cells reactivate hTERT expression by alleviating transcriptional repression through diverse genetic and epigenetic mechanisms. Transcription-activating hTERT promoter mutations have been found to occur at high frequencies in multiple cancer types. These mutations have been shown to form new transcription factor binding sites that drive hTERT expression, but this model cannot fully account for differences in wild-type (WT) and mutant promoter activation and has not yet enabled a selective therapeutic strategy. Here, we demonstrate a novel mechanism by which promoter mutations activate hTERT transcription, which also sheds light on a unique therapeutic opportunity. Promoter mutations occur in a core promoter region that forms tertiary structures consisting of a pair of G-quadruplexes involved in transcriptional silencing. We show that promoter mutations exert a detrimental effect on the folding of one of these G-quadruplexes, resulting in a nonfunctional silencer element that alleviates transcriptional repression. We have also identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, GTC365, that acts at an early step in the G-quadruplex folding pathway to redirect mutant promoter G-quadruplex misfolding, partially reinstate the correct folding pathway, and reduce hTERT activity through transcriptional repression. This transcription-mediated repression produces cancer cell death through multiple routes including both induction of apoptosis through inhibition of hTERT's role in regulating apoptosis-related proteins and induction of senescence by decreasing telomerase activity and telomere length. We demonstrate the selective therapeutic potential of this strategy in melanoma cells that overexpress hTERT.
• Kendrick, S., Kang, H. J., Alam, M. P., Madathil, M. M., Agrawal, P., Gokhale, V., Yang, D., Hecht, S. M., & Hurley, L. H. (2016). Correction to "The Dynamic Character of the BCL2 Promoter i-Motif Provides a Mechanism for Modulation of Gene Expression by Compounds That Bind Selectively to the Alternative DNA Hairpin Structure". Journal of the American Chemical Society, 138(35), 11408.
• Roy, B., Talukder, P., Kang, H. J., Tsuen, S. S., Alam, M. P., Hurley, L. H., & Hecht, S. M. (2016). Interaction of Individual Structural Domains of hnRNP LL with the BCL2 Promoter i-Motif DNA. Journal of the American Chemical Society, 138(34), 10950-62.
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  The recently discovered role of the BCL2 (B-cell lymphoma 2 gene) promoter i-motif DNA in modulation of gene expression via interaction with the ribonucleoprotein hnRNP L-like (hnRNP LL) has prompted a more detailed study of the nature of this protein-DNA interaction. The RNA recognition motifs (RRMs) of hnRNP LL were expressed individually, and both RRM1 and RRM2 were found to bind efficiently to the BCL2 i-motif DNA, as well as being critical for transcriptional activation, whereas RRM3-4 bound only weakly to this DNA. Binding was followed by unfolding of the DNA as monitored by changes in the CD spectrum. Mutational analysis of the i-motif DNA revealed that binding involved primarily the lateral loops of the i-motif. The kinetics of binding of the DNA with RRM1 was explored by recording CD spectra at predetermined times following admixture of the protein and DNA. The change in molar ellipticity was readily apparent after 30 s and largely complete within 1 min. A more detailed view of protein-DNA interaction was obtained by introducing the fluorescence donor 6-CNTrp in RRM1 at position 137, and the acceptor 4-aminobenzo[g]quinazoline-2-one (Cf) in lieu of cytidine22 in the i-motif DNA. The course of binding of the two species was monitored by FRET, which reflected a steady increase in energy transfer over a period of several minutes. The FRET signal could be diminished by the further addition of (unlabeled) RRM2, no doubt reflecting competition for binding to the i-motif DNA. These experiments using the individual RRM domains from hnRNP LL confirm the role of this transcription factor in activation of BCL2 transcription via the i-motif in the promoter element.
• Sutherland, C., Cui, Y., Mao, H., & Hurley, L. H. (2016). A Mechanosensor Mechanism Controls the G-Quadruplex/i-Motif Molecular Switch in the MYC Promoter NHE III1. Journal of the American Chemical Society.
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  MYC is overexpressed in many different cancer types and is an intensively studied oncogene because of its contributions to tumorigenesis. The regulation of MYC is complex, and the NHE III1 and FUSE elements rely upon noncanonical DNA structures and transcriptionally induced negative superhelicity. In the NHE III1 only the G-quadruplex has been extensively studied, whereas the role of the i-motif, formed on the opposite C-rich strand, is much less understood. We demonstrate here that the i-motif is formed within the 4CT element and is recognized by hnRNP K, which leads to a low level of transcription activation. For maximal hnRNP K transcription activation, two additional cytosine runs, located seven bases downstream of the i-motif-forming region, are also required. To access these additional runs of cytosine, increased negative superhelicity is necessary, which leads to a thermodynamically stable complex between hnRNP K and the unfolded i-motif. We also demonstrate mutual exclusivity between the MYC G-quadruplex and i-motif, providing a rationale for a molecular switch mechanism driven by SP1-induced negative superhelicity, where relative hnRNP K and nucleolin expression shifts the equilibrium to the on or off state.
• Weldon, C., Behm-Ansmant, I., Hurley, L. H., Burley, G. A., Branlant, C., Eperon, I. C., & Dominguez, C. (2016). Identification of G-quadruplexes in long functional RNAs using 7-deazaguanine RNA. Nature chemical biology, 13(1), 18-20.
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  RNA G-quadruplex (G4) structures are thought to affect biological processes, including translation and pre-mRNA splicing, but it is not possible at present to demonstrate that they form naturally at specific sequences in long functional RNA molecules. We developed a new strategy, footprinting of long 7-deazaguanine-substituted RNAs (FOLDeR), that allows the formation of G4s to be confirmed in long RNAs and under functional conditions.
• Cui, Y., Koirala, D., Kang, H., Dhakal, S., Yangyuoru, P., Hurley, L. H., & Mao, H. (2014). Molecular population dynamics of DNA structures in a bcl-2 promoter sequence is regulated by small molecules and the transcription factor hnRNP LL. Nucleic acids research, 42(9), 5755-64.
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  Minute difference in free energy change of unfolding among structures in an oligonucleotide sequence can lead to a complex population equilibrium, which is rather challenging for ensemble techniques to decipher. Herein, we introduce a new method, molecular population dynamics (MPD), to describe the intricate equilibrium among non-B deoxyribonucleic acid (DNA) structures. Using mechanical unfolding in laser tweezers, we identified six DNA species in a cytosine (C)-rich bcl-2 promoter sequence. Population patterns of these species with and without a small molecule (IMC-76 or IMC-48) or the transcription factor hnRNP LL are compared to reveal the MPD of different species. With a pattern recognition algorithm, we found that IMC-48 and hnRNP LL share 80% similarity in stabilizing i-motifs with 60 s incubation. In contrast, IMC-76 demonstrates an opposite behavior, preferring flexible DNA hairpins. With 120-180 s incubation, IMC-48 and hnRNP LL destabilize i-motifs, which has been previously proposed to activate bcl-2 transcriptions. These results provide strong support, from the population equilibrium perspective, that small molecules and hnRNP LL can modulate bcl-2 transcription through interaction with i-motifs. The excellent agreement with biochemical results firmly validates the MPD analyses, which, we expect, can be widely applicable to investigate complex equilibrium of biomacromolecules.
• Kang, H., Kendrick, S., Hecht, S. M., & Hurley, L. H. (2014). The transcriptional complex between the BCL2 i-motif and hnRNP LL is a molecular switch for control of gene expression that can be modulated by small molecules. Journal of the American Chemical Society, 136(11), 4172-85.
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  In a companion paper (DOI: 10.021/ja410934b) we demonstrate that the C-rich strand of the cis-regulatory element in the BCL2 promoter element is highly dynamic in nature and can form either an i-motif or a flexible hairpin. Under physiological conditions these two secondary DNA structures are found in an equilibrium mixture, which can be shifted by the addition of small molecules that trap out either the i-motif (IMC-48) or the flexible hairpin (IMC-76). In cellular experiments we demonstrate that the addition of these molecules has opposite effects on BCL2 gene expression and furthermore that these effects are antagonistic. In this contribution we have identified a transcriptional factor that recognizes and binds to the BCL2 i-motif to activate transcription. The molecular basis for the recognition of the i-motif by hnRNP LL is determined, and we demonstrate that the protein unfolds the i-motif structure to form a stable single-stranded complex. In subsequent experiments we show that IMC-48 and IMC-76 have opposite, antagonistic effects on the formation of the hnRNP LL-i-motif complex as well as on the transcription factor occupancy at the BCL2 promoter. For the first time we propose that the i-motif acts as a molecular switch that controls gene expression and that small molecules that target the dynamic equilibrium of the i-motif and the flexible hairpin can differentially modulate gene expression.
• Kendrick, S., Kang, H. J., Alam, M. P., Madathil, M. M., Agrawal, P., Gokhale, V., Yang, D., Hecht, S. M., & Hurley, L. H. (2014). The dynamic character of the BCL2 promoter i-motif provides a mechanism for modulation of gene expression by compounds that bind selectively to the alternative DNA hairpin structure. Journal of the American Chemical Society, 136(11), 4161-71.
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  It is generally accepted that DNA predominantly exists in duplex form in cells. However, under torsional stress imposed by active transcription, DNA can assume nonduplex structures. The BCL2 promoter region forms two different secondary DNA structures on opposite strands called the G-quadruplex and the i-motif. The i-motif is a highly dynamic structure that exists in equilibrium with a flexible hairpin species. Here we identify a pregnanol derivative and a class of piperidine derivatives that differentially modulate gene expression by stabilizing either the i-motif or the flexible hairpin species. Stabilization of the i-motif structure results in significant upregulation of the BCL2 gene and associated protein expression; in contrast, stabilization of the flexible hairpin species lowers BCL2 levels. The BCL2 levels reduced by the hairpin-binding compound led to chemosensitization to etoposide in both in vitro and in vivo models. Furthermore, we show antagonism between the two classes of compounds in solution and in cells. For the first time, our results demonstrate the principle of small molecule targeting of i-motif structures in vitro and in vivo to modulate gene expression.
• Kendrick, S., Kang, H., Alam, M. P., Madathil, M. M., Agrawal, P., Gokhale, V., Yang, D., Hecht, S. M., & Hurley, L. H. (2014). The dynamic character of the BCL2 promoter i-motif provides a mechanism for modulation of gene expression by compounds that bind selectively to the alternative DNA hairpin structure. Journal of the American Chemical Society, 136(11), 4161-4171.
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  Abstract: It is generally accepted that DNA predominantly exists in duplex form in cells. However, under torsional stress imposed by active transcription, DNA can assume nonduplex structures. The BCL2 promoter region forms two different secondary DNA structures on opposite strands called the G-quadruplex and the i-motif. The i-motif is a highly dynamic structure that exists in equilibrium with a flexible hairpin species. Here we identify a pregnanol derivative and a class of piperidine derivatives that differentially modulate gene expression by stabilizing either the i-motif or the flexible hairpin species. Stabilization of the i-motif structure results in significant upregulation of the BCL2 gene and associated protein expression; in contrast, stabilization of the flexible hairpin species lowers BCL2 levels. The BCL2 levels reduced by the hairpin-binding compound led to chemosensitization to etoposide in both in vitro and in vivo models. Furthermore, we show antagonism between the two classes of compounds in solution and in cells. For the first time, our results demonstrate the principle of small molecule targeting of i-motif structures in vitro and in vivo to modulate gene expression. © 2014 American Chemical Society.
• Hurley, L., Kaiser, C. E., Gokhale, V., Yang, D., & Hurley, L. -. (2013). Gaining Insights into the Small Molecule Targeting of the G-Quadruplex in the c-MYC Promoter Using NMR and an Allele-Specific Transcriptional Assay. Topics in current chemistry, 330.
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  G-quadruplexes (four-stranded DNA secondary structures) are showing promise as new targets for anticancer therapies. Specifically, G-quadruplexes in the proximal promoter region of regulatory genes have the potential to act as silencer elements and thereby turn off transcription. Thus, compounds that are capable of binding to and stabilizing G-quadruplexes would be of great benefit. In this chapter we describe two recent studies from our labs. In the first case, we use NMR to elucidate the structure of a 2:1 complex between a small molecule and the G-quadruplex in the c-MYC promoter. In the second case, we use an allele-specific transcription assay to demonstrate that the effect of a G-quadruplex-interactive compound is mediated directly through the G-quadruplex. Finally, we use this information to propose models for the interaction of various small molecules with the c-MYC G-quadruplex.
• Kaiser, C. E., Gokhale, V., Yang, D., & Hurley, L. H. (2013). Gaining insights into the small molecule targeting of the G-QUADRUPLEX in the c-MYC promoter using NMR and an allele-specific transcriptional assay. Topics in Current Chemistry, 330, 1-22.
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  PMID: 22752577;Abstract: G-quadruplexes (four-stranded DNA secondary structures) are showing promise as new targets for anticancer therapies. Specifically, G-quadruplexes in the proximal promoter region of regulatory genes have the potential to act as silencer elements and thereby turn off transcription. Thus, compounds that are capable of binding to and stabilizing G-quadruplexes would be of great benefit. In this chapter we describe two recent studies from our labs. In the first case, we use NMR to elucidate the structure of a 2:1 complex between a small molecule and the G-quadruplex in the c-MYC promoter. In the second case, we use an allele-specific transcription assay to demonstrate that the effect of a G-quadruplex-interactive compound is mediated directly through the G-quadruplex. Finally, we use this information to propose models for the interaction of various small molecules with the c-MYC G-quadruplex. © 2013 Springer-Verlag Berlin Heidelberg.
• Siddiqui-Jain, A., & Hurley, L. H. (2013). DNA structure: Visualizing the quadruplex. Nature Chemistry, 5(3), 153-155.
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  PMID: 23422553;
• Siddiqui-Jain, A., & Hurley, L. H. (2013). DNA structure: Visualizing the quadruplex. Nature chemistry, 5(3), 153-5.
• Chen, Y., Agrawal, P., Brown, R. V., Hatzakis, E., Hurley, L., & Yang, D. (2012). The major G-quadruplex formed in the human platelet-derived growth factor receptor β promoter adopts a novel broken-strand structure in K+ solution. Journal of the American Chemical Society, 134(32), 13220-3.
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  Overexpression of platelet-derived growth factor receptor β (PDGFR-β) has been associated with cancers and vascular and fibrotic disorders. PDGFR-β has become an attractive target for the treatment of cancers and fibrotic disorders. DNA G-quadruplexes formed in the GC-rich nuclease hypersensitivity element of the human PDGFR-β gene promoter have been found to inhibit PDGFR-β transcriptional activity. Here we determined the major G-quadruplex formed in the PDGFR-β promoter. Instead of using four continuous runs with three or more guanines, this G-quadruplex adopts a novel folding with a broken G-strand to form a primarily parallel-stranded intramolecular structure with three 1 nucleotide (nt) double-chain-reversal loops and one additional lateral loop. The novel folding of the PDGFR-β promoter G-quadruplex emphasizes the robustness of parallel-stranded structural motifs with a 1 nt loop. Considering recent progress on G-quadruplexes formed in gene-promoter sequences, we suggest the 1 nt looped G(i)NG(j) motif may have been evolutionarily selected to serve as a stable foundation upon which the promoter G-quadruplexes can build. The novel folding of the PDGFR-β promoter G-quadruplex may be attractive for small-molecule drugs that specifically target this secondary structure and modulate PDGFR-β gene expression.
• Chen, Y., Agrawal, P., Brown, R. V., Hatzakis, E., Hurley, L., & Yang, D. (2012). The major G-quadruplex formed in the human platelet-derived growth factor receptor β promoter adopts a novel broken-strand structure in K⁺ solution. Journal of the American Chemical Society, 134(32), 13220-13223.
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  PMID: 22866911;PMCID: PMC3428200;Abstract: Overexpression of platelet-derived growth factor receptor β (PDGFR-β) has been associated with cancers and vascular and fibrotic disorders. PDGFR-β has become an attractive target for the treatment of cancers and fibrotic disorders. DNA G-quadruplexes formed in the GC-rich nuclease hypersensitivity element of the human PDGFR-β gene promoter have been found to inhibit PDGFR-β transcriptional activity. Here we determined the major G-quadruplex formed in the PDGFR-β promoter. Instead of using four continuous runs with three or more guanines, this G-quadruplex adopts a novel folding with a broken G-strand to form a primarily parallel-stranded intramolecular structure with three 1 nucleotide (nt) double-chain-reversal loops and one additional lateral loop. The novel folding of the PDGFR-β promoter G-quadruplex emphasizes the robustness of parallel-stranded structural motifs with a 1 nt loop. Considering recent progress on G-quadruplexes formed in gene-promoter sequences, we suggest the 1 nt looped GiNGj motif may have been evolutionarily selected to serve as a stable foundation upon which the promoter G-quadruplexes can build. The novel folding of the PDGFR-β promoter G-quadruplex may be attractive for small-molecule drugs that specifically target this secondary structure and modulate PDGFR-β gene expression. © 2012 American Chemical Society.
• Hurley, L., Boddupally, P. V., Hahn, S., Beman, C., De, B., Brooks, T. A., Gokhale, V., & Hurley, L. -. (2012). Anticancer activity and cellular repression of c-MYC by the G-quadruplex-stabilizing 11-piperazinylquindoline is not dependent on direct targeting of the G-quadruplex in the c-MYC promoter. Journal of medicinal chemistry, 55(13).
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  This G-rich region of the c-MYC promoter has been shown to form a G-quadruplex structure that acts as a silencer element for c-MYC transcriptional control. In the present work, we have synthesized a series of 11-substituted quindoline analogues as c-MYC G-quadruplex-stabilizing compounds, and the cell-free and in vitro activity of these compounds were evaluated. Two lead compounds (4 and 12) demonstrated good cell-free profiles, and compound 4 (2-(4-(10H-indolo[3,2-b]quinolin-11-yl)piperazin-1-yl)-N,N-dimethylethanamine) significantly down-regulated c-MYC expression. However, despite the good cell-free activity and the effect of these compounds on c-MYC gene expression, we have demonstrated, using a cellular assay in a Burkitt's lymphoma cell line (CA46-specific), that these effects were not mediated through targeting of the c-MYC G-quadruplex. Thus, caution should be used in assigning the effects of G-quadruplex-interactive compounds that lower c-MYC to direct targeting of these promoter elements unless this assay, or similar ones, demonstrates direct targeting of the G-quadruplex in cells.
• V., P., Hahn, S., Beman, C., Biswanath, D. e., Brooks, T. A., Gokhale, V., & Hurley, L. H. (2012). Anticancer activity and cellular repression of c-MYC by the G-quadruplex-stabilizing 11-piperazinylquindoline is not dependent on direct targeting of the G-quadruplex in the c-MYC promoter. Journal of Medicinal Chemistry, 55(13), 6076-6086.
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  PMID: 22691117;PMCID: PMC3395776;Abstract: This G-rich region of the c-MYC promoter has been shown to form a G-quadruplex structure that acts as a silencer element for c-MYC transcriptional control. In the present work, we have synthesized a series of 11-substituted quindoline analogues as c-MYC G-quadruplex-stabilizing compounds, and the cell-free and in vitro activity of these compounds were evaluated. Two lead compounds (4 and 12) demonstrated good cell-free profiles, and compound 4 (2-(4-(10H-indolo[3,2-b]quinolin-11-yl)piperazin-1-yl)-N,N-dimethylethanamine) significantly down-regulated c-MYC expression. However, despite the good cell-free activity and the effect of these compounds on c-MYC gene expression, we have demonstrated, using a cellular assay in a Burkitts lymphoma cell line (CA46-specific), that these effects were not mediated through targeting of the c-MYC G-quadruplex. Thus, caution should be used in assigning the effects of G-quadruplex-interactive compounds that lower c-MYC to direct targeting of these promoter elements unless this assay, or similar ones, demonstrates direct targeting of the G-quadruplex in cells. © 2012 American Chemical Society.
• Yu, Z., Gaerig, V., Cui, Y., Kang, H., Gokhale, V., Zhao, Y., Hurley, L. H., & Mao, H. (2012). Tertiary DNA structure in the single-stranded hTERT promoter fragment unfolds and refolds by parallel pathways via cooperative or sequential events. Journal of the American Chemical Society, 134(11), 5157-64.
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  The discovery of G-quadruplexes and other DNA secondary elements has increased the structural diversity of DNA well beyond the ubiquitous double helix. However, it remains to be determined whether tertiary interactions can take place in a DNA complex that contains more than one secondary structure. Using a new data analysis strategy that exploits the hysteresis region between the mechanical unfolding and refolding traces obtained by a laser-tweezers instrument, we now provide the first convincing kinetic and thermodynamic evidence that a higher order interaction takes place between a hairpin and a G-quadruplex in a single-stranded DNA fragment that is found in the promoter region of human telomerase. During the hierarchical unfolding or refolding of the DNA complex, a 15-nucleotide hairpin serves as a common species among three intermediates. Moreover, either a mutant that prevents this hairpin formation or the addition of a DNA fragment complementary to the hairpin destroys the cooperative kinetic events by removing the tertiary interaction mediated by the hairpin. The coexistence of the sequential and the cooperative refolding events provides direct evidence for a unifying kinetic partition mechanism previously observed only in large proteins and complex RNA structures. Not only does this result rationalize the current controversial observations for the long-range interaction in complex single-stranded DNA structures, but also this unexpected complexity in a promoter element provides additional justification for the biological function of these structures in cells.
• Zhongbo, Y. u., Gaerig, V., Cui, Y., Kang, H., Gokhale, V., Zhao, Y., Hurley, L. H., & Mao, H. (2012). Tertiary DNA structure in the single-stranded hTERT promoter fragment unfolds and refolds by parallel pathways via cooperative or sequential events. Journal of the American Chemical Society, 134(11), 5157-5164.
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  PMID: 22372563;PMCID: PMC3336359;Abstract: The discovery of G-quadruplexes and other DNA secondary elements has increased the structural diversity of DNA well beyond the ubiquitous double helix. However, it remains to be determined whether tertiary interactions can take place in a DNA complex that contains more than one secondary structure. Using a new data analysis strategy that exploits the hysteresis region between the mechanical unfolding and refolding traces obtained by a laser-tweezers instrument, we now provide the first convincing kinetic and thermodynamic evidence that a higher order interaction takes place between a hairpin and a G-quadruplex in a single-stranded DNA fragment that is found in the promoter region of human telomerase. During the hierarchical unfolding or refolding of the DNA complex, a 15-nucleotide hairpin serves as a common species among three intermediates. Moreover, either a mutant that prevents this hairpin formation or the addition of a DNA fragment complementary to the hairpin destroys the cooperative kinetic events by removing the tertiary interaction mediated by the hairpin. The coexistence of the sequential and the cooperative refolding events provides direct evidence for a unifying kinetic partition mechanism previously observed only in large proteins and complex RNA structures. Not only does this result rationalize the current controversial observations for the long-range interaction in complex single-stranded DNA structures, but also this unexpected complexity in a promoter element provides additional justification for the biological function of these structures in cells. © 2012 American Chemical Society.
• Balasubramanian, S., Hurley, L. H., & Neidle, S. (2011). Targeting G-quadruplexes in gene promoters: A novel anticancer strategy?. Nature Reviews Drug Discovery, 10(4), 261-275.
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  PMID: 21455236;PMCID: PMC3119469;Abstract: G-quadruplexes are four-stranded DNA structures that are over-represented in gene promoter regions and are viewed as emerging therapeutic targets in oncology, as transcriptional repression of oncogenes through stabilization of these structures could be a novel anticancer strategy. Many gene promoter G-quadruplexes have physicochemical properties and structural characteristics that might make them druggable, and their structural diversity suggests that a high degree of selectivity might be possible. Here, we describe the evidence for G-quadruplexes in gene promoters and discuss their potential as therapeutic targets, as well as progress in the development of strategies to harness this potential through intervention with small-molecule ligands. © 2011 Macmillan Publishers Limited. All rights reserved.
• Balasubramanian, S., Hurley, L. H., & Neidle, S. (2011). Targeting G-quadruplexes in gene promoters: a novel anticancer strategy?. Nature reviews. Drug discovery, 10(4), 261-75.
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  G-quadruplexes are four-stranded DNA structures that are over-represented in gene promoter regions and are viewed as emerging therapeutic targets in oncology, as transcriptional repression of oncogenes through stabilization of these structures could be a novel anticancer strategy. Many gene promoter G-quadruplexes have physicochemical properties and structural characteristics that might make them druggable, and their structural diversity suggests that a high degree of selectivity might be possible. Here, we describe the evidence for G-quadruplexes in gene promoters and discuss their potential as therapeutic targets, as well as progress in the development of strategies to harness this potential through intervention with small-molecule ligands.
• Brown, R. V., & Hurley, L. H. (2011). DNA acting like RNA. Biochemical Society Transactions, 39(2), 635-640.
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  PMID: 21428953;PMCID: PMC3074478;Abstract: Over the last decade or so, secondary non-B-DNA structures such as G-quadruplexes and i-motifs have come into focus as biologically functioning moieties that are potentially involved in telomeric interactions and the control of gene expression. In the present short review, we first describe the structural and dynamic parallels with complex RNA structures, including the importance of sequence and ions in folding, and then we describe the biological consequences of the folded structures. We conclude that there are considerable parallels between secondary and tertiary structures in RNA and DNA from both the folding and the biological perspectives. ©The Authors Journal compilation ©2011 Biochemical Society.
• Brown, R. V., Danford, F. L., Gokhale, V., Hurley, L. H., & Brooks, T. A. (2011). Demonstration that drug-targeted down-regulation of MYC in non-Hodgkins lymphoma is directly mediated through the promoter G-quadruplex. Journal of Biological Chemistry, 286(47), 41018-41027.
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  PMID: 21956115;PMCID: PMC3220475;Abstract: Most transcription of the MYC proto-oncogene initiates in the near upstream promoter, within which lies the nuclease hypersensitive element (NHE) III 1 region containing the CT-element. This dynamic stretch of DNA can form at least three different topologies: single-stranded DNA, double-stranded DNA, or higher order secondary structures that silence transcription. In the current report, we identify the ellipticine analog GQC-05 (NSC338258) as a high affinity, potent, and selective stabilizer of the MYC G-quadruplex (G4). In cells, GQC-05 induced cytotoxicity with corresponding decreased MYC mRNA and altered protein binding to the NHE III 1 region, in agreement with a G4 stabilizing compound. We further describe a unique feature of the Burkitt's lymphoma cell line CA46 that allowed us to clearly demonstrate the mechanism and location of action of GQC-05 within this region of DNA and through the G4. Most importantly, these data present, as far as we are aware, the most direct evidence of intracellular G4-mediated control of a particular promoter. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
• Brown, R. V., Danford, F. L., Gokhale, V., Hurley, L. H., & Brooks, T. A. (2011). Demonstration that drug-targeted down-regulation of MYC in non-Hodgkins lymphoma is directly mediated through the promoter G-quadruplex. The Journal of biological chemistry, 286(47), 41018-27.
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  Most transcription of the MYC proto-oncogene initiates in the near upstream promoter, within which lies the nuclease hypersensitive element (NHE) III(1) region containing the CT-element. This dynamic stretch of DNA can form at least three different topologies: single-stranded DNA, double-stranded DNA, or higher order secondary structures that silence transcription. In the current report, we identify the ellipticine analog GQC-05 (NSC338258) as a high affinity, potent, and selective stabilizer of the MYC G-quadruplex (G4). In cells, GQC-05 induced cytotoxicity with corresponding decreased MYC mRNA and altered protein binding to the NHE III(1) region, in agreement with a G4 stabilizing compound. We further describe a unique feature of the Burkitt's lymphoma cell line CA46 that allowed us to clearly demonstrate the mechanism and location of action of GQC-05 within this region of DNA and through the G4. Most importantly, these data present, as far as we are aware, the most direct evidence of intracellular G4-mediated control of a particular promoter.
• Dai, J., Carver, M., Hurley, L. H., & Yang, D. (2011). Solution structure of a 2:1 quindoline-c-MYC G-quadruplex: Insights into G-quadruplex-interactive small molecule drug design. Journal of the American Chemical Society, 133(44), 17673-17680.
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  PMID: 21967482;PMCID: PMC3207019;Abstract: Unimolecular parallel-stranded G-quadruplex structures are found to be prevalent in gene promoters. The nuclease hypersensitivity element III 1 (NHE III 1) of the c-MYC promoter can form transcriptionally active and silenced forms, and the formation of DNA G-quadruplex structures has been shown to be critical for c-MYC transcriptional silencing. The solution structure of a 2:1 quindoline-G-quadruplex complex has been solved and shows unexpected features, including the drug-induced reorientation of the flanking sequences to form a new binding pocket. While both 3′ and 5′ complexes show overall similar features, there are identifiable differences that emphasize the importance of both stacking and electronic interactions. For the first time, we describe the importance of the shape of the ligand as well as the two flanking bases in determining drug binding specificity. These structures provide important insights for the structure-based rational design of drugs that bind to unimolecular parallel G-quadruplexes commonly found in promoter elements. © 2011 American Chemical Society.
• Dai, J., Carver, M., Hurley, L. H., & Yang, D. (2011). Solution structure of a 2:1 quindoline-c-MYC G-quadruplex: insights into G-quadruplex-interactive small molecule drug design. Journal of the American Chemical Society, 133(44), 17673-80.
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  Unimolecular parallel-stranded G-quadruplex structures are found to be prevalent in gene promoters. The nuclease hypersensitivity element III(1) (NHE III(1)) of the c-MYC promoter can form transcriptionally active and silenced forms, and the formation of DNA G-quadruplex structures has been shown to be critical for c-MYC transcriptional silencing. The solution structure of a 2:1 quindoline-G-quadruplex complex has been solved and shows unexpected features, including the drug-induced reorientation of the flanking sequences to form a new binding pocket. While both 3' and 5' complexes show overall similar features, there are identifiable differences that emphasize the importance of both stacking and electronic interactions. For the first time, we describe the importance of the shape of the ligand as well as the two flanking bases in determining drug binding specificity. These structures provide important insights for the structure-based rational design of drugs that bind to unimolecular parallel G-quadruplexes commonly found in promoter elements.
• Hahn, T., Bradley-Dunlop, D. J., Hurley, L. H., Von-Hoff, D., Gately, S., Mary, D. L., Hailing, L. u., Penichet, M. L., Besselsen, D. G., Cole, B. B., Meeuwsen, T., Walker, E., & Akporiaye, E. T. (2011). The vitamin E analog, alpha-tocopheryloxyacetic acid enhances the anti-tumor activity of trastuzumab against HER2/neu-expressing breast cancer. BMC Cancer, 11.
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  PMID: 22044845;PMCID: PMC3217981;Abstract: Background: HER2/neu is an oncogene that facilitates neoplastic transformation due to its ability to transduce growth signals in a ligand-independent manner, is over-expressed in 20-30% of human breast cancers correlating with aggressive disease and has been successfully targeted with trastuzumab (Herceptin®). Because trastuzumab alone achieves only a 15-30% response rate, it is now commonly combined with conventional chemotherapeutic drugs. While the combination of trastuzumab plus chemotherapy has greatly improved response rates and increased survival, these conventional chemotherapy drugs are frequently associated with gastrointestinal and cardiac toxicity, bone marrow and immune suppression. These drawbacks necessitate the development of new, less toxic drugs that can be combined with trastuzumab. Recently, we reported that orally administered alpha-tocopheryloxyacetic acid (α-TEA), a novel ether derivative of alpha-tocopherol, dramatically suppressed primary tumor growth and reduced the incidence of lung metastases both in a transplanted and a spontaneous mouse model of breast cancer without discernable toxicity.Methods: In this study we examined the effect of α-TEA plus HER2/neu-specific antibody treatment on HER2/neu-expressing breast cancer cells in vitro and in a HER2/neu positive human xenograft tumor model in vivo.Results: We show in vitro that α-TEA plus anti-HER2/neu antibody has an increased cytotoxic effect against murine mammary tumor cells and human breast cancer cells and that the anti-tumor effect of α-TEA is independent of HER2/neu status. More importantly, in a human breast cancer xenograft model, the combination of α-TEA plus trastuzumab resulted in faster tumor regression and more tumor-free animals than trastuzumab alone.Conclusion: Due to the cancer cell selectivity of α-TEA, and because α-TEA kills both HER2/neu positive and HER2/neu negative breast cancer cells, it has the potential to be effective and less toxic than existing chemotherapeutic drugs when used in combination with HER2/neu antibody. © 2011 Hahn et al; licensee BioMed Central Ltd.
• Hahn, T., Bradley-Dunlop, D. J., Hurley, L. H., Von-Hoff, D., Gately, S., Mary, D. L., Lu, H., Penichet, M. L., Besselsen, D. G., Cole, B. B., Meeuwsen, T., Walker, E., & Akporiaye, E. T. (2011). The vitamin E analog, alpha-tocopheryloxyacetic acid enhances the anti-tumor activity of trastuzumab against HER2/neu-expressing breast cancer. BMC cancer, 11, 471.
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  HER2/neu is an oncogene that facilitates neoplastic transformation due to its ability to transduce growth signals in a ligand-independent manner, is over-expressed in 20-30% of human breast cancers correlating with aggressive disease and has been successfully targeted with trastuzumab (Herceptin®). Because trastuzumab alone achieves only a 15-30% response rate, it is now commonly combined with conventional chemotherapeutic drugs. While the combination of trastuzumab plus chemotherapy has greatly improved response rates and increased survival, these conventional chemotherapy drugs are frequently associated with gastrointestinal and cardiac toxicity, bone marrow and immune suppression. These drawbacks necessitate the development of new, less toxic drugs that can be combined with trastuzumab. Recently, we reported that orally administered alpha-tocopheryloxyacetic acid (α-TEA), a novel ether derivative of alpha-tocopherol, dramatically suppressed primary tumor growth and reduced the incidence of lung metastases both in a transplanted and a spontaneous mouse model of breast cancer without discernable toxicity.
• Hurley, L., Brown, R. V., & Hurley, L. -. (2011). DNA acting like RNA. Biochemical Society transactions, 39(2).
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  Over the last decade or so, secondary non-B-DNA structures such as G-quadruplexes and i-motifs have come into focus as biologically functioning moieties that are potentially involved in telomeric interactions and the control of gene expression. In the present short review, we first describe the structural and dynamic parallels with complex RNA structures, including the importance of sequence and ions in folding, and then we describe the biological consequences of the folded structures. We conclude that there are considerable parallels between secondary and tertiary structures in RNA and DNA from both the folding and the biological perspectives.
• Nichol, G. S., Boddupally, P. V., Biswanath, D. e., & Hurley, L. H. (2011). 3-[4-(10H-Indolo[3,2-b]quinolin-11-yl)piperazin-1-yl]propan-1-ol. Acta Crystallographica Section E: Structure Reports Online, 67(12), o3465-o3466.
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  PMID: 22199941;PMCID: PMC3239093;Abstract: In the title compound, C22H24N4O, the aromatic moiety is essentially planar (r.m.s. deviation of a least-squares plane fitted through all non-H atoms = 0.0386 Å) and is rotated by 89.98 (4)°from the piperazine ring, which adopts the expected chair conformation. The propanol chain is not fully extended away from the piperazine ring. In the crystal, there are two unique hydrogen-bonding interactions. One is an O-H⋯N interaction which, together with an inversion-related symmetry equivalent, forms a ring motif. The second is an N-H⋯N interaction which links adjacent molecules by means of a chain motif which propagates in the c-axis direction. Overall, a two-dimensional hydrogen-bonded structure is formed.
• Nichol, G. S., Boddupally, P. V., De, B., & Hurley, L. H. (2011). 3-[4-(10H-Indolo[3,2-b]quinolin-11-yl)piperazin-1-yl]propan-1-ol. Acta crystallographica. Section E, Structure reports online, 67(Pt 12), o3465-6.
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  In the title compound, C(22)H(24)N(4)O, the aromatic moiety is essentially planar (r.m.s. deviation of a least-squares plane fitted through all non-H atoms = 0.0386 Å) and is rotated by 89.98 (4)° from the piperazine ring, which adopts the expected chair conformation. The propanol chain is not fully extended away from the piperazine ring. In the crystal, there are two unique hydrogen-bonding inter-actions. One is an O-H⋯N inter-action which, together with an inversion-related symmetry equivalent, forms a ring motif. The second is an N-H⋯N inter-action which links adjacent mol-ecules by means of a chain motif which propagates in the c-axis direction. Overall, a two-dimensional hydrogen-bonded structure is formed.
• Brooks, T. A., Kendrick, S., & Hurley, L. (2010). Making sense of G-quadruplex and i-motif functions in oncogene promoters. FEBS Journal, 277(17), 3459-3469.
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  PMID: 20670278;PMCID: PMC2971675;Abstract: The presence and biological importance of DNA secondary structures in eukaryotic promoters are becoming increasingly recognized among chemists and biologists as bioinformatics in vitro and in vivo evidence for these structures in the c-Myc, c-Kit, KRAS, PDGF-A, hTERT, Rb, RET and Hif-1 promoters accumulates. Nevertheless, the evidence remains largely circumstantial. This minireview differs from previous ones in that here we examine the diversity of G-quadruplex and i-motif structures in promoter elements and attempt to categorize the different types of arrangements in which they are found. For the c-Myc G-quadruplex and Bcl-2 i-motif, we summarize recent biological and structural studies. © 2010 FEBS.
• Dai, J., Hatzakis, E., Hurley, L. H., & Yang, D. (2010). I-motif structures formed in the human c-MYC promoter are highly dynamic--insights into sequence redundancy and I-motif stability. PloS one, 5(7), e11647.
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  The GC-rich nuclease hypersensitivity element III1 (NHE III1) of the c-MYC promoter largely controls the transcriptional activity of the c-MYC oncogene. The C-rich strand in this region can form I-motif DNA secondary structures. We determined the folding pattern of the major I-motif formed in the NHE III1, which can be formed at near-neutral pH. While we find that the I-motif formed in the four 3' consecutive runs of cytosines appears to be the most favored, our results demonstrate that the C-rich strand of the c-MYC NHE III1 exhibits a high degree of dynamic equilibration. Using a trisubstituted oligomer of this region, we determined the formation of two equilibrating loop isomers, one of which contains a flipped-out cytosine. Our results indicate that the intercalative cytosine+-cytosine base pairs are not always necessary for an intramolecular I-motif. The dynamic character of the c-MYC I-motif is intrinsic to the NHE III1 sequence and appears to provide stability to the c-MYC I-motif.
• Dai, J., Hatzakis, E., Hurley, L. H., & Yang, D. (2010). I-motif structures formed in the human c-MYC promoter are highly dynamic-insights into sequence redundancy and I-motif stability. PLoS ONE, 5(7).
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  PMID: 20657837;PMCID: PMC2906509;Abstract: The GC-rich nuclease hypersensitivity element III1 (NHE III1) of the c-MYC promoter largely controls the transcriptional activity of the c-MYC oncogene. The C-rich strand in this region can form I-motif DNA secondary structures. We determined the folding pattern of the major I-motif formed in the NHE III1, which can be formed at near-neutral pH. While we find that the I-motif formed in the four 3′ consecutive runs of cytosines appears to be the most favored, our results demonstrate that the C-rich strand of the c-MYC NHE III1 exhibits a high degree of dynamic equilibration. Using a trisubstituted oligomer of this region, we determined the formation of two equilibrating loop isomers, one of which contains a flipped-out cytosine. Our results indicate that the intercalative cytosine+-cytosine base pairs are not always necessary for an intramolecular Imotif. The dynamic character of the c-MYC I-motif is intrinsic to the NHE III1 sequence and appears to provide stability to the c-MYC I-motif. © 2010 Dai et al.
• Dietrich, J., Gokhale, V., Wang, X., Hurley, L. H., & Flynn, G. A. (2010). Application of a novel [3+2] cycloaddition reaction to prepare substituted imidazoles and their use in the design of potent DFG-out allosteric B-Raf inhibitors. Bioorganic & medicinal chemistry, 18(1), 292-304.
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  B-Raf protein kinase, which is a key signaling molecule in the RAS-RAF-MEK-ERK signaling pathway, plays an important role in many cancers. The B-Raf V600E mutation represents the most frequent oncogenic kinase mutation known and is responsible for increased kinase activity in approximately 7% of all human cancers, establishing B-Raf as an important therapeutic target for inhibition. Through the use of an iterative program that utilized a chemocentric approach and a rational structure based design, we have developed novel, potent, and specific DFG-out allosteric inhibitors of B-Raf kinase. Here, we present efficient and versatile chemistry that utilizes a key one pot, [3+2] cycloaddition reaction to obtain highly substituted imidazoles and their application in the design of allosteric B-Raf inhibitors. Inhibitors based on this scaffold display subnanomolar potency and a favorable kinase profile.
• Dietrich, J., Gokhale, V., Wang, X., Hurley, L. H., & Flynn, G. A. (2010). Application of a novel [3+2] cycloaddition reaction to prepare substituted imidazoles and their use in the design of potent DFG-out allosteric B-Raf inhibitors. Bioorganic and Medicinal Chemistry, 18(1), 292-304.
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  PMID: 19962319;Abstract: B-Raf protein kinase, which is a key signaling molecule in the RAS-RAF-MEK-ERK signaling pathway, plays an important role in many cancers. The B-Raf V600E mutation represents the most frequent oncogenic kinase mutation known and is responsible for increased kinase activity in approximately 7% of all human cancers, establishing B-Raf as an important therapeutic target for inhibition. Through the use of an iterative program that utilized a chemocentric approach and a rational structure based design, we have developed novel, potent, and specific DFG-out allosteric inhibitors of B-Raf kinase. Here, we present efficient and versatile chemistry that utilizes a key one pot, [3+2] cycloaddition reaction to obtain highly substituted imidazoles and their application in the design of allosteric B-Raf inhibitors. Inhibitors based on this scaffold display subnanomolar potency and a favorable kinase profile. © 2009 Elsevier Ltd. All rights reserved.
• Gonzalez, V., & Hurley, L. H. (2010). The c-MYC NHE III₁: Function and regulation. Annual Review of Pharmacology and Toxicology, 50, 111-129.
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  PMID: 19922264;Abstract: c-MYC is an important regulator of a wide array of cellular processes necessary for normal cell growth and differentiation, and its dysregulation is one of the hallmarks of many cancers. Consequently, understanding c-MYC transcriptional activation is critical for understanding developmental and cancer biology, as well as for the development of new anticancer drugs. The nuclease hypersensitive element (NHE) III1 region of the c-MYC promoter has been shown to be particularly important in regulating c-MYC expression. Specifically, the formation of a G-quadruplex structure appears to promote repression of c-MYC transcription. This review focuses on what is known about the formation of a G-quadruplex in the NHE III1 region of the c-MYC promoter, as well as on those factors that are known to modulate its formation. Last, we discuss the development of small molecules that stabilize or induce the formation of G-quadruplex structures and could potentially be used as anticancer agents. Copyright © 2010 by Annual Reviews. All rights reserved.
• González, V., & Hurley, L. H. (2010). The C-terminus of nucleolin promotes the formation of the c-MYC G-quadruplex and inhibits c-MYC promoter activity. Biochemistry, 49(45), 9706-9714.
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  PMID: 20932061;PMCID: PMC2976822;Abstract: Nucleolin, the most abundant nucleolar phosphoprotein of eukaryotic cells, is known primarily for its role in ribosome biogenesis and cell proliferation. It is, however, a multifunctional protein that, depending on the cellular context, can drive either cell proliferation or apoptosis. Our laboratory recently demonstrated that nucleolin can function as a repressor of c-MYC transcription by binding to and stabilizing the formation of a G-quadruplex structure in a region of the c-MYC promoter responsible for controlling 85-90% of c-MYC's transcriptional activity. In this study, we investigate the structural elements of nucleolin that are required for c-MYC repression. The effect of nucleolin deletion mutants on the formation and stability of the c-MYC G-quadruplex, as well as c-MYC transcriptional activity, was assessed by circular dichroism spectropolarimetry, thermal stability, and in vitro transcription. Here we report that nucleolin's RNA binding domains 3 and 4, as well as the arginine-glycine-glycine (RGG) domain, are required to repress c-MYC transcription. © 2010 American Chemical Society.
• Hurley, L., Brooks, T. A., & Hurley, L. -. (2010). Targeting MYC Expression through G-Quadruplexes. Genes & cancer, 1(6).
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  In this review, the authors describe a novel mechanism for control of MYC expression that involves a four-stranded DNA structure, termed a G-quadruplex, amenable to small molecule targeting. The DNA element involved in this mechanism, the nuclease hypersensitive element III(1) (NHE III(1)), is just upstream of the P1 promoter and is subjected to dynamic stress (negative superhelicity) resulting from transcription. This is sufficient to convert the duplex DNA to a G-quadruplex on the purine-rich strand and an i-motif of the pyrimidine-rich strand, which displaces the activating transcription factors to silence gene expression. Specific proteins have been identified, NM23-H2 and nucleolin, that resolve and fold the G-quadruplex to activate and silence MYC expression, respectively. Inhibition of the activity of NM23-H2 molecules that bind to the G-quadruplex silences gene expression, and redistribution of nucleolin from the nucleolus to the nucleoplasm is expected to inhibit MYC. The authors also describe the mechanism of action of Quarfloxin, a first-in-class G-quadruplex-interactive compound that involves the redistribution of nucleolin from the nucleolus to the nucleoplasm. G-quadruplexes have been best known as test-tube oddities for more than four decades. However, during the past decade, they have emerged as likely players in a number of important biological processes, including transcriptional control. Only time will tell if these odd DNA structures will assume the role of an established receptor class, but it is clear from the scientific literature that there is a dramatic increase in interest in this little-known area in the past few years.
• Hurley, L., Brooks, T. A., Kendrick, S., & Hurley, L. -. (2010). Making sense of G-quadruplex and i-motif functions in oncogene promoters. The FEBS journal, 277(17).
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  The presence and biological importance of DNA secondary structures in eukaryotic promoters are becoming increasingly recognized among chemists and biologists as bioinformatics in vitro and in vivo evidence for these structures in the c-Myc, c-Kit, KRAS, PDGF-A, hTERT, Rb, RET and Hif-1alpha promoters accumulates. Nevertheless, the evidence remains largely circumstantial. This minireview differs from previous ones in that here we examine the diversity of G-quadruplex and i-motif structures in promoter elements and attempt to categorize the different types of arrangements in which they are found. For the c-Myc G-quadruplex and Bcl-2 i-motif, we summarize recent biological and structural studies.
• Hurley, L., Dietrich, J., Hulme, C., & Hurley, L. -. (2010). The design, synthesis, and evaluation of 8 hybrid DFG-out allosteric kinase inhibitors: a structural analysis of the binding interactions of Gleevec, Nexavar, and BIRB-796. Bioorganic & medicinal chemistry, 18(15).
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  The majority of kinase inhibitors developed to date are competitive inhibitors that target the ATP binding site; however, recent crystal structures of Gleevec (imatinib mesylate, STI571, PDB: 1IEP), Nexavar (Sorafenib tosylate, BAY 43-9006, PDB: 1UWJ), and BIRB-796 (PDB: 1KV2) have revealed a secondary binding site adjacent to the ATP binding site known as the DFG-out allosteric binding site. The recent successes of Gleevec and Nexavar for the treatment of chronic myeloid leukemia and renal cell carcinoma has generated great interest in the development of other kinase inhibitors that target this secondary binding site. Here, we present a structural comparison of the important and similar interactions necessary for Gleevec(R), Nexavar, and BIRB-796 to bind to their respective DFG-out allosteric binding pockets and the selectivity of each with respect to c-Abl, B-Raf, and p38alpha. A structural analysis of their selectivity profiles has been generated from the synthesis and evaluation of 8 additional DFG-out allosteric inhibitors that were developed directly from fragments of these successful scaffolds.
• Hurley, L., González, V., & Hurley, L. -. (2010). The C-terminus of nucleolin promotes the formation of the c-MYC G-quadruplex and inhibits c-MYC promoter activity. Biochemistry, 49(45).
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  Nucleolin, the most abundant nucleolar phosphoprotein of eukaryotic cells, is known primarily for its role in ribosome biogenesis and cell proliferation. It is, however, a multifunctional protein that, depending on the cellular context, can drive either cell proliferation or apoptosis. Our laboratory recently demonstrated that nucleolin can function as a repressor of c-MYC transcription by binding to and stabilizing the formation of a G-quadruplex structure in a region of the c-MYC promoter responsible for controlling 85-90% of c-MYC's transcriptional activity. In this study, we investigate the structural elements of nucleolin that are required for c-MYC repression. The effect of nucleolin deletion mutants on the formation and stability of the c-MYC G-quadruplex, as well as c-MYC transcriptional activity, was assessed by circular dichroism spectropolarimetry, thermal stability, and in vitro transcription. Here we report that nucleolin's RNA binding domains 3 and 4, as well as the arginine-glycine-glycine (RGG) domain, are required to repress c-MYC transcription.
• Hurley, L., González, V., & Hurley, L. -. (2010). The c-MYC NHE III(1): function and regulation. Annual review of pharmacology and toxicology, 50.
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  c-MYC is an important regulator of a wide array of cellular processes necessary for normal cell growth and differentiation, and its dysregulation is one of the hallmarks of many cancers. Consequently, understanding c-MYC transcriptional activation is critical for understanding developmental and cancer biology, as well as for the development of new anticancer drugs. The nuclease hypersensitive element (NHE) III(1) region of the c-MYC promoter has been shown to be particularly important in regulating c-MYC expression. Specifically, the formation of a G-quadruplex structure appears to promote repression of c-MYC transcription. This review focuses on what is known about the formation of a G-quadruplex in the NHE III(1) region of the c-MYC promoter, as well as on those factors that are known to modulate its formation. Last, we discuss the development of small molecules that stabilize or induce the formation of G-quadruplex structures and could potentially be used as anticancer agents.
• Hurley, L., Kendrick, S., & Hurley, L. -. (2010). The role of G-quadruplex/i-motif secondary structures as cis-acting regulatory elements. Pure and applied chemistry. Chimie pure et appliquee, 82(8).
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  The nature of DNA has captivated scientists for more than fifty years. The discovery of the double-helix model of DNA by Watson and Crick in 1953 not only established the primary structure of DNA, but also provided the mechanism behind DNA function. Since then, researchers have continued to further the understanding of DNA structure and its pivotal role in transcription. The demonstration of DNA secondary structure formation has allowed for the proposal that the dynamics of DNA itself can function to modulate transcription. This review presents evidence that DNA can exist in a dynamic equilibrium between duplex and secondary conformations. In addition, data demonstrating that intracellular proteins as well as small molecules can shift this equilibrium in either direction to alter gene transcription will be discussed, with a focus on the modulation of proto-oncogene expression.
• Hurley, L., Qin, Y., Fortin, J. S., Tye, D., Gleason-Guzman, M., Brooks, T. A., & Hurley, L. -. (2010). Molecular cloning of the human platelet-derived growth factor receptor beta (PDGFR-beta) promoter and drug targeting of the G-quadruplex-forming region to repress PDGFR-beta expression. Biochemistry, 49(19).
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  To understand the mechanisms controlling platelet-derived growth factor receptor beta (PDGFR-beta) expression in malignancies, we have cloned and characterized the first functional promoter of the human PDGFR-beta gene, which has been confirmed by luciferase reporter gene assays. The transcription initiation sites were mapped by primer extension. Promoter deletion experiments demonstrate that the proximal, highly GC-rich region (positions -165 to -139) of the human PDGFR-beta promoter is crucial for basal promoter activity. This region is sensitive to S1 nuclease and likely to assume a non-B-form DNA secondary structure within the supercoiled plasmid. The G-rich strand in this region contains a series of runs of three or more guanines that can form multiple different G-quadruplex structures, which have been subsequently assessed by circular dichroism. A Taq polymerase stop assay has shown that three different G-quadruplex-interactive drugs can each selectively stabilize different G-quadruplex structures of the human PDGFR-beta promoter. However, in transfection experiments, only telomestatin significantly reduced the human PDGFR-beta basal promoter activity relative to the control. Furthermore, the PDGFR-beta mRNA level in Daoy cells was significantly decreased after treatment with 1 muM telomestatin for 24 h. Therefore, we propose that ligand-mediated stabilization of specific G-quadruplex structures in the human PDGFR-beta promoter can modulate its transcription.
• Hurley, L., Sun, D., & Hurley, L. -. (2010). Biochemical techniques for the characterization of G-quadruplex structures: EMSA, DMS footprinting, and DNA polymerase stop assay. Methods in molecular biology (Clifton, N.J.), 608.
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  The proximal promoter region of many human growth-related genes contains a polypurine/polypyrimidine tract that serves as multiple binding sites for Sp1 or other transcription factors. These tracts often contain a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif known for the formation of an intramolecular G-quadruplex. Recent results provide strong evidence that specific G-quadruplex structures form naturally within these polypurine/polypyrimidine tracts in many human promoter regions, raising the possibility that the transcriptional control of these genes can be modulated by G-quadruplex-interactive agents. In this chapter, we describe three general biochemical methodologies, electrophoretic mobility shift assay (EMSA), dimethylsulfate (DMS) footprinting, and the DNA polymerase stop assay, which can be useful for initial characterization of G-quadruplex structures formed by G-rich sequences.
• Kendrick, S., & Hurley, L. H. (2010). The role of G-quadruplex/i-motif secondary structures as cis-acting regulatory elements. Pure and Applied Chemistry, 82(8), 1609-1621.
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  Abstract: The nature of DNA has captivated scientists for more than 50 years. The discovery of the double-helix model of DNA by Watson and Crick in 1953 not only established the primary structure of DNA, but also provided the mechanism behind DNA function. Since then, researchers have continued to further the understanding of DNA structure and its pivotal role in transcription. The demonstration of DNA secondary structure formation has allowed for the proposal that the dynamics of DNA itself can function to modulate transcription. This review presents evidence that DNA can exist in a dynamic equilibrium between duplex and secondary conformations. In addition, data demonstrating that intracellular proteins as well as small molecules can shift this equilibrium in either direction to alter gene transcription will be discussed, with a focus on the modulation of proto-oncogene expression. © 2010 IUPAC.
• Qin, Y., Fortin, J. S., Tye, D., Gleason-Guzman, M., Brooks, T. A., & Hurley, L. H. (2010). Molecular cloning of the human platelet-derived growth factor receptor β (PDGFR-β) promoter and drug targeting of the g-quadruplex-forming region to repress PDGFR-β expression. Biochemistry, 49(19), 4208-4219.
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  PMID: 20377208;PMCID: PMC2868086;Abstract: To understand the mechanisms controlling platelet-derived growth factor receptor β (PDGFR-β) expression in malignancies, we have cloned and characterized the first functional promoter of the human PDGFR-β gene, which has been confirmed by luciferase reporter gene assays. The transcription initiation sites were mapped by primer extension. Promoter deletion experiments demonstrate that the proximal, highly GC-rich region (positions -165 to -139) of the human PDGFR-β promoter is crucial for basal promoter activity. This region is sensitive to S1 nuclease and likely to assume a non-B-form DNA secondary structure within the supercoiled plasmid. The G-rich strand in this region contains a series of runs of three or more guanines that can form multiple different G-quadruplex structures, which have been subsequently assessed by circular dichroism. A Taq polymerase stop assay has shown that three different G-quadruplex-interactive drugs can each selectively stabilize different G-quadruplex structures of the human PDGFR-β promoter. However, in transfection experiments, only telomestatin significantly reduced the human PDGFR-β basal promoter activity relative to the control. Furthermore, the PDGFR-β mRNA level in Daoy cells was significantly decreased after treatment with 1 μM telomestatin for 24 h. Therefore, we propose that ligand-mediated stabilization of specific G-quadruplex structures in the human PDGFR-β promoter can modulate its transcription. © 2010 American Chemical Society.
• Sun, D., & Hurley, L. H. (2010). Biochemical techniques for the characterization of G-quadruplex structures: EMSA, DMS footprinting, and DNA polymerase stop assay.. Methods in molecular biology (Clifton, N.J.), 608, 65-79.
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  PMID: 20012416;PMCID: PMC2797547;Abstract: The proximal promoter region of many human growth-related genes contains a polypurine/polypyrimidine tract that serves as multiple binding sites for Sp1 or other transcription factors. These tracts often contain a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif known for the formation of an intramolecular G-quadruplex. Recent results provide strong evidence that specific G-quadruplex structures form naturally within these polypurine/polypyrimidine tracts in many human promoter regions, raising the possibility that the transcriptional control of these genes can be modulated by G-quadruplex-interactive agents. In this chapter, we describe three general biochemical methodologies, electrophoretic mobility shift assay (EMSA), dimethylsulfate (DMS) footprinting, and the DNA polymerase stop assay, which can be useful for initial characterization of G-quadruplex structures formed by G-rich sequences.
• Brooks, T. A., & Hurley, L. H. (2009). The role of supercoiling in transcriptional control of MYC and its importance in molecular therapeutics. Nature Reviews Cancer, 9(12), 849-861.
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  PMID: 19907434;Abstract: MYC is deregulated in most tumour types, but an effective means to selectively target its aberrant expression is not yet available. Supercoiling that is induced by transcription has been demonstrated to have dynamic effects on DNA in the MYC promoter element: it converts duplex DNA to non-duplex DNA structures, even at considerable distances from the transcriptional start site. These non-duplex DNA structures, which control both turning on and off of transcription and the rate of transcription firing, are amenable to small-molecule targeting. This dynamic system provides a unique opportunity for the treatment of tumours in which MYC is an important oncogene. © 2009 Macmillan Publishers Limited. All rights reserved.
• Dai, J., Ambrus, A., Hurley, L. H., & Yang, D. (2009). A direct and nondestructive approach to determine the folding structure of the I-motif DNA secondary structure by NMR. Journal of the American Chemical Society, 131(17), 6102-6104.
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  PMID: 19400591;PMCID: PMC2749488;Abstract: I-motifs are four-stranded DNA secondary structures formed in C-rich DNA sequences and consist of parallel-stranded DNA duplexes zipped together in an antiparallel orientation by intercalated, hemiprotonated cytosine +-cytosine base pairs. I-motif structures have been indicated to form in various regions of the human genome as well as in nanotechnological applications. While NMR is a major tool for structural studies of I-motifs, the determination of the folding topologies of unimolecular I-motifs has been a challenging and arduous task using conventional NMR spectral assignment strategies, due to the inherent sequence redundancy of the C-rich strands in the formation of unimolecular I-motif structures. We report here a direct and nondestructive method that can be utilized to unambiguously determine the hemiprotonated C +-C base pairs and thus the folding topology of unimolecular I-motif structures formed from native C-rich DNA sequences. The reported approach uses affordable low-enrichment site-specific labeling. More significantly, the reported method can directly and unambiguously determine the equilibrating multiple conformations coexisting in a single DNA sequence, which would be a very difficult task using conventional assignment strategies. Additionally, this method can be applied to the direct detection of the base-paired thymines that are involved in the capping structures. © 2009 American Chemical Society.
• González, V., Guo, K., Hurley, L., & Sun, D. (2009). Identification and characterization of nucleolin as a c-myc G-quadruplex-binding protein. Journal of Biological Chemistry, 284(35), 23622-23635.
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  PMID: 19581307;PMCID: PMC2749137;Abstract: myc is a proto-oncogene that plays an important role in the promotion of cellular growth and proliferation. Understanding the regulation of c-myc is important in cancer biology, as it is overexpressed in a wide variety of human cancers, including most gynecological, breast, and colon cancers. We previously demonstrated that a guanine-rich region upstream of the P1 promoter of c-myc that controls 85-90% of the transcriptional activation of this gene can form an intramolecular G-quadruplex (G4) that functions as a transcriptional repressor element. In this study, we used an affinity column to purify proteins that selectively bind to the human c-myc G-quadruplex. We found that nucleolin, a multifunctional phosphoprotein, binds in vitro to the c-myc G-quadruplex structure with high affinity and selectivity when compared with other known quadruplex structures. In addition, we demonstrate that upon binding, nucleolin facilitates the formation and increases the stability of the c-myc G-quadruplex structure. Furthermore, we provide evidence that nucleolin overexpression reduces the activity of a c-myc promoter in plasmid presumably by inducing and stabilizing the formation of the c-myc G-quadruplex. Finally, we show that nucleolin binds to the c-myc promoter in HeLa cells, which indicates that this interaction occurs in vivo. In summary, nucleolin may induce c-myc G4 formation in vivo. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
• Hahn, T., Fried, K., Hurley, L. H., & Akporiaye, E. T. (2009). Orally active α-tocopheryloxyacetic acid suppresses tumor growth and multiplicity of spontaneous murine breast cancer. Molecular Cancer Therapeutics, 8(6), 1570-1578.
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  PMID: 19509249;PMCID: PMC3693733;Abstract: We recently demonstrated the antitumor efficacy of orally administered α-tocopheryloxyacetic acid (α-TEA), a redox silent and nonhydrolyzable derivative of naturally occurring vitamin E. In order to move α-TEA closer to the clinic to benefit patients with breast cancer, the present study had two goals. First, to determine the minimal effective treatment dose; and second, to test the efficacy of dietary administration of α-TEA in the clinically relevant MMTV-PyMT mouse model of spontaneous breast cancer that more closely resembles human disease. The minimal effective dose of α-TEA was evaluated in the transplantable 4T1 tumor model and we show a dose-dependent decrease of primary tumor growth and reduction of metastatic spread to the lung. Six-week-old MMTV-PyMT mice were treated with oral α-TEA for 9 weeks, with no apparent signs of drug toxicity. The α-TEA treatment delayed tumor development and significantly slowed tumor progression, resulting in a 6-fold reduction of the average cumulative tumor size. In addition, oral α-TEA caused an 80% reduction in spontaneous metastases. In situ analysis of tumor tissue identified apoptosis as an important mechanism of α-TEA-mediated tumor suppression in addition to inhibition of tumor cell proliferation. This study shows, for the first time, the ability of orally administered α-TEA to delay tumor onset and to inhibit the progression and metastatic spread of a clinically relevant model of spontaneous breast cancer. Our finding of the high efficacy in this tumor model highlights the translational potential of oral α-TEA therapy. Copyright © 2009 American Association for Cancer Research.
• Henderson, M. C., Shaw, Y. Y., Wang, H., Han, H., Hurley, L. H., Flynn, G., Dorr, R. T., & D., D. (2009). UA62784, a novel inhibitor of centromere protein E kinesin-like protein. Molecular Cancer Therapeutics, 8(1), 36-44.
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  PMID: 19139111;PMCID: PMC2634858;Abstract: Pancreatic carcinoma is the fourth leading cause of death from cancer. Novel targets and therapeutic options are needed to aid in the treatment of pancreatic cancer. The compound UA62784 is a novel fluorenone with inhibitory activity against the centromere protein E (CENP-E) kinesin-like protein. UA62784 was isolated due to its selectivity in isogenic pancreatic carcinoma cell lines with a deletion of the DPC4 gene. UA62784 causes mitotic arrest by inhibiting chromosome congression at the metaphase plate likely through inhibition of the microtubule-associated ATPase activity of CENP-E. Furthermore, CENP-E binding to kinetochores duringmitosis is not affected by UA62784, suggesting that the target lies within the motor domain of CENP-E. UA62784 is a novel specific inhibitor of CENP-E and its activity suggests a potential role for antimitotic drugs in treating pancreatic carcinomas. Copyright © 2009 American Association for Cancer Research.
• Hurley, L., Brooks, T. A., & Hurley, L. -. (2009). The role of supercoiling in transcriptional control of MYC and its importance in molecular therapeutics. Nature reviews. Cancer, 9(12).
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  MYC is deregulated in most tumour types, but an effective means to selectively target its aberrant expression is not yet available. Supercoiling that is induced by transcription has been demonstrated to have dynamic effects on DNA in the MYC promoter element: it converts duplex DNA to non-duplex DNA structures, even at considerable distances from the transcriptional start site. These non-duplex DNA structures, which control both turning on and off of transcription and the rate of transcription firing, are amenable to small-molecule targeting. This dynamic system provides a unique opportunity for the treatment of tumours in which MYC is an important oncogene.
• Hurley, L., Palumbo, S. L., Ebbinghaus, S. W., & Hurley, L. -. (2009). Formation of a unique end-to-end stacked pair of G-quadruplexes in the hTERT core promoter with implications for inhibition of telomerase by G-quadruplex-interactive ligands. Journal of the American Chemical Society, 131(31).
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  The hTERT core promoter contains a G-rich region of 12 consecutive G-tracts, embracing 3 Sp1 binding sites, and has the potential to form multiple G-quadruplexes. From the 12 runs of guanines, 9 putative hTERT G-quadruplex-forming sequences were selected to assay for G-quadruplex formation and stability using circular dichroism and a Taq polymerase stop assay. Results from biophysical and chemical assays demonstrate an approximate inverse correlation between total loop size and structure stability. Investigation of the full-length hTERT G-rich sequence using a Taq polymerase stop assay and dimethyl sulfate footprinting revealed the formation of a unique end-to-end stacked G-quadruplex structure from this sequence. This structure consists of an all parallel G-quadruplex, formed by four consecutive G-tracts, linked to another, atypical G-quadruplex, formed by two pairs of consecutive G-tracts separated by a 26-base loop. This 26-base loop likely forms a stable hairpin structure, which would explain the unexpected stability of this G-quadruplex. Significantly, the formation of this tandem G-quadruplex structure in the full-length sequence masks all three Sp1 binding sites, which is predicted to produce significant inhibition of hTERT promoter activity. Furthermore, our study implies that inhibition of telomerase activity by some G-quadruplex ligands is not only produced by targeting telomeric G-quadruplexes but also by stabilization of the hTERT promoter G-quadruplexes.
• Hurley, L., Sun, D., & Hurley, L. -. (2009). The importance of negative superhelicity in inducing the formation of G-quadruplex and i-motif structures in the c-Myc promoter: implications for drug targeting and control of gene expression. Journal of medicinal chemistry, 52(9).
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  The importance of DNA supercoiling in transcriptional regulation has been known for many years, and more recently, transcription itself has been shown to be a source of this superhelicity. To mimic the effect of transcriptionally induced negative superhelicity, the G-quadruplex/i-motif-forming region in the c-Myc promoter was incorporated into a supercoiled plasmid. We show, using enzymatic and chemical footprinting, that negative superhelicity facilitates the formation of secondary DNA structures under physiological conditions. Significantly, these structures are not the same as those formed in single-stranded DNA templates. Together with the recently demonstrated role of transcriptionally induced superhelicity in maintaining a mechanosensor mechanism for controlling the firing rate of the c-Myc promoter, we provide a more complete picture of how c-Myc transcription is likely controlled. Last, these physiologically relevant G-quadruplex and i-motif structures, along with the mechanosensor mechanism for control of gene expression, are proposed as novel mechanisms for small molecule targeting of transcriptional control of c-Myc.
• Hurley, N. E., Schildmeyer, L. A., Bosworth, K. A., Sakurai, Y., Eskin, S. G., Hurley, L. H., & McIntire, L. V. (2009). Modulating the functional contributions of c-Myc to the human endothelial cell cyclic strain response. Journal of Vascular Research, 47(1), 80-90.
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  PMID: 19729955;PMCID: PMC2855285;Abstract: This study addresses whether pathological levels of cyclic strain activate the c-Myc promoter, leading to c-Myc transcription and downstream gene induction in human umbilical vein endothelial cells (HUVEC) or human aortic endothelial cells (HAEC). mRNA and protein expression of c-Myc under physiological (6-10%) and pathological cyclic strain conditions (20%) were studied. Both c-Myc mRNA and protein expression increased 2-3-fold in HUVEC cyclically strained at 20%. c-Myc protein increased 4-fold in HAEC. In HUVEC, expression of mRNA peaked at 1.5-2 h. Subsequently, the effect of modulating c-Myc on potential downstream gene targets was determined. A small molecular weight compound that binds to and stabilizes the silencer element in the c-Myc promoter attenuates cyclic strain-induced c-Myc transcription by about 50%. This compound also modulates c-Myc downstream gene targets that may be instrumental in induction of vascular disease. Cyclic strain-induced gene expression of vascular endothelial growth factor, proliferating cell nuclear antigen and heat shock protein 60 are attenuated by this compound. These results offer a possible mechanism and promising clinical treatment for vascular diseases initiated by increased cyclic strain. Copyright © 2009 S. Karger AG, Basel.
• Kendrick, S., Akiyama, Y., Hecht, S. M., & Hurley, L. H. (2009). The i-motif in the bcl-2 P1 promoter forms an unexpectedly stable structure with a unique 8:5:7 loop folding pattern. Journal of the American Chemical Society, 131(48), 17667-17676.
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  PMID: 19908860;PMCID: PMC2787777;Abstract: Transcriptional regulation of the bcl-2 proto-oncogene is highly complex, with the majority of transcription driven by the P1 promoter site and the interaction of multiple regulatory proteins. A guanine- and cytosine-rich (GC-rich) region directly upstream of the P1 site has been shown to be integral to bcl-2 promoter activity, as deletion or mutation of this region significantly increases transcription. This GC-rich element consists of six contiguous runs of guanines and cytosines that have the potential to adopt DNA secondary structures, the G-quadruplex and i-motif, respectively. Our laboratory has previously demonstrated that the polypurine-rich strand of the bcl-2 promoter can form a mixture of three different G-quadruplex structures. In this current study, we demonstrate that the complementary polypyrimidine-rich strand is capable of forming one major intramolecular i-motif DNA secondary structure with a transition pH of 6.6. Characterization of the i-motif folding pattern using mutational studies coupled with circular dichroic spectra and thermal stability analyses revealed an 8:5:7 loop conformation as the predominant structure at pH 6.1. The folding pattern was further supported by chemical footprinting with bromine. In addition, a novel assay involving the sequential incorporation of a fluorescent thymine analog at each thymine position provided evidence of a capping structure within the top loop region of the i-motif. The potential of the GC-rich element within the bcl-2 promoter region to form DNA secondary structures suggests that the transition from the B-DNA to non-B-DNA conformation may play an important role in bcl-2 transcriptional regulation. Furthermore, the two adjacent large lateral loops in the i-motif structure provide an unexpected opportunity for protein and small molecule recognition. © 2009 American Chemical Society.
• Kendrick, S., Akiyama, Y., Hecht, S. M., & Hurley, L. H. (2009). The i-motif in the bcl-2 P1 promoter forms an unexpectedly stable structure with a unique 8:5:7 loop folding pattern. Journal of the American Chemical Society, 131(48), 17667-76.
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  Transcriptional regulation of the bcl-2 proto-oncogene is highly complex, with the majority of transcription driven by the P1 promoter site and the interaction of multiple regulatory proteins. A guanine- and cytosine-rich (GC-rich) region directly upstream of the P1 site has been shown to be integral to bcl-2 promoter activity, as deletion or mutation of this region significantly increases transcription. This GC-rich element consists of six contiguous runs of guanines and cytosines that have the potential to adopt DNA secondary structures, the G-quadruplex and i-motif, respectively. Our laboratory has previously demonstrated that the polypurine-rich strand of the bcl-2 promoter can form a mixture of three different G-quadruplex structures. In this current study, we demonstrate that the complementary polypyrimidine-rich strand is capable of forming one major intramolecular i-motif DNA secondary structure with a transition pH of 6.6. Characterization of the i-motif folding pattern using mutational studies coupled with circular dichroic spectra and thermal stability analyses revealed an 8:5:7 loop conformation as the predominant structure at pH 6.1. The folding pattern was further supported by chemical footprinting with bromine. In addition, a novel assay involving the sequential incorporation of a fluorescent thymine analog at each thymine position provided evidence of a capping structure within the top loop region of the i-motif. The potential of the GC-rich element within the bcl-2 promoter region to form DNA secondary structures suggests that the transition from the B-DNA to non-B-DNA conformation may play an important role in bcl-2 transcriptional regulation. Furthermore, the two adjacent large lateral loops in the i-motif structure provide an unexpected opportunity for protein and small molecule recognition.
• Palumbo, S. L., Ebbinghaus, S. W., & Hurley, L. H. (2009). Formation of a unique end-to-end stacked pair of G-quadruplexes in the hTERT core promoter with implications for inhibition of telomerase by G-quadruplex-interactive ligands. Journal of the American Chemical Society, 131(31), 10878-10891.
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  PMID: 19601575;PMCID: PMC2761083;Abstract: The hTERT core promoter contains a G-rich region of 12 consecutive G-tracts, embracing 3 Sp1 binding sites, and has the potential to form multiple G-quadruplexes. From the 12 runs of guanines, 9 putative hTERT G-quadruplex-forming sequences were selected to assay for G-quadruplex formation and stability using circular dichroism and a Taq polymerase stop assay. Results from biophysical and chemical assays demonstrate an approximate inverse correlation between total loop size and structure stability. Investigation of the full-length hTERT G-rich sequence using a Taq polymerase stop assay and dimethyl sulfate footprinting revealed the formation of a unique end-to-end stacked G-quadruplex structure from this sequence. This structure consists of an all parallel G-quadruplex, formed by four consecutive G-tracts, linked to another, atypical G-quadruplex, formed by two pairs of consecutive G-tracts separated by a 26-base loop. This 26-base loop likely forms a stable hairpin structure, which would explain the unexpected stability of this G-quadruplex. Significantly, the formation of this tandem G-quadruplex structure in the full-length sequence masks all three Sp1 binding sites, which is predicted to produce significant inhibition of hTERT promoter activity. Furthermore, our study implies that inhibition of telomerase activity by some G-quadruplex ligands is not only produced by targeting telomeric G-quadruplexes but also by stabilization of the hTERT promoter G-quadruplexes. © 2009 American Chemical Society.
• Shaw, A. Y., Henderson, M. C., Flynn, G., Samulitis, B., Han, H., Stratton, S. P., Chow, H. S., Hurley, L. H., & Dorr, R. T. (2009). Characterization of novel diaryl oxazole-based compounds as potential agents to treat pancreatic cancer. Journal of Pharmacology and Experimental Therapeutics, 331(2), 636-647.
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  PMID: 19657049;PMCID: PMC2775253;Abstract: A series of diaryl- and fluorenone-based analogs of the lead compound UA-62784 [4-(5-(4-methoxyphenyl)oxazol-2-yl)-9H-fluoren-9-one] was synthesized with the intention of improving upon the selective cytotoxicity of UA-62784 against human pancreatic cancer cell lines with a deletion of the tumor suppressor gene deleted in pancreas cancer locus 4 (DPC-4, SMAD-4). Over 80 analogs were synthesized and tested for antitumor activity against pancreatic cancer (PC) cell lines (the PC series). Despite a structural relationship to UA-62784, which inhibits the mitotic kinesin centromere protein E (CENP-E), none of the analogs was selective for DPC-4-deleted pancreatic cancer cell lines. Furthermore, none of the analogs was a potent or selective inhibitor of four different mitotic kinesins (mitotic kinesin-5, CENP-E, mitotic kinesin-like protein-1, and mitotic centromere-associated kinesin). Therefore, other potential mechanisms of action were evaluated. A diaryl oxazole lead analog from this series, PC-046 [5-(4-methoxyphenyl)-2-(3-(3-methoxyphenyl)pyridin-4-yl) oxazole], was shown to potently inhibit several protein kinases that are overexpressed in human pancreatic cancers, including tyrosine receptor kinase B, interleukin-1 receptor-associated kinase-4, and proto-oncogene Pim-1. Cells exposed to PC-046 exhibit a cell cycle block in the S-phase followed by apoptotic death and necrosis. PC-046 effectively reduced MiaPaca-2 tumor growth in severe combined immunodeficiency mice by 80% compared with untreated controls. The plasma half-life was 7.5 h, and cytotoxic drug concentrations of >3 μM were achieved in vivo in mice. The diaryl oxazole series of compounds represent a new chemical class of anticancer agents that inhibit several types of cancer-relevant protein kinases. Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics.
• Sun, D., & Hurley, L. H. (2009). The importance of negative superhelicity in inducing the formation of G-quadruplex and i-motif structures in the c-Myc promoter: Implications for drug targeting and control of gene expression. Journal of Medicinal Chemistry, 52(9), 2863-2874.
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  PMID: 19385599;PMCID: PMC2757002;Abstract: The importance of DNA supercoiling in transcriptional regulation has been known for many years, and more recently, transcription itself has been shown to be a source of this superhelicity. To mimic the effect of transcriptionally induced negative superhelicity, the G-quadruplex/i-motif-forming region in the c-Myc promoter was incorporated into a supercoiled plasmid. We show, using enzymatic and chemical footprinting, that negative superhelicity facilitates the formation of secondary DNA structures under physiological conditions. Significantly, these structures are not the same as those formed in single-stranded DNA templates. Together with the recently demonstrated role of transcriptionally induced superhelicity in maintaining a mechanosensor mechanism for controlling the firing rate of the c-Myc promoter, we provide a more complete picture of how c-Myc transcription is likely controlled. Last, these physiologically relevant G-quadruplex and i-motif structures, along with the mechanosensor mechanism for control of gene expression, are proposed as novel mechanisms for small molecule targeting of transcriptional control of c-Myc. © 2009 American Chemical Society.
• Carey, S. S., Gleason-Guzman, M., Gokhale, V., & Hurley, L. H. (2008). Psorospermin structural requirements for P-glycoprotein resistance reversal. Molecular Cancer Therapeutics, 7(11), 3617-3623.
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  PMID: 19001443;PMCID: PMC2586895;Abstract: Resistance to chemotherapy reduces its effectiveness, resulting in increased mortality. Psorospermin, a natural product, is a topoisomerase II-directed DNA alkylating agent active against multidrug-resistant (MDR) cell lines, including multiple myeloma. In this study, the mechanism of the P-glycoprotein (P-gp) modulation activity of psorospermin and that of its associated pharmacophore were examined. Flow cytometry shows that doxorubicin-resistant multiple myeloma cells (8226/D40) pretreated with psorospermin enhance intracellular retention of doxorubicin compared with control (75% versus 38%). Because the overexpression of P-gp is the primary cause of drug resistance in the 8226/D40 cells, psorospermin-induced sensitization was likely due to mdr1/P-gp expressional or functional inhibition. As shown by PCR and Western blot, neither transcription of mdr1 nor translation of P-gp was down-regulated by psorospermin treatment. Therefore, the mechanism of psorospermin-induced resistance reversal is most likely through a direct interaction between psorospermin and P-gp. Furthermore, because only the (2′R,3′R) isomer of psorospermin showed any resistance reversal activity, the side chain of psorospermin is apparently a crucial moiety for resistance reversal. By understanding the mechanism of psorospermin-induced MDR modulation, psorospermin and similar compounds can be combined with other chemotherapies to treat resistant cancers. Copyright © 2008 American Association for Cancer Research.
• Cashman, D. J., Buscaglia, R., Freyer, M. W., Dettler, J., Hurley, L. H., & Lewis, E. A. (2008). Molecular modeling and biophysical analysis of the c-MYC NHE-III₁ silencer element. Journal of Molecular Modeling, 14(2), 93-101.
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  PMID: 18087730;Abstract: G-Quadruplex and i-Motif-forming sequences in the promoter regions of several oncogenes show promise as targets for the regulation of oncogenes. In this study, molecular models were created for the c-MYC NHE-III1 (nuclease hypersensitivity element III1) from two 39-base complementary sequences. The NHE modeled here consists of single folded conformers of the polypurine intramolecular G-Quadruplex and the polypyrimidine intramolecular i-Motif structures, flanked by short duplex DNA sequences. The G-Quadruplex was based on published NMR structural data for the c-MYC 1:2:1 loop isomer. The i-Motif structure is theoretical (with five cytosine-cytosine pairs), where the central intercalated cytosine core interactions are based on NMR structural data obtained for a tetramolecular [d(A2 C4)4] model i-Motif. The loop structures are in silico predictions of the c-MYC i-motif loops. The porphyrin meso-tetra(N-methyl-4-pyridyl)porphine (TMPyP4), as well as the ortho and meta analogs TMPyP2 and TMPyP3, were docked to six different locations in the complete c-MYC NHE. Comparisons are made for drug binding to the NHE and the isolated G-Quadruplex and i-Motif structures. NHE models both with and without bound cationic porphyrin were simulated for 100 ps using molecular dynamics techniques, and the non-bonded interaction energies between the DNA and porphyrins calculated for all of the docking interactions. © Springer-Verlag 2007.
• Guo, K., Gokhale, V., Hurley, L. H., & Sun, D. (2008). Intramolecularly folded G-quadruplex and i-motif structures in the proximal promoter of the vascular endothelial growth factor gene. Nucleic Acids Research, 36(14), 4598-4608.
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  PMID: 18614607;PMCID: PMC2504309;Abstract: A polyguanine/polycytosine (polyG/polyC) tract in the proximal promoter of the vascular endothelial growth factor (VEGF) gene is essential for transcriptional activation. The guanine-rich (G-rich) and cytosine-rich (C-rich) strands on this tract are shown to form specific secondary structures, characterized as G-quadruplexes and i-motifs, respectively. Mutational analysis of the G-rich strand combined with dimethyl sulfate (DMS) footprinting, a polymerase stop assay, and circular dichroism (CD) spectroscopy revealed that the G-quadruplex containing a 1:4:1 double-chain reversal loop is the most thermodynamically stable conformation that this strand readily adopts. These studies provide strong evidence that the size of loop regions plays a critical role in determining the most favored folding pattern of a G-quadruplex. The secondary structure formed on the complementary C-rich strand was also determined by mutational analysis combined with Br2 footprinting and CD spectroscopy. Our results reveal that at a pH of 5.9 this strand is able to form an intramolecular i-motif structure that involves six C-C+ base pairs and a 2:3:2 loop configuration. Taken together, our results demonstrate that the G-quadruplex and i-motif structures are able to form on the G- and C-rich strands, respectively, of the polyG/polyC tract in the VEGF proximal promoter under conditions that favor the transition from B-DNA to non-B-DNA conformations. © 2008 The Author(s).
• Hurley, L., Carey, S. S., Gleason-Guzman, M., Gokhale, V., & Hurley, L. -. (2008). Psorospermin structural requirements for P-glycoprotein resistance reversal. Molecular cancer therapeutics, 7(11).
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  Resistance to chemotherapy reduces its effectiveness, resulting in increased mortality. Psorospermin, a natural product, is a topoisomerase II-directed DNA alkylating agent active against multidrug-resistant (MDR) cell lines, including multiple myeloma. In this study, the mechanism of the P-glycoprotein (P-gp) modulation activity of psorospermin and that of its associated pharmacophore were examined. Flow cytometry shows that doxorubicin-resistant multiple myeloma cells (8226/D40) pretreated with psorospermin enhance intracellular retention of doxorubicin compared with control (75% versus 38%). Because the overexpression of P-gp is the primary cause of drug resistance in the 8226/D40 cells, psorospermin-induced sensitization was likely due to mdr1/P-gp expressional or functional inhibition. As shown by PCR and Western blot, neither transcription of mdr1 nor translation of P-gp was down-regulated by psorospermin treatment. Therefore, the mechanism of psorospermin-induced resistance reversal is most likely through a direct interaction between psorospermin and P-gp. Furthermore, because only the (2'R,3'R) isomer of psorospermin showed any resistance reversal activity, the side chain of psorospermin is apparently a crucial moiety for resistance reversal. By understanding the mechanism of psorospermin-induced MDR modulation, psorospermin and similar compounds can be combined with other chemotherapies to treat resistant cancers.
• Hurley, L., Qin, Y., & Hurley, L. -. (2008). Structures, folding patterns, and functions of intramolecular DNA G-quadruplexes found in eukaryotic promoter regions. Biochimie, 90(8).
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  In its simplest form, a DNA G-quadruplex is a four-stranded DNA structure that is composed of stacked guanine tetrads. G-quadruplex-forming sequences have been identified in eukaryotic telomeres, as well as in non-telomeric genomic regions, such as gene promoters, recombination sites, and DNA tandem repeats. Of particular interest are the G-quadruplex structures that form in gene promoter regions, which have emerged as potential targets for anticancer drug development. Evidence for the formation of G-quadruplex structures in living cells continues to grow. In this review, we examine recent studies on intramolecular G-quadruplex structures that form in the promoter regions of some human genes in living cells and discuss the biological implications of these structures. The identification of G-quadruplex structures in promoter regions provides us with new insights into the fundamental aspects of G-quadruplex topology and DNA sequence-structure relationships. Progress in G-quadruplex structural studies and the validation of the biological role of these structures in cells will further encourage the development of small molecules that target these structures to specifically modulate gene transcription.
• Palumbo, S. L., Memmott, R. M., Uribe, D. J., Krotova-Khan, Y., Hurley, L. H., & Ebbinghaus, S. W. (2008). A novel G-quadruplex-forming GGA repeat region in the c-myb promoter is a critical regulator of promoter activity. Nucleic Acids Research, 36(6), 1755-1769.
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  PMID: 18252774;PMCID: PMC2330228;Abstract: The c-myb promoter contains multiple GGA repeats beginning 17 bp downstream of the transcription initiation site. GGA repeats have been previously shown to form unusual DNA structures in solution. Results from chemical footprinting, circular dichroism and RNA and DNA polymerase arrest assays on oligonucleotides representing the GGA repeat region of the c-myb promoter demonstrate that the element is able to form tetrad:heptad:heptad:tetrad (T:H:H:T) G-quadruplex structures by stacking two tetrad:heptad G-quadruplexes formed by two of the three (GGA)4 repeats. Deletion of one or two (GGA)4 motifs destabilizes this secondary structure and increases c-myb promoter activity, indicating that the G-quadruplexes formed in the c-myb GGA repeat region may act as a negative regulator of the c-myb promoter. Complete deletion of the c-myb GGA repeat region abolishes c-myb promoter activity, indicating dual roles of the c-myb GGA repeat element as both a transcriptional repressor and an activator. Furthermore, we demonstrated that Myc-associated zinc finger protein (MAZ) represses c-myb promoter activity and binds to the c-myb T:H:H:T G-quadruplexes. Our findings show that the T:H:H:T G-quadruplex-forming region in the c-myb promoter is a critical cis-acting element and may repress c-myb promoter activity through MAZ interaction with G-quadruplexes in the c-myb promoter. © 2008 The Author(s).
• Qin, Y., & Hurley, L. H. (2008). Structures, folding patterns, and functions of intramolecular DNA G-quadruplexes found in eukaryotic promoter regions. Biochimie, 90(8), 1149-1171.
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  PMID: 18355457;PMCID: PMC2585383;Abstract: In its simplest form, a DNA G-quadruplex is a four-stranded DNA structure that is composed of stacked guanine tetrads. G-quadruplex-forming sequences have been identified in eukaryotic telomeres, as well as in non-telomeric genomic regions, such as gene promoters, recombination sites, and DNA tandem repeats. Of particular interest are the G-quadruplex structures that form in gene promoter regions, which have emerged as potential targets for anticancer drug development. Evidence for the formation of G-quadruplex structures in living cells continues to grow. In this review, we examine recent studies on intramolecular G-quadruplex structures that form in the promoter regions of some human genes in living cells and discuss the biological implications of these structures. The identification of G-quadruplex structures in promoter regions provides us with new insights into the fundamental aspects of G-quadruplex topology and DNA sequence-structure relationships. Progress in G-quadruplex structural studies and the validation of the biological role of these structures in cells will further encourage the development of small molecules that target these structures to specifically modulate gene transcription. © 2008 Elsevier Masson SAS. All rights reserved.
• Sun, D., Liu, W., Guo, K., Rusche, J. J., Ebbinghaus, S., Gokhale, V., & Hurley, L. H. (2008). The proximal promoter region of the human vascular endothelial growth factor gene has a G-quadruplex structure that can be targeted by G-quadruplex-interactive agents. Molecular Cancer Therapeutics, 7(4), 880-889.
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  PMID: 18413801;PMCID: PMC2367258;Abstract: Previous studies on the functional analysis of the human vascular endothelial growth factor (VEGF) promoter using the full-length VEGF promoter reporter revealed that the proximal 36-bp region (-85 to -50 relative to transcription initiation site) is essential for basal or inducible VEGF promoter activity in several human cancer cells. This region consists of a polypurine (guanine) tract that contains four runs of at least three contiguous guanines separated by one or more bases, thus conforming to a general motif capable of forming an intramolecular G-quadruplex. Here, we show that the G-rich strand in this region is able to form an intramolecular propeller-type parallel-stranded G-quadruplex structure in vitro by using the electrophoretic mobility shift assay, dimethyl sulfate footprinting technique, the DNA polymerase stop assay, circular dichroism spectroscopy, and computer-aided molecular modeling. Two well-known G-quadruplex-interactive agents, TMPyP4and Se2SAP, stabilize G-quadruplex structures formed by this sequence in the presence of a potassium ion, although Se2SAP is at least 10-fold more effective in binding to the G-quadruplex than TMPyP4. Between these two agents, Se2SAP better suppresses VEGF transcription in different cancer cell lines, including HEC1A and MDA-MB-231. Collectively, our results provide evidence that specific G-quadruplex structures can be formed in the VEGF promoter region, and that the transcription of this gene can be controlled by ligand-mediated G-quadruplex stabilization. Our results also provide further support for the idea that G-quadruplex structures may play structural roles in vivo and therefore might provide insight into novel methodologies for rational drug design. Copyright © 2008 American Association for Cancer Research.
• Freyer, M. W., Buscaglia, R., Kaplan, K., Cashman, D., Hurley, L. H., & Lewis, E. A. (2007). Biophysical studies of the c-MYC NHE III₁ promoter: Model quadruplex interactions with a cationic porphyrin. Biophysical Journal, 92(6), 2007-2015.
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  PMID: 17172304;PMCID: PMC1861781;Abstract: Regulation of the structural equilibrium of G-quadruplex-forming sequences located in the promoter regions of oncogenes by the binding of small molecules has shown potential as a new avenue for cancer chemotherapy. In this study, microcalorimetry (isothermal titration calorimetry and differential scanning calorimetry), electronic spectroscopy (ultraviolet-visible and circular dichroism), and molecular modeling were used to probe the complex interactions between a cationic porphryin mesotetra (N-methyl-4-pyridyl) porphine (TMPyP4) and the c-MYC PU 27-mer quadruplex. The stoichiometry at saturation is 4:1 mol of TMPyP4/c-MYC PU 27-mer G-quadruplex as determined by isothermal titration calorimetry, circular dichroism, and ultraviolet-visible spectroscopy. The four independent TMPyP4 binding sites fall into one of two modes. The two binding modes are different with respect to affinity, enthalpy change, and entropy change for formation of the 1:1 and 2:1, or 3:1 and 4:1 complexes. Binding of TMPyP4, at or near physiologic ionic strength ([K+] = 0.13 M), is described by a "two-independent-sites model." The two highest-affinity sites exhibit a K1 of 1.6 x 107 M-1 and the two lowest-affinity sites exhibit a K2 of 4.2 x 105 M -1. Dissection of the free-energy change into the enthalpy- and entropy-change contributions for the two modes is consistent with both "intercalative" and "exterior" binding mechanisms. An additional complexity is that there may be as many as six possible conformational quadruplex isomers based on the sequence. Differential scanning calorimetry experiments demonstrated two distinct melting events (Tm1 = 74.7°C and Tm2 = 91.2°C) resulting from a mixture of at least two conformers for the c-MYC PU 27-mer in solution. © 2007 by the Biophysical Society.
• Guo, K., Pourpak, A., Beetz-Rogers, K., Gokhale, V., Sun, D., & Hurley, L. H. (2007). Formation of pseudosymmetrical G-quadruplex and i-motif structures in the proximal promoter region of the RET oncogene. Journal of the American Chemical Society, 129(33), 10220-10228.
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  PMID: 17672459;PMCID: PMC2566970;Abstract: A polypurine (guanine)/polypyrimidine (cytosine)-rich sequence within the proximal promoter region of the human RET oncogene has been shown to be essential for RET basal transcription. Specifically, the G-rich strand within this region consists of five consecutive runs of guanines, which is consistent with the general motif capable of forming intramolecular G-quadruplexes. Here we demonstrate that, in the presence of 100 mM K+, this G-rich strand has the ability to adopt two intramolecular G-quadruplex structures in vitro. Moreover, comparative circular dichroism (CD) and DMS footprinting studies have revealed that the 3′-G-quadruplex structure is a parallel-type intramolecular structure containing three G-tetrads. The G-quadruplex- interactive agents TMPyP4 and telomestatin further stabilize this G-quadruplex structure. In addition, we demonstrate that the complementary C-rich strand forms an i-motif structure in vitro, as shown by CD spectroscopy and chemical footprinting. This 19-mer duplex sequence is predicted to form stable intramolecular G-quadruplex and i-motif species having minimum symmetrical loop sizes of 1:3:1 and 2:3: 2, respectively. Together, our results indicate that stable G-quadruplex and i-motif structures can form within the proximal promoter region of the human RET oncogene, suggesting that these secondary structures play an important role in transcriptional regulation of this gene. © 2007 American Chemical Society.
• Hurley, L., Guo, K., Pourpak, A., Beetz-Rogers, K., Gokhale, V., Sun, D., & Hurley, L. -. (2007). Formation of pseudosymmetrical G-quadruplex and i-motif structures in the proximal promoter region of the RET oncogene. Journal of the American Chemical Society, 129(33).
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  A polypurine (guanine)/polypyrimidine (cytosine)-rich sequence within the proximal promoter region of the human RET oncogene has been shown to be essential for RET basal transcription. Specifically, the G-rich strand within this region consists of five consecutive runs of guanines, which is consistent with the general motif capable of forming intramolecular G-quadruplexes. Here we demonstrate that, in the presence of 100 mM K+, this G-rich strand has the ability to adopt two intramolecular G-quadruplex structures in vitro. Moreover, comparative circular dichroism (CD) and DMS footprinting studies have revealed that the 3'-G-quadruplex structure is a parallel-type intramolecular structure containing three G-tetrads. The G-quadruplex-interactive agents TMPyP4 and telomestatin further stabilize this G-quadruplex structure. In addition, we demonstrate that the complementary C-rich strand forms an i-motif structure in vitro, as shown by CD spectroscopy and chemical footprinting. This 19-mer duplex sequence is predicted to form stable intramolecular G-quadruplex and i-motif species having minimum symmetrical loop sizes of 1:3:1 and 2:3:2, respectively. Together, our results indicate that stable G-quadruplex and i-motif structures can form within the proximal promoter region of the human RET oncogene, suggesting that these secondary structures play an important role in transcriptional regulation of this gene.
• Hurley, L., Qin, Y., Rezler, E. M., Gokhale, V., Sun, D., & Hurley, L. -. (2007). Characterization of the G-quadruplexes in the duplex nuclease hypersensitive element of the PDGF-A promoter and modulation of PDGF-A promoter activity by TMPyP4. Nucleic acids research, 35(22).
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  The proximal 5'-flanking region of the human platelet-derived growth factor A (PDGF-A) promoter contains one nuclease hypersensitive element (NHE) that is critical for PDGF-A gene transcription. On the basis of circular dichroism (CD) and electrophoretic mobility shift assay (EMSA), we have shown that the guanine-rich (G-rich) strand of the DNA in this region can form stable intramolecular parallel G-quadruplexes under physiological conditions. A Taq polymerase stop assay has shown that the G-rich strand of the NHE can form two major G-quadruplex structures, which are in dynamic equilibrium and differentially stabilized by three G-quadruplex-interactive drugs. One major parallel G-quadruplex structure of the G-rich strand DNA of NHE was identified by CD and dimethyl sulfate (DMS) footprinting. Surprisingly, CD spectroscopy shows a stable parallel G-quadruplex structure formed within the duplex DNA of the NHE at temperatures up to 100 degrees C. This structure has been characterized by DMS footprinting in the double-stranded DNA of the NHE. In transfection experiments, 10 microM TMPyP4 reduced the activity of the basal promoter of PDGF-A approximately 40%, relative to the control. On the basis of these results, we have established that ligand-mediated stabilization of G-quadruplex structures within the PDGF-A NHE can silence PDGF-A expression.
• Qin, Y., Rezler, E. M., Gokhale, V., Sun, D., & Hurley, L. H. (2007). Characterization of the G-quadruplexes in the duplex nuclease hypersensitive element of the PDGF-A promoter and modulation of PDGF-A promoter activity by TMPyP4. Nucleic Acids Research, 35(22), 7698-7713.
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  PMID: 17984069;PMCID: PMC2190695;Abstract: The proximal 5′-flanking region of the human platelet-derived growth factor A (PDGF-A) promoter contains one nuclease hypersensitive element (NHE) that is critical for PDGF-A gene transcription. On the basis of circular dichroism (CD) and electrophoretic mobility shift assay (EMSA), we have shown that the guanine-rich (G-rich) strand of the DNA in this region can form stable intramolecular parallel G-quadruplexes under physiological conditions. A Taq polymerase stop assay has shown that the G-rich strand of the NHE can form two major G-quadruplex structures, which are in dynamic equilibrium and differentially stabilized by three G-quadruplex-interactive drugs. One major parallel G-quadruplex structure of the G-rich strand DNA of NHE was identified by CD and dimethyl sulfate (DMS) footprinting. Surprisingly, CD spectroscopy shows a stable parallel G-quadruplex structure formed within the duplex DNA of the NHE at temperatures up to 100 °. This structure has been characterized by DMS footprinting in the double-stranded DNA of the NHE. In transfection experiments, 10 μ M TMPyP4 reduced the activity of the basal promoter of PDGF-A ∼ 40%, relative to the control. On the basis of these results, we have established that ligand-mediated stabilization of G-quadruplex structures within the PDGF-A NHE can silence PDGF-A expression. © 2007 The Author(s).
• Zhu, M., Gokhale, V. M., Szabo, L., Munoz, R. M., Baek, H., Bashyam, S., Hurley, L. H., D., D., & Han, H. (2007). Identification of a novel inhibitor of urokinase-type plasminogen activator. Molecular Cancer Therapeutics, 6(4), 1348-1356.
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  PMID: 17431113;Abstract: Urokinase-type plasminogen activator (uPA), a highly restricted serine protease, plays an important role in the regulation of diverse physiologic and pathologic processes. Strong clinical and experimental evidence has shown that elevated uPA expression is associated with cancer progression, metastasis, and shortened survival in patients. uPA has been considered as a promising molecular target for development of anticancer drugs. Here, we report the identification of several new uPA inhibitors using a high-throughput screen from a chemical library. From these uPA inhibitors, molecular modeling and docking studies identified 4-oxazolidinone as a novel lead pharmacophore. Optimization of the 4-oxazolidinone pharmacophore resulted in a series of structurally modified compounds with improved potency and selectivity. One of the 4-oxazolidinone analogues, UK 122, showed the highest inhibition of uPA activity. The IC50 of UK 122 in a cell-free indirect uPA assay is 0.2 μmol/L. This compound also showed no or little inhibition of other serine proteases such as thrombin, trypsin, plasmin, and the tissue-type plasminogen activator, indicating its high specificity against uPA. Moreover, UK122 showed little cytotoxicity against CFPAC-1 cells (IC50 > 100 μmol/L) but significantly inhibited the migration and invasion of this pancreatic cancer cell line. Our data show that UK122 could potentially be developed as a new anticancer agent that prevents the invasion and metastasis of pancreatic cancer. Copyright © 2007 American Association for Cancer Research.
• Dai, J., Chen, D., Jones, R. A., Hurley, L. H., & Yang, D. (2006). NMR solution structure of the major G-quadruplex structure formed in the human BCL2 promoter region. Nucleic Acids Research, 34(18), 5133-5144.
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  PMID: 16998187;PMCID: PMC1636422;Abstract: BCL2 protein functions as an inhibitor of cell apoptosis and has been found to be aberrantly expressed in a wide range of human diseases. A highly GC-rich region upstream of the P1 promoter plays an important role in the transcriptional regulation of BCL2. Here we report the NMR solution structure of the major intramolecular G-quadruplex formed on the G-rich strand of this region in K+ solution. This well-defined mixed parallel/antiparallel-stranded G-quadruplex structure contains three G-tetrads of mixed G-arrangements, which are connected with two lateral loops and one side loop, and four grooves of different widths. The three loops interact with the core G-tetrads in a specific way that defines and stabilizes the overall G-quadruplex structure. The loop conformations are in accord with the experimental mutation and footprinting data. The first 3-nt loop adopts a lateral loop conformation and appears to determine the overall folding of the BCL2 G-quadruplex. The third 1-nt double-chain-reversal loop defines another example of a stable parallel-stranded structural motif using the G3 NG3 sequence. Significantly, the distinct major BCL2 promoter G-quadruplex structure suggests that it can be specifically involved in gene modulation and can be an attractive target for pathway-specific drug design. © 2006 Oxford University Press.
• Dexheimer, T. S., Sun, D., & Hurley, L. H. (2006). Deconvoluting the structural and drug-recognition complexity of the G-quadruplex-forming region upstream of the bcl-2 P1 promoter. Journal of the American Chemical Society, 128(16), 5404-5415.
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  PMID: 16620112;PMCID: PMC2580050;Abstract: The human bcl-2 gene contains a GC-rich region upstream of the P1 promoter that has been shown to be critically involved in the regulation of bcl-2 gene expression. We have demonstrated that the guanine-rich strand of the DNA in this region can form any one of three distinct intramolecular G-quadruplex structures. Mutation and deletion analysis permitted isolation and identification of three overlapping DNA sequences within this element that formed the three individual G-quadruplexes. Each of these was characterized using nondenaturing gel analysis, DMS footprinting, and circular dichroism. The central G-quadruplex, which is the most stable, forms a mixed parallel/antiparallel structure consisting of three tetrads connected by loops of one, seven, and three bases. Three different G-quadruplex-interactive agents were found to further stabilize these structures, with individual selectivity toward one or more of these G-quadruplexes. Collectively, these results suggest that the multiple G-quadruplexes identified in the promoter region of the bcl-2gene are likely to play a similar role to the G-quadruplexes in the c-myc promoter in that their formation could serve to modulate gene transcription. Last, we demonstrate that the complexity of the G-quadruplexes in the bcl-2 promoter extends beyond the ability to form any one of three separate G-quadruplexes to each having the capacity to form either three or six different loop isomers. These results are discussed in relation to the biological significance of this G-quadruplex-forming element in modulation of bcl-2 gene expression and the inherent complexity of the system where different G-quadruplexes and loop isomers are possible. © 2006 American Chemical Society.
• Hahn, T., Szabo, L., Gold, M., Ramanathapuram, L., Hurley, L. H., & Akporiaye, E. T. (2006). Dietary administration of the proapoptotic vitamin E analogue α-tocopheryloxyacetic acid inhibits metastatic murine breast cancer. Cancer Research, 66(19), 9374-9378.
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  PMID: 17018590;Abstract: The ability of the vitamin E (RRR-α-tocopherol) derivatives α-tocopheryl succinate (α-TOS) and α-tocopheryloxyacetic acid (α-TEA) to suppress tumor growth in preclinical animal models has recently led to increased interest in their potential use for treating human cancer. To make the use of these vitamin E analogues more clinically relevant, we compared the antitumor efficacy of orally and i.p. delivered forms of α-TEA and α-TOS against a murine mammary cancer (4T1) that bears resemblance to human breast cancer because of its poor immunogenicity and high metastatic potential. In cell culture studies, we showed that both compounds inhibited tumor colony formation and induced apoptotic death of tumor cells. To avoid solubility concerns associated with the hydrophobicity of α-TEA and α-TOS, we used the vesiculated forms of α-TEA (Vα-TEA) and α-TOS (Vα-TOS) for the in vivo tumor studies. Both compounds inhibited the growth of preestablished 4T1 tumors when given i.p. However, when given by oral gavage, only the esterase-resistant Vα-TEA was able to suppress primary tumor growth and reduce lung metastasis. To make this approach more translatable to the clinic, α-TEA was incorporated into the diet and fed to tumor-bearing mice. We report here for the first time that dietary α-TEA delivery significantly inhibited primary tumor growth and dramatically reduced spontaneous metastatic spread to the lung in prophylactic and therapeutic settings. This study suggests that dietary α-TEA could prove useful as a relatively easy and effective modality for treating metastatic breast cancer. ©2006 American Association for Cancer Research.
• Hurley, L. H., D., D., Siddiqui-Jain, A., & Yang, D. (2006). Drug Targeting of the c-MYC Promoter to Repress Gene Expression via a G-Quadruplex Silencer Element. Seminars in Oncology, 33(4), 498-512.
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  PMID: 16890804;Abstract: In this review, we describe the evidence for a parallel-stranded G-quadruplex in the purine-rich strand of the nuclease hypersensitivity element III1 (NHE III1) of the promoter of c-MYC upstream of the P1 and P2 promoters. This biologically relevant G-quadruplex is a mixture of four loop isomers. The folding pattern of a nuclear magnetic resonance (NMR)-derived structure for the predominant loop isomer of this G-quadruplex has been obtained. This G-quadruplex has been demonstrated to be a silencer element, and the cationic porphyrin TMPyP4 has been shown to stabilize this G-quadruplex. Furthermore, TMPyP4 has been shown to repress c-MYC expression, and this effect is mediated through the silencer element. Last, the in vivo activity of TMPyP4 in xenograph models is presented. © 2006 Elsevier Inc. All rights reserved.
• Hurley, L., Dexheimer, T. S., Sun, D., & Hurley, L. -. (2006). Deconvoluting the structural and drug-recognition complexity of the G-quadruplex-forming region upstream of the bcl-2 P1 promoter. Journal of the American Chemical Society, 128(16).
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  The human bcl-2 gene contains a GC-rich region upstream of the P1 promoter that has been shown to be critically involved in the regulation of bcl-2 gene expression. We have demonstrated that the guanine-rich strand of the DNA in this region can form any one of three distinct intramolecular G-quadruplex structures. Mutation and deletion analysis permitted isolation and identification of three overlapping DNA sequences within this element that formed the three individual G-quadruplexes. Each of these was characterized using nondenaturing gel analysis, DMS footprinting, and circular dichroism. The central G-quadruplex, which is the most stable, forms a mixed parallel/antiparallel structure consisting of three tetrads connected by loops of one, seven, and three bases. Three different G-quadruplex-interactive agents were found to further stabilize these structures, with individual selectivity toward one or more of these G-quadruplexes. Collectively, these results suggest that the multiple G-quadruplexes identified in the promoter region of the bcl-2 gene are likely to play a similar role to the G-quadruplexes in the c-myc promoter in that their formation could serve to modulate gene transcription. Last, we demonstrate that the complexity of the G-quadruplexes in the bcl-2 promoter extends beyond the ability to form any one of three separate G-quadruplexes to each having the capacity to form either three or six different loop isomers. These results are discussed in relation to the biological significance of this G-quadruplex-forming element in modulation of bcl-2 gene expression and the inherent complexity of the system where different G-quadruplexes and loop isomers are possible.
• Hurley, L., Warner, S. L., Bashyam, S., Vankayalapati, H., Bearss, D. J., Han, H., Mahadevan, D., Von Hoff, D. D., & Hurley, L. -. (2006). Identification of a lead small-molecule inhibitor of the Aurora kinases using a structure-assisted, fragment-based approach. Molecular cancer therapeutics, 5(7).
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  Aurora A and Aurora B are potential targets for anticancer drug development due to their roles in tumorigenesis and disease progression. To identify small-molecule inhibitors of the Aurora kinases, we undertook a structure-based design approach that used three-dimensional structural models of the Aurora A kinase and molecular docking simulations of chemical entities. Based on these computational methods, a new generation of inhibitors derived from quinazoline and pyrimidine-based tricyclic scaffolds were synthesized and evaluated for Aurora A kinase inhibitory activity, which led to the identification of 4-(6,7-dimethoxy-9H-1,3,9-triaza-fluoren-4-yl)-piperazine-1-carbothioic acid [4-(pyrimidin-2-ylsulfamoyl)-phenyl]-amide. The lead compound showed selectivity for the Aurora kinases when it was evaluated against a panel of diverse kinases. Additionally, the compound was evaluated in cell-based assays, showing a dose-dependent decrease in phospho-histone H3 levels and an arrest of the cell cycle in the G(2)-M fraction. Although biological effects were observed only at relatively high concentrations, this chemical series provides an excellent starting point for drug optimization and further development.
• Hurley, L., Yang, D., & Hurley, L. -. (2006). Structure of the biologically relevant G-quadruplex in the c-MYC promoter. Nucleosides, nucleotides & nucleic acids, 25(8).
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  The nuclease hypersensitivity element III1 (NHE III1) in the c-MYC promoter controls up to 80-90% of the transcriptional activity of this gene. We have demonstrated that the guanine-rich strand of the NHE III1 forms a G-quadruplex consisting of a mixture of four biologically relevant loop isomers that function as a silencer element. NMR studies have shown that these G-quadruplexes are propeller-type parallel structures consisting of three stacked G-tetrads and three double-chain reversal loops. An NMR-derived solution structure for this quadruplex provides insight into the unusual stability of the structure. This structure is a target for small molecule inhibitors of c-MYC gene expression.
• Warner, S. L., Bashyam, S., Vankayalapati, H., Bearss, D. J., Han, H., D., D., & Hurley, L. H. (2006). Identification of a lead small-molecule inhibitor of the Aurora kinases using a structure-assisted, fragment-based approach. Molecular Cancer Therapeutics, 5(7), 1764-1773.
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  PMID: 16891462;Abstract: Aurora A and Aurora B are potential targets for anticancer drug development due to their roles in tumorigenesis and disease progression. To identify small-molecule inhibitors of the Aurora kinases, we undertook a structure-based design approach that used three-dimensional structural models of the Aurora A kinase and molecular docking simulations of chemical entities. Based on these computational methods, a new generation of inhibitors derived from quinazoline and pyrimidine-based tricyclic scaffolds were synthesized and evaluated for Aurora A kinase inhibitory activity, which led to the identification of 4-(6,7-dimethoxy-9H-1, 3,9-triaza-fluoren-4-yl)-piperazine-1-carbothioic acid [4-(pyrimidin-2-ylsulfamoyl)-phenyl]-amide. The lead compound showed selectivity for the Aurora kinases when it was evaluated against a panel of diverse kinases. Additionally, the compound was evaluated in cell-based assays, showing a dose-dependent decrease in phospho-histone H3 levels and an arrest of the cell cycle in the G2-M fraction. Although biological effects were observed only at relatively high concentrations, this chemical series provides an excellent starting point for drug optimization and further development. Copyright © 2006 American Association for Cancer Research.
• Warner, S. L., Munoz, R. M., Stafford, P., Koller, E., Hurley, L. H., D., D., & Han, H. (2006). Comparing Aurora A and Aurora B as molecular targets for growth inhibition of pancreatic cancer cells. Molecular Cancer Therapeutics, 5(10), 2450-2458.
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  PMID: 17041088;Abstract: To address the increased need to understand the similarities and differences in targeting Aurora A or Aurora B for the treatment of cancer, we systematically evaluated the relative importance of Aurora A and/or Aurora B as molecular targets using antisense oligonucleotides. It was found that perturbations in Aurora A and Aurora B signaling result in growth arrest and a apoptosis preferentially in cancer cells. The biological fingerprints of Aurora A and Aurora B inhibition were compared and contrasted in efforts to identify the superior therapeutic target. Due to the different biological responses, we conclude that each Aurora kinase should be treated as autonomous drug targets, which can be targeted independently or in combination. We observed no advantages to targeting both kinases simultaneously and feel that an Aurora A-targeted therapy may have some beneficial consequences over an Aurora B -targeted therapy, such as mitotic arrest and the rapid induction of apoptosis. Copyright © 2006 American Association for Cancer Research.
• Yang, D., & Hurley, L. (2006). Structure of the biologically relevant g-quadruplex in the c-MYC promoter. Nucleosides, Nucleotides and Nucleic Acids, 25(8), 951-968.
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  PMID: 16901825;Abstract: The nuclease hypersensitivity element III1 (NHE III1) in the c-MYC promoter controls up to 80-90% of the transcriptional activity of this gene. We have demonstrated that the guanine-rich strand of the NHE III 1 forms a G-quadruplex consisting of a mixture of four biologically relevant loop isomers that function as a silencer element. NMR studies have shown that these G-quadruplexes are propeller-type parallel structures consisting of three stacked G-tetrads and three double-chain reversal loops. An NMR-derived solution structure for this quadruplex provides insight into the unusual stability of the structure. This structure is a target for small molecule inhibitors of c-MYC gene expression. Copyright © Taylor & Francis Group, LLC.
• Armond, R. D., Wood, S., Sun, D., Hurley, L. H., & Ebbinghaus, S. W. (2005). Evidence for the presence of a guanine quadruplex forming region within a polypurine tract of the hypoxia inducible factor 1α promoter. Biochemistry, 44(49), 16341-16350.
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  PMID: 16331995;Abstract: The promoter of the hypoxia inducible factor 1 alpha (HIF-1α) gene has a polypurine/polypyrimidine tract (-65 to -85) overlapping or adjacent to several putative transcription factor binding sites, and we found that mutagenesis of this region diminished basal HIF-Iα expression. Oligonucleotides representing this region of the HIF-1α promoter were analyzed by electrophoretic mobility shift, chemical probing, circular dichroism, and DNA polymerase arrest assays. The guanine-rich strand was found to form a parallel, unimolecular quadruplex in the presence of potassium that was further stabilized by two known quadruplex binding compounds, the cationic porphyrin TmPyP4 and the natural product telomestatin, while TmPyP2, a positional isomer of TmPyP4, did not stabilize quadruplex formation. These data suggest that a quadruplex structure may form in a region of the HIF-1α promoter that regulates basal HIF-1α expression. © 2005 American Chemical Society.
• Fellows, I. M., Schwaebe, M., Dexheimer, T. S., Vankayalapati, H., Gleason-Guzman, M., Whitten, J. P., & Hurley, L. H. (2005). Determination of the importance of the stereochemistry of psorospermin in topoisomerase II-induced alkylation of DNA and in vitro and in vivo biological activity. Molecular Cancer Therapeutics, 4(11), 1729-1739.
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  PMID: 16275994;Abstract: Psorospermin is a natural product that has been shown to have activity against drug-resistant leukemia lines and AIDS-related lymphoma. It has also been shown to alkylate DNA through an epoxide-mediated electrophilic attack, and this alkylation is greatly enhanced at specific sites by topoisomerase II. In this article, we describe the synthesis of the two diastereomers of O5-methyl psorospermin and their in vitro activity against a range of solid and hematopoietic tumors. The diastereomeric pair (±)-(2′ R,3′ R) having the naturally occurring enantiomer (2′ R,3′ R) is the most active across all the cell lines and shows approximately equal activity in both drug-sensitive and drug-resistant cell lines. In subsequent studies using all four enantiomers of O5-methyl psorospermin, the order of biological potency is (2′ R,3′ R) > (2′ R,3′ S) = (2′ S,3′ R) > (2′ S,3′ S). This order of potency is also found in the topoisomerase II-induced alkylation of O5-methyl psorospermin and can be rationalized by molecular modeling of the psorospermin-duplex binding complex. Therefore, this study defines the optimum stereochemical requirements for both the topoisomerase II-induced alkylation of DNA and the biological activity by psorospermin and its O5-methyl derivatives. Finally, (2′ R,3′ R) psorospermin was found to be as effective as gemcitabine in slowing tumor growth in vivo in a MiaPaCa pancreatic cancer model. In addition, (2′ R,3′ R) psorospermin in combination with gemcitabine was found to show an at least additive effect in slowing tumor growth of MiaPaCa. Copyright © 2005 American Association for Cancer Research.
• Gerner, E. W., Ignatenko, N. A., Lance, P., & Hurley, L. H. (2005). A comprehensive strategy to combat colon cancer targeting the adenomatous polyposis coli tumor suppressor gene. Annals of the New York Academy of Sciences, 1059, 97-105.
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  PMID: 16382048;Abstract: Somatic cells in the majority of colorectal polyps and cancers contain mutations/deletions in the adenomatous polyposis coli (APC) tumor suppressor gene. APC is involved in normal intestinal development and acts to influence a variety of cellular processes. Loss of APC function leads to intestinal neoplasia in both mice and humans. APC influences expression of specific genes, including the c-Myc oncogene, which functions as a transcriptional activator. Loss of APC function leads to alterations in c-Myc-regulated genes including ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis. A single nucleotide polymorphism (SNP) in the ODC promoter affecting c-Myc-dependent expression has been associated with risk of colorectal and other cancers. Pharmaceuticals that target structural features of the c-Myc promoter, and suppress expression of c-Myc and other genes regulated by similar promoter elements, are being developed as potential colorectal cancer chemotherapies. Difluoromethylornithine (DFMO), a selective inhibitor of ODC, is under clinical evaluation as a colorectal cancer chemopreventive agent. APC and APC-dependent genes, such as c-Myc and ODC, may be useful as genetic markers of risk and as targets for chemoprevention and therapy for colorectal cancer. © 2005 New York Academy of Sciences.
• Grand, C. L., Powell, T. J., Nagle, R. B., Bearss, D. J., Tye, D., Gleason-Guzman, M., & Hurley, L. H. (2005). Erratum (Retracted Article): Mutations in the G-quadruplex silencer element and their relationship to c-MYC overexpression, NM23 repression, and therapeutic rescue (Proceedings of the National Academy of Sciences of the United States of America (April 20, 2004) 101:16 (6140-6145)). Proceedings of the National Academy of Sciences of the United States of America, 102(2), 516-.
• Grand, C. L., Powell, T. J., Nagle, R. B., Bearss, D. J., Tye, D., Gleason-Guzman, M., & Hurley, L. H. (2005). Mutations in the G-quadruplex silencer element and their relationship to c-MYC overexpression, NM23 repression, and therapeutic rescue.. Proceedings of the National Academy of Sciences of the United States of America, 102(2), 516-.
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  PMID: 15696627;PMCID: PMC544325;
• Heald, R. A., Dexheimer, T. S., Vankayalapati, H., Siddiqui-Jain, A., Szabo, L. Z., Gleason-Guzman, M. C., & Hurley, L. H. (2005). Conformationally restricted analogues of psorospermin: Design, synthesis, and bioactivity of natural-product-related bisfuranoxanthones. Journal of Medicinal Chemistry, 48(8), 2993-3004.
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  PMID: 15828838;Abstract: The antileukemic xanthone psorospermin is a topoisomerase II-dependent DNA alkylator in advanced preclinical development. Efforts have been made to further understand the structural requirements of its mechanism of action through the synthesis of ring-constrained analogues, based on the skeleton of the bisfuranoxanthone natural products. Molecules were designed that contain the bisfuran and xanthone portions of naturally occurring psorofebrins, and molecular modeling was used to assess their DNA alkylating potential and to refine the structures. A short, diastereoselective synthetic process to access bisfuranoxanthones was developed, culminating in the first total synthesis of (±)-isohydroxypsorofebrin. Two compounds designed and synthesized were of particular interest, chlorohydrin 7 and epoxide 6, which are reactive analogues of the natural product isohydroxypsorofebrin. The chlorohydrin retains the psorospermin-like DNA alkylation characteristics despite its rigid structure and high innate affinity for DNA. Molecular modeling has been used to rationalize the increased activity of the chlorohydrin. The chlorohydrin and epoxide show increased cytotoxicity compared to isohydroxypsorofebrin against a range of human tumor cell lines. © 2005 American Chemical Society.
• Hurley, L., Gerner, E. W., Ignatenko, N. A., Lance, P., & Hurley, L. -. (2005). A comprehensive strategy to combat colon cancer targeting the adenomatous polyposis coli tumor suppressor gene. Annals of the New York Academy of Sciences, 1059.
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  Somatic cells in the majority of colorectal polyps and cancers contain mutations/deletions in the adenomatous polyposis coli (APC) tumor suppressor gene. APC is involved in normal intestinal development and acts to influence a variety of cellular processes. Loss of APC function leads to intestinal neoplasia in both mice and humans. APC influences expression of specific genes, including the c-Myc oncogene, which functions as a transcriptional activator. Loss of APC function leads to alterations in c-Myc-regulated genes including ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis. A single nucleotide polymorphism (SNP) in the ODC promoter affecting c-Myc-dependent expression has been associated with risk of colorectal and other cancers. Pharmaceuticals that target structural features of the c-Myc promoter, and suppress expression of c-Myc and other genes regulated by similar promoter elements, are being developed as potential colorectal cancer chemotherapies. Difluoromethylornithine (DFMO), a selective inhibitor of ODC, is under clinical evaluation as a colorectal cancer chemopreventive agent. APC and APC-dependent genes, such as c-Myc and ODC, may be useful as genetic markers of risk and as targets for chemoprevention and therapy for colorectal cancer.
• Hurley, L., Heald, R. A., Dexheimer, T. S., Vankayalapati, H., Siddiqui-Jain, A., Szabo, L. Z., Gleason-Guzman, M. C., & Hurley, L. -. (2005). Conformationally restricted analogues of psorospermin: design, synthesis, and bioactivity of natural-product-related bisfuranoxanthones. Journal of medicinal chemistry, 48(8).
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  The antileukemic xanthone psorospermin is a topoisomerase II-dependent DNA alkylator in advanced preclinical development. Efforts have been made to further understand the structural requirements of its mechanism of action through the synthesis of ring-constrained analogues, based on the skeleton of the bisfuranoxanthone natural products. Molecules were designed that contain the bisfuran and xanthone portions of naturally occurring psorofebrins, and molecular modeling was used to assess their DNA alkylating potential and to refine the structures. A short, diastereoselective synthetic process to access bisfuranoxanthones was developed, culminating in the first total synthesis of (+/-)-isohydroxypsorofebrin. Two compounds designed and synthesized were of particular interest, chlorohydrin 7 and epoxide 6, which are reactive analogues of the natural product isohydroxypsorofebrin. The chlorohydrin retains the psorospermin-like DNA alkylation characteristics despite its rigid structure and high innate affinity for DNA. Molecular modeling has been used to rationalize the increased activity of the chlorohydrin. The chlorohydrin and epoxide show increased cytotoxicity compared to isohydroxypsorofebrin against a range of human tumor cell lines.
• Hurley, L., Liu, W., Sun, D., & Hurley, L. -. (2005). Binding of G-quadruplex-interactive agents to distinct G-quadruplexes induces different biological effects in MiaPaCa cells. Nucleosides, nucleotides & nucleic acids, 24(10-12).
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  Our previous studies have demonstrated the preference of telomestatin for intramolecular, rather than the intermolecular, G-quadruplex structures, while TiMPyP4 has selectivity for intermolecular over intramolecular G-quadruplex structures. However, it was not clear whether the difference in the selectivity between two different G-quadruplex-interactive agents could determine the corresponding biological effects in cultured human tumor cells. Here we evaluated the biological effects of both TMPyP4 and telomestatin in the human pancreatic carcinoma cell line (MiaPaCa) using subtoxic and cytotoxic concentrations. The cytotoxicity of these agents against MiaPaCa cells is quite different, and the IC50 of telomestatin (0.5 microM) is about 100 times less than that of TMPyP4 (50 microM). At IC50 concentrations, TMPyP4 induced anaphase bridge formation in MiaPaCa cells, while telomestatin failed to induce anaphase bridge formation. At subtoxic concentrations, TMPyP4 induced MiaPaCa cell growth arrest, senescence, apoptosis, and telomere length shortening within 5 weeks, while similar biological effects were evident after 12 weeks following treatment with telomestatin. Our data suggest that binding of G-quadruplex-interactive agents to distinct G-quadruplexes could induce different biological effects in human cancer cells.
• Hurley, L., Rezler, E. M., Seenisamy, J., Bashyam, S., Kim, M., White, E., Wilson, W. D., & Hurley, L. -. (2005). Telomestatin and diseleno sapphyrin bind selectively to two different forms of the human telomeric G-quadruplex structure. Journal of the American Chemical Society, 127(26).
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  The human telomeric sequence d[T(2)AG(3)](4) has been demonstrated to form different types of G-quadruplex structures, depending upon the incubation conditions. For example, in sodium (Na(+)), a basket-type G-quadruplex structure is formed. In this investigation, using circular dichroism (CD), biosensor-surface plasmon resonance (SPR), and a polymerase stop assay, we have examined how the addition of different G-quadruplex-binding ligands affects the conformation of the telomeric G-quadruplex found in solution. The results show that while telomestatin binds preferentially to the basket-type G-quadruplex structure with a 2:1 stoichiometry, 5,10,15,20-[tetra-(N-methyl-3-pyridyl)]-26-28-diselena sapphyrin chloride (Se2SAP) binds to a different form with a 1:1 stoichiometry in potassium (K(+)). CD studies suggest that Se2SAP binds to a hybrid G-quadruplex that has strong parallel and antiparallel characteristics, suggestive of a structure containing both propeller and lateral, or edgewise, loops. Telomestatin is unique in that it can induce the formation of the basket-type G-quadruplex from a random coil human telomeric oligonucleotide, even in the absence of added monovalent cations such as K(+) or Na(+). In contrast, in the presence of K(+), Se2SAP was found to convert the preformed basket G-quadruplex to the hybrid structure. The significance of these results is that the presence of different ligands can determine the type of telomeric G-quadruplex structures formed in solution. Thus, the biochemical and biological consequences of binding of ligands to G-quadruplex structures found in telomeres and promoter regions of certain important oncogenes go beyond mere stabilization of these structures.
• Hurley, L., Seenisamy, J., Bashyam, S., Gokhale, V., Vankayalapati, H., Sun, D., Siddiqui-Jain, A., Streiner, N., Shin-Ya, K., White, E., Wilson, W. D., & Hurley, L. -. (2005). Design and synthesis of an expanded porphyrin that has selectivity for the c-MYC G-quadruplex structure. Journal of the American Chemical Society, 127(9).
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  Cationic porphyrins are known to bind to and stabilize different types of G-quadruplexes. Recent studies have shown the biological relevance of the intramolecular parallel G-quadruplex as a transcriptional silencer in the c-MYC promoter. TMPyP4 also binds to this G-quadruplex and most likely converts it to a mixed parallel/antiparallel G-quadruplex with two external lateral loops and one internal propeller loop, suppressing c-MYC transcriptional activation. To achieve therapeutic selectivity by targeting G-quadruplexes, it is necessary to synthesize drugs that can differentiate among the different types of G-quadruplexes. We have designed and synthesized a core-modified expanded porphyrin analogue, 5,10,15,20-[tetra(N-methyl-3-pyridyl)]-26,28-diselenasapphyrin chloride (Se2SAP). Se2SAP converts the parallel c-MYC G-quadruplex into a mixed parallel/antiparallel G-quadruplex with one external lateral loop and two internal propeller loops, resulting in strong and selective binding to this G-quadruplex. A Taq polymerase stop assay was used to evaluate the binding of TMPyP4 and Se2SAP to G-quadruplex DNA. Compared to TMPyP4, Se2SAP shows a greater selectivity for and a 40-fold increase in stabilization of the single lateral-loop hybrid. Surface plasmon resonance and competition experiments with duplex DNA and other G-quadruplexes further confirmed the selectivity of Se2SAP for the c-MYC G-quadruplex. Significantly, Se2SAP was found to be less photoactive and noncytotoxic in comparison to TMPyP4. From this study, we have identified an expanded porphyrin that selectively binds with the c-MYC G-quadruplex in the presence of duplex DNA and other G-quadruplexes.
• Hurley, L., Sun, D., Guo, K., Rusche, J. J., & Hurley, L. -. (2005). Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents. Nucleic acids research, 33(18).
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  The proximal promoter region of the human vascular endothelial growth factor (VEGF) gene contains a polypurine/polypyrimidine tract that serves as a multiple binding site for Sp1 and Egr-1 transcription factors. This tract contains a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif for the formation of an intramolecular G-quadruplex. In this study, we observed the progressive unwinding of the oligomer duplex DNA containing this region into single-stranded forms in the presence of KCl and the G-quadruplex-interactive agents TMPyP4 and telomestatin, suggesting the dynamic nature of this tract under conditions which favor the formation of the G-quadruplex structures. Subsequent footprinting studies with DNase I and S1 nucleases using a supercoiled plasmid DNA containing the human VEGF promoter region also revealed a long protected region, including the guanine-rich sequences, in the presence of KCl and telomestatin. Significantly, a striking hypersensitivity to both nucleases was observed at the 3'-side residue of the predicted G-quadruplex-forming region in the presence of KCl and telomestatin, indicating altered conformation of the human VEGF proximal promoter region surrounding the guanine-rich sequence. In contrast, when specific point mutations were introduced into specific guanine residues within the G-quadruplex-forming region (Sp1 binding sites) to abolish G-quadruplex-forming ability, the reactivity of both nucleases toward the mutated human VEGF proximal promoter region was almost identical, even in the presence of telomestatin with KCl. This comparison of wild-type and mutant sequences strongly suggests that the formation of highly organized secondary structures such as G-quadruplexes within the G-rich region of the human VEGF promoter region is responsible for observed changes in the reactivity of both nucleases within the polypurine/polypyrimidine tract of the human VEGF gene. The formation of the G-quadruplex structures from this G-rich sequence in the human VEGF promoter is further confirmed by the CD experiments. Collectively, our results provide strong evidence that specific G-quadruplex structures can naturally be formed by the G-rich sequence within the polypurine/polypyrimidine tract of the human VEGF promoter region, raising the possibility that the transcriptional control of the VEGF gene can be modulated by G-quadruplex-interactive agents.
• Liu, W., Sun, D., & Hurley, L. H. (2005). Binding of G-quadruplex-interactive agents to distinct G-quadruplexes induces different biological effects in MiaPaCa cells. Nucleosides, Nucleotides and Nucleic Acids, 24(10-12), 1801-1815.
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  PMID: 16438049;Abstract: □ Our previous studies have demonstrated the preference of telomestatin for intramolecular, rather than the intermolecular, G-quadruplex structures, while TMPyP4 has selectivity for intermolecular over intramolecular G-quadruplex structures. However, it was not clear whether the difference in the selectivity between two different G-quadruplex-interactive agents could determine the corresponding biological effects in cultured human tumor cells. Here we evaluated the biological effects of both TMPyP4 and telomestatin in the human pancreatic carcinoma cell line (MiaPaCa) using subtoxic and cytotoxic concentrations. The cytotoxicity of these agents against MiaPaCa cells is quite different, and the IC50 of telomestatin (0.5 μM) is about 100 times less than that of TMPyP4 (50 μM). At IC50 concentrations, TMPyP4 induced anaphase bridge formation in MiaPaCa cells, while telomestatin failed to induce anaphase bridge formation. At subtoxic concentrations, TMPyP4 induced MiaPaCa cell growth arrest, senescence, apoptosis, and telomere length shortening within 5 weeks, while similar biological effects were evident after 12 weeks following treatment with telomestatin. Our data suggest that binding of G-quadruplex-interactive agents to distinct G-quadruplexes could induce different biological effects in human cancer cells. Copyright © Taylor & Francis Group, LLC.
• Rezler, E. M., Seenisamy, J., Bashyam, S., Kim, M., White, E., Wilson, W. D., & Hurley, L. H. (2005). Telomestatin and diseleno sapphyrin bind selectively to two different forms of the human telomeric G-quadruplex structure. Journal of the American Chemical Society, 127(26), 9439-9447.
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  PMID: 15984871;Abstract: The human telomeric sequence d[T2AG3]4 has been demonstrated to form different types of G-quadruplex structures, depending upon the incubation conditions. For example, in sodium (Na+), a basket-type G-quadruplex structure is formed. In this investigation, using circular dichroism (CD), biosensor-surface plasmon resonance (SPR), and a polymerase stop assay, we have examined how the addition of different G-quadruplex-binding ligands affects the conformation of the telomeric G-quadruplex found in solution. The results show that while telomestatin binds preferentially to the basket-type G-quadruplex structure with a 2:1 stoichiometry, 5,10,15,20-[tetra-(N-methyl-3-pyridyl)]-26-28-diselena sapphyrin chloride (Se2SAP) binds to a different form with a 1:1 stoichiometry in potassium (K+). CD studies suggest that Se2SAP binds to a hybrid G-quadruplex that has strong parallel and antiparallel characteristics, suggestive of a structure containing both propeller and lateral, or edgewise, loops. Telomestatin is unique in that it can induce the formation of the basket-type G-quadruplex from a random coil human telomeric oligonucleotide, even in the absence of added monovalent cations such as K+ or Na +. In contrast, in the presence of K+, Se2SAP was found to convert the preformed basket G-quadruplex to the hybrid structure. The significance of these results is that the presence of different ligands can determine the type of telomeric G-quadruplex structures formed in solution. Thus, the biochemical and biological consequences of binding of ligands to G-quadruplex structures found in telomeres and promoter regions of certain important oncogenes go beyond mere stabilization of these structures. © 2005 American Chemical Society.
• Seenisamy, J., Bashyam, S., Gokhale, V., Vankayalapati, H., Sun, D., Siddiqui-Jain, A., Streiner, N., Shin-ya, K., White, E., Wilson, W. D., & Hurley, L. H. (2005). Design and synthesis of an expanded porphyrin that has selectivity for the c-MYC G-quadruplex structure. Journal of the American Chemical Society, 127(9), 2944-2959.
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  PMID: 15740131;Abstract: Cationic porphyrins are known to bind to and stabilize different types of G-quadruplexes. Recent studies have shown the biological relevance of the intramolecular parallel G-quadruplex as a transcriptional silencer in the c-MYC promoter. TMPyP4 also binds to this G-quadruplex and most likely converts it to a mixed parallel/antiparallel G-quadruplex with two external lateral loops and one internal propeller loop, suppressing c-MYC transcriptional activation. To achieve therapeutic selectivity by targeting G-quadruplexes, it is necessary to synthesize drugs that can differentiate among the different types of G-quadruplexes. We have designed and synthesized a core-modified expanded porphyrin analogue, 5,10,15,20-[tetra(N-methyl-3-pyridyl)]-26,28- diselenasapphyrin chloride (Se2SAP). Se2SAP converts the parallel c-MYC G-quadruplex into a mixed parallel/antiparallel G-quadruplex with one external lateral loop and two internal propeller loops, resulting in strong and selective binding to this G-quadruplex. A Taq polymerase stop assay was used to evaluate the binding of TMPyP4 and Se2SAP to G-quadruplex DNA. Compared to TMPyP4, Se2SAP shows a greater selectivity for and a 40-fold increase in stabilization of the single lateral-loop hybrid. Surface plasmon resonance and competition experiments with duplex DNA and other G-quadruplexes further confirmed the selectivity of Se2SAP for the c-MYC G-quadruplex. Significantly, Se2SAP was found to be less photoactive and noncytotoxic in comparison to TMPyP4. From this study, we have identified an expanded porphyrin that selectively binds with the c-MYC G-quadruplex in the presence of duplex DNA and other G-quadruplexes. © 2005 American Chemical Society.
• Sun, D., Guo, K., Rusche, J. J., & Hurley, L. H. (2005). Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents. Nucleic Acids Research, 33(18), 6070-6080.
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  PMID: 16239639;PMCID: PMC1266068;Abstract: The proximal promoter region of the human vascular endothelial growth factor (VEGF) gene contains a polypurine/polypyrimidine tract that serves as a multiple binding site for Sp1 and Egr-1 transcription factors. This tract contains a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif for the formation of an intramolecular G-quadruplex. In this study, we observed the progressive unwinding of the oligomer duplex DNA containing this region into single-stranded forms in the presence of KCl and the G-quadruplex-interactive agents TMPyP4 and telomestatin, suggesting the dynamic nature of this tract under conditions which favor the formation of the G-quadruplex structures. Subsequent footprinting studies with DNase I and S1 nucleases using a supercoiled plasmid DNA containing the human VEGF promoter region also revealed a long protected region, including the guanine-rich sequences, in the presence of KCl and telomestatin. Significantly, a striking hypersensitivity to both nucleases was observed at the 3′-side residue of the predicted G-quadruplex-forming region in the presence of KCl and telomestatin, indicating altered conformation of the human VEGF proximal promoter region surrounding the guanine-rich sequence. In contrast, when specific point mutations were introduced into specific guanine residues within the G-quadruplex-forming region (Sp1 binding sites) to abolish G-quadruplex-forming ability, the reactivity of both nucleases toward the mutated human VEGF proximal promoter region was almost identical, even in the presence of telomestatin with KCl. This comparison of wild-type and mutant sequences strongly suggests that the formation of highly organized secondary structures such as G-quadruplexes within the G-rich region of the human VEGF promoter region is responsible for observed changes in the reactivity of both nucleases within the polypurine/polypyrimidine tract of the human VEGF gene. The formation of the G-quadruplex structures from this G-rich sequence in the human VEGF promoter is further confirmed by the CD experiments. Collectively, our results provide strong evidence that specific G-quadruplex structures can naturally be formed by the G-rich sequence within the polypurine/ polypyrimidine tract of the human VEGF promoter region, raising the possibility that the transcriptional control of the VEGF gene can be modulated by G-quadruplex-interactive agents. © The Author 2005. Published by Oxford University Press. All rights reserved.
• Tao, L. u., Shi, D., Sun, D., Han, H., & Hurley, L. H. (2005). Design, synthesis and biological activity of cationic porphyrins bearing mixed 3-quinolyl and 4-pyridyl meso groups. Journal of China Pharmaceutical University, 36(5), 393-397.
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  Abstract: Aim: To search for the potent telomerase inhibitors with structures of cationic porphyrins to improve the interactions between G-quadruplex and porphyrins by systematically varying the meso substituents. Methods: Porphyrins bearing mixed 3-quinolyl/4-pyridyl meso groups were synthesized using the Adler-Longo method by condensation of aldehydes with pyrrole, and then followed by methylation and ion exchange. The compounds were tested for the telomerase inhibitory activity and c-Myc inhibitory activity. Results: All compounds were found to be potent and approximately equivalent in terms of their ability to inhibit the action of telomerase in a cell-free assay. Compound 4 had the best inhibitory activity on c-Myc. Conclusion: Cationic porphyrins would be the potential anticancer candidates.
• Grand, C. L., Powell, T. J., Nagle, R. B., Bearss, D. J., Tye, D., Gleason-Guzman, M., & Hurley, L. H. (2004). Mutations in the G-quadruplex silencer element and their relationship to c-MYC overexpression, NM23 repression, and therapeutic rescue. Proceedings of the National Academy of Sciences of the United States of America, 101(16), 6140-6145.
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  PMID: 15079086;PMCID: PMC395936;Abstract: We have demonstrated that a parallel G-quadruplex structure in the c-MYC promoter functions as a transcriptional repressor element. Furthermore, a specific G-to-A mutation in this element results in destabilization of the G-quadruplex repressor element and an increase in basal transcriptional activity. To validate this model in an in vivo context, we have examined the sequence of this region in human colorectal tumors and the surrounding normal tissue. We have found that ≈30% of tumors contain one of two specific G-to-A mutations, not present in the surrounding normal tissue, that destabilize the parallel G-quadruplex, which would be expected to give rise to abnormally high expression of c-MYC in these cells. In contrast, G-quadruplex-disruptive mutations were absent in 20 colon adenomas, suggesting that these mutations occur late in tumorigenesis. We have also demonstrated that these same mutations are found in established colorectal cell lines. NM23-H2 levels are lower in cancer tissues and cell lines that harbor these mutations. In cells with repressed levels of NM23-H2, the mutated and destabilized G-quadruplex silencer element can be reinstated by the addition of G-quadruplex-stabilizing compounds, providing an opportunity for therapeutic intervention for patients carrying these mutations.
• Hurley, L., Seenisamy, J., Rezler, E. M., Powell, T. J., Tye, D., Gokhale, V., Joshi, C. S., Siddiqui-Jain, A., & Hurley, L. -. (2004). The dynamic character of the G-quadruplex element in the c-MYC promoter and modification by TMPyP4. Journal of the American Chemical Society, 126(28).
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  The nuclease hypersensitivity element III1 (NHE III1) upstream of the P1 and P2 promoters of c-MYC controls 80-90% of the transcriptional activity of this gene. The purine-rich strand in this region can form a G-quadruplex structure that is a critical part of the silencer element for this promoter. We have demonstrated that this G-quadruplex structure can form a mixture of four biologically relevant parallel-loop isomers, which upon interaction with the cationic porphyrin TMPyP4 are converted to mixed parallel/antiparallel G-quadruplex structures.
• Mehta, A. K., Shayo, Y., Vankayalapati, H., Hurley, L. H., & Schaefer, J. (2004). Structure of a quinobenzoxazine-G-quadruplex complex by REDOR NMR. Biochemistry, 43(38), 11953-11958.
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  PMID: 15379535;Abstract: Rotational-echo double resonance solid-state 31P{19F} and 13C{19F} NMR spectra have been used to locate the binding of a fluoroquinobenzoxazine to a DNA G-quadruplex labeled by phosphorothioation and [methyl-13C]thymidine.
• Seenisamy, J., Rezler, E. M., Powell, T. J., Tye, D., Gokhale, V., Joshi, C. S., Siddiqui-Jain, A., & Hurley, L. H. (2004). The dynamic character of the G-quadruplex element in the c-MYC promoter and modification by TMPyP4. Journal of the American Chemical Society, 126(28), 8702-8709.
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  PMID: 15250722;Abstract: The nuclease hypersensitivity element III1 (NHE III1) upstream of the P1 and P2 promoters of c-MYC controls 80-90% of the transcriptional activity of this gene. The purine-rich strand in this region can form a G-quadruplex structure that is a critical part of the silencer element for this promoter. We have demonstrated that this G-quadruplex structure can form a mixture of four biologically relevant parallel-loop isomers, which upon interaction with the cationic porphyrin TMPyP4 are converted to mixed parallel/antiparallel G-quadruplex structures.
• Shammas, M. A., J., R., Cheng, L. i., Koley, H., Hurley, L. H., Anderson, K. C., & Munshi, N. C. (2004). Telomerase Inhibition and Cell Growth Arrest after Telomestatin Treatment in Multiple Myeloma. Clinical Cancer Research, 10(2), 770-776.
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  PMID: 14760100;Abstract: Purpose: The aim of this study was to test the efficacy of telomestatin, an intramolecular G-quadruplex intercalating drug with specificity for telomeric sequences, as a potential therapeutic agent for multiple myeloma. Experimental Design: We treated ARD, ARP, and MM1S myeloma cells with various concentrations of telomestatin for 7 days and evaluated for telomerase activity. Myeloma cells were treated with the minimal effective telomestatin concentration for 3-5 weeks. Every 7th day the fraction of live cells was determined by trypan blue exclusion, aliquots of cells were removed for various molecular assays, and the remaining cells were replated at the same cell number and at the same concentration of telomestatin. Telomere length, apoptosis, and gene expression changes were monitored as described in detail in "Materials and Methods." Results: Telomestatin treatment led to inhibition of telomerase activity, reduction in telomere length, and apoptotic cell death in ARD, MMLS, and ARP myeloma cells. Gene expression profile after 1 and 7 days of telomestatin treatment revealed ≥2-fold change in only 6 (0.027%) and 51 (0.23%) of 33,000 genes surveyed, respectively. No changes were seen in expression of genes involved in cell cycle, apoptosis, DNA repair, or recombination. Conclusions: These results demonstrate that telomestatin exerts its antiproliferative and proapoptotic effects in myeloma cells via inhibition of telomerase and subsequent reduction in telomere length. We conclude that telomerase is an important potential therapeutic target for multiple myeloma therapy, and G-quadruplex interacting agents with specificity for binding to telomeric sequences can be important agents for additional evaluation.
• Tao, L. u., & Hurley, L. H. (2004). Synthesis of 5,10,15,20-tetra(N-methyl-6-quinolyl)-21,23-dithiaporphyrin chloride as cationic core-modified porphyrin. Chinese Chemical Letters, 15(11), 1261-1264.
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  Abstract: First cationic 6-quinolyl substituted dithiaporphyrin was synthesized using Skraup quinoline methodology from thiaporphyrin bearing 4-acetamidophenyl prepared by condensation reaction of aromatic aldehyde with pyrrole.
• Zewail-Foote, M., Li, V., Kohn, H., Bearss, D., Guzman, M., & Hurley, L. H. (2004). Erratum: The inefficiency of incisions of ecteinascidin 743-DNA adducts by the UvrABC nuclease and the unique structural feature of the DNA adducts can be used to explain the repair-dependent toxicities of this antitumor agent (Chemistry and Biology 8 (1033-1049)). Chemistry and Biology, 11(2), 283-.
• Zhilina, Z. V., Ziemba, A. J., Trent, J. O., Reed, M. W., Gorn, V., Zhou, Q., Duan, W., Hurley, L., & Ebbinghaus, S. W. (2004). Synthesis and evaluation of a triplex-forming oligonucleotide- pyrrolobenzodiazepine conjugate. Bioconjugate Chemistry, 15(6), 1182-1192.
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  PMID: 15546183;Abstract: In most cases, unmodified oligonucleotides designed as antigene molecules are incapable of binding to DNA with sufficient stability to prevent gene expression. To stabilize binding to a polypurine tract in the HER-2/neu promoter, a triplex forming oligonucleotide (TFO) was conjugated to a pyrrolo[1,4]-benzodiazepine (PBD), desmethyltomaymycin, and site-specific DNA binding was evaluated. An activated ester of the PBD moiety was conjugated by an acylation reaction to a free primary amine on a 50-atom aliphatic linker at the 5' end of the TFO. This long aliphatic linker was designed to provide a bridge from the major groove binding site of the TFO to the minor groove binding site of the PBD. Triplex formation by the resulting TFO-PBD conjugate occurred more slowly and with a nearly 30-fold lower affinity compared to an unconjugated TFO. PBD binding to the triplex target was demonstrated by protection from restriction enzyme digestion, and covalent binding to the exocyclic amino group of guanine was inferred by substituting specific guanines with inosines. Although the binding of the TFO was less efficient, this report demonstrates that in principle, TFOs can be used to direct the binding of a PBD to specific location. Further optimization of TFO-PBD conjugate design, likely involving optimization of the linker and perhaps placing a PBD at both ends of the TFO, will be needed to make gene modification robust.
• Hurley, L., Kim, M., Duan, W., Gleason-Guzman, M., & Hurley, L. -. (2003). Design, synthesis, and biological evaluation of a series of fluoroquinoanthroxazines with contrasting dual mechanisms of action against topoisomerase II and G-quadruplexes. Journal of medicinal chemistry, 46(4).
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  Topoisomerase inhibitors are important and clinically effective drugs, while G-quadruplex-interactive compounds that disrupt telomere maintenance mechanisms have yet to be proven useful in the clinic. If G-quadruplex-interactive compounds are to be clinically useful, it will most likely be in combination with more established cytotoxic agents. We have previously reported on a family of topoisomerase II inhibitors that also interact with G-quadruplexes. On the basis of previously established structure-activity relationships (SARs) for compounds that are able to inhibit topoisomerase II or interact with G-quadruplex to varying degrees, we have now designed and synthesized four new fluoroquinoanthroxazines (FQAs) that have different profiles of mixed topoisomerase II poisoning effects and G-quadruplex interactions. The biological profiles of the four new compounds were determined with respect to G-quadruplex interaction (polymerase stop and photocleavage assays) and topoisomerase II interaction (DNA cleavage and kDNA decatenation assays), alongside cytotoxicity tests with matched pairs of topoisomerase II-resistant and topoisomerase II-sensitive cells and with telomerase (+) and ALT (+) cell lines (ALT = alternative lengthening of telomeres). From this study, we have identified two FQAs with sharply contrasting profiles of potent G-quadruplex interaction with a weak topoisomerase II poisoning effect, and vice versa, for further evaluation to determine the optimum combination of these activities in subsequent in vivo studies.
• Hurley, L., Kim, M., Gleason-Guzman, M., Izbicka, E., Nishioka, D., & Hurley, L. -. (2003). The different biological effects of telomestatin and TMPyP4 can be attributed to their selectivity for interaction with intramolecular or intermolecular G-quadruplex structures. Cancer research, 63(12).
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  Demonstration of the existence of G-quadruplex structures in telomeres of Stylonychia macronuclei and in the promoter of c-myc in human cells has validated these secondary DNA structures as potential targets for drug design. The next important issue is the selectivity of G-quadruplex-interactive agents for the different types of G-quadruplex structures. In this study, we have taken an important step in associating specific biological effects of these drugs with selective interaction with either intermolecular or intramolecular G-quadruplex structures formed in telomeres. Telomestatin is a natural product isolated from Streptomyces anulatus 3533-SV4 and has been shown to be a very potent telomerase inhibitor through its G-quadruplex interaction. We have demonstrated that telomestatin interacts preferentially with intramolecular versus intermolecular G-quadruplex structures and also has a 70-fold selectivity for intramolecular G-quadruplex structures over duplex DNA. Telomestatin is able to stabilize G-quadruplex structures that are formed from duplex human telomeric DNA as well as from single-stranded DNA. Importantly, telomestatin stabilizes these G-quadruplex structures in the absence of monovalent cations, which is a unique characteristic among G-quadruplex-interactive compounds. At noncytotoxic concentrations, telomestatin suppresses the proliferation of telomerase-positive cells within several weeks. In contrast, TMPyP4, a compound that preferentially facilitates the formation of intermolecular G-quadruplex structures, suppresses the proliferation of alternative lengthening of telomeres (ALT)-positive cells as well as telomerase-positive cells. We have also demonstrated that TMPyP4 induces anaphase bridges in sea urchin embryos, whereas telomestatin did not have this effect, leading us to conclude that the selectivity of telomestatin for intramolecular G-quadruplex structures and TMPyP4 for intermolecular G-quadruplex structures is important in mediating different biological effects: stabilization of intramolecular G-quadruplex structures produces telomerase inhibition and accelerated telomere shortening, whereas facilitation of the formation of intermolecular G-quadruplex structures induces the formation of anaphase bridges.
• Hurley, L., Kim, M., Na, Y., Vankayalapati, H., Gleason-Guzman, M., & Hurley, L. -. (2003). Design, synthesis, and evaluation of psorospermin/quinobenzoxazine hybrids as structurally novel antitumor agents. Journal of medicinal chemistry, 46(14).
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  Topoisomerase II, an enzyme that catalyzes changes in the topology of DNA, plays several key roles in DNA metabolism and chromosome structure, and it is the primary cytotoxic target for a number of clinically important DNA intercalating agents such as doxorubicin. It seems likely that if these intercalating topoisomerase II poisons are structurally modified to also be DNA alkylating agents, they will have increased dwell time on the topoisomerase II-DNA complex and increased potency and selectivity for cancer cells. On the basis of insights into the mechanisms of action of psorospermin and the quinobenzoxazine A-62176 and molecular modeling studies of these compounds with duplex DNA, we have designed and synthesized a series of novel hybrid DNA-interactive compounds that alkylate DNA most efficiently at sequences directed by topoisomerase II. The epoxydihydrofuran ring of psorospermin was used as a DNA alkylating moiety, and this was fused to the pyridobenzophenoxazine ring of A-62176. The chlorohydrin ring opened form of the epoxide was also prepared and tested. These hybrid compounds showed enhanced DNA alkylating activity in the presence of topoisomerase II, exhibited significant activity against all the cancer cells tested at submicromolar concentrations, and were more potent than both parent compounds. However, the biochemical assays indicated that they lost some of the topoisomerase II and Mg(2+) dependency for reaction with DNA that is associated with psorospermin and A-62176, respectively.
• Hurley, L., Rezler, E. M., Bearss, D. J., & Hurley, L. -. (2003). Telomere inhibition and telomere disruption as processes for drug targeting. Annual review of pharmacology and toxicology, 43.
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  The components and cofactors of the holoenzyme telomerase and its substrate telomeric DNA are attractive targets for anticancer agents that act by inhibiting the activity of telomerase. This review outlines recent advances in telomerase inhibition that have been achieved using antisense oligonucleotides and ribozymes that target the telomerase mRNA or its hTR RNA template. Although these are potent catalytic inhibitors of telomerase, they are challenging to implement in the clinic due to their delayed effectiveness. Drugs that directly bind to the telomeres, the complex structures that are associated at the telomeric ends, and stabilize secondary DNA structures such as G-quadruplexes are also potent inhibitors of telomerase. Special focus is given here to the telomeres, the biological machinery that works in tandem with telomerase to elongate telomeres, the causes of telomere disruption or dysfunction, and the consequences of disruption/dysfunction on the activity and design of anticancer agents.
• Kim, M., Duan, W., Gleason-Guzman, M., & Hurley, L. H. (2003). Design, synthesis, and biological evaluation of a series of fluoroquinoanthroxazines with contrasting dual mechanisms of action against topoisomerase II and G-quadruplexes. Journal of Medicinal Chemistry, 46(4), 571-583.
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  PMID: 12570378;Abstract: Topoisomerase inhibitors are important and clinically effective drugs, while G-quadruplex-interactive compounds that disrupt telomere maintenance mechanisms have yet to be proven useful in the clinic. If G-quadruplex-interactive compounds are to be clinically useful, it will most likely be in combination with more established cytotoxic agents. We have previously reported on a family of topoisomerase II inhibitors that also interact with G-quadruplexes. On the basis of previously established structure-activity relationships (SARs) for compounds that are able to inhibit topoisomerase II or interact with G-quadruplex to varying degrees, we have now designed and synthesized four new fluoroquinoanthroxazines (FQAs) that have different profiles of mixed topoisomerase II poisoning effects and G-quadruplex interactions. The biological profiles of the four new compounds were determined with respect to G-quadruplex interaction (polymerase stop and photocleavage assays) and topoisomerase II interaction (DNA cleavage and kDNA decatenation assays), alongside cytotoxicity tests with matched pairs of topoisomerase II-resistant and topoisomerase II-sensitive cells and with telomerase (+) and ALT (+) cell lines (ALT = alternative lengthening of telomeres). From this study, we have identified two FQAs with sharply contrasting profiles of potent G-quadruplex interaction with a weak topoisomerase II poisoning effect, and vice versa, for further evaluation to determine the optimum combination of these activities in subsequent in vivo studies.
• Kim, M., Gleason-Guzman, M., Izbicka, E., Nishioka, D., & Hurley, L. H. (2003). The different biological effects of Telomestatin and TMPyP4 can be attributed to their selectivity for interaction with intramolecular or intermolecular G-quadruplex structures. Cancer Research, 63(12), 3247-3256.
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  PMID: 12810655;Abstract: Demonstration of the existence of G-quadruplex structures in telomeres of Stylonychia macronuclei and In the promoter of c-myc in human cells has validated these secondary DNA structures as potential targets for drug design. The next important issue is the selectivity of G-quadruplex. interactive agents for the different types of G-quadruplex structures. In this study, we have taken an important step in associating specific biological effects of these drugs with selective interaction with either intermolecular or intramolecular G-quadruplex structures formed in telomeres. Telomestatin is a natural product isolated from Streptomyces anulatus 3533-SV4 and has been shown to be a very potent telomerase inhibitor through its G-quadruplex interaction. We have demonstrated that telomestatin interacts preferentially with intramolecular versus intermolecular G-quadruplex structures and also has a 70-fold selectivity for intramolecular G-quadruplex structures over duplex DNA. Telomestatin is able to stabilize G-quadruplex structures that are formed from duplex human telomeric DNA as well as from single-stranded DNA. Importantly, telomestatin stabilizes these G-quadruplex structures in the absence of monovalent cations, which is a unique characteristic among G-quadruplex-interactive compounds. At noncytotoxic concentrations, telomestatin suppresses the proliferation of telomerase-positive cells within several weeks. In contrast, TMPyP4, a compound that preferentially facilitates the formation of intermolecular G-quadruplex structures, suppresses the proliferation of alternative lengthening of telomeres (ALT)-positive cells as well as telomerase-positive cells. We have also demonstrated that TMPyP4 induces anaphase bridges in sea urchin embryos, whereas telomestatin did not have this effect, leading us to conclude that the selectivity of telomestatin for intramolecular G-quadruplex structures and TMPyP4 for Intermolecular G-quadruplex structures is important in mediating different biological effects: stabilization of intramolecular G-quadruplex structures produces telomerase inhibition and accelerated telomere shortening, whereas facilitation of the formation of intermolecular G-quadruplex structures induces the formation of anaphase bridges.
• Kim, M., Younghwa, N. a., Vankayalapati, H., Gleason-Guzman, M., & Hurley, L. H. (2003). Design, synthesis, and evaluation of psorospermin/quinobenzoxazine hybrids as structurally novel antitumor agents. Journal of Medicinal Chemistry, 46(14), 2958-2972.
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  PMID: 12825936;Abstract: Topoisomerase II, an enzyme that catalyzes changes in the topology of DNA, plays several key roles in DNA metabolism and chromosome structure, and it is the primary cytotoxic target for a number of clinically important DNA intercalating agents such as doxorubicin. It seems likely that if these intercalating topoisomerase II poisons are structurally modified to also be DNA alkylating agents, they will have increased dwell time on the topoisomerase II-DNA complex and increased potency and selectivity for cancer cells, On the basis of insights into the mechanisms of action of psorospermin and the quinobenzoxazine A-62176 and molecular modeling studies of these compounds with duplex DNA, we have designed and synthesized a series of novel hybrid DNA-interactive compounds that alkylate DNA most efficiently at sequences directed by topoisomerase II. The epoxydihydrofuran ring of psorospermin was used as a DNA alkylating moiety, and this was fused to the pyridobenzophenoxazine ring of A-62176. The chlorohydrin ring opened form of the epoxide was also prepared and tested. These hybrid compounds showed enhanced DNA alkylating activity in the presence of topoisomerase II, exhibited significant activity against all the cancer cells tested at submicromolar concentrations, and were more potent than both parent compounds. However, the biochemical assays indicated that they lost some of the topoisomerase II and Mg2+ dependency for reaction with DNA that is associated with psorospermin and A-62176, respectively.
• Rezler, E. M., Bearss, D. J., & Hurley, L. H. (2003). Telomere Inhibition and Telomere Disruption as Processes for Drug Targeting. Annual Review of Pharmacology and Toxicology, 43, 359-379.
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  PMID: 12540745;Abstract: The components and cofactors of the holoenzyme telomerase and its substrate telomeric DNA are attractive targets for anticancer agents that act by inhibiting the activity of telomerase. This review outlines recent advances in telomerase inhibition that have been achieved using antisense oligonucleotides and ribozymes that target the telomerase mRNA or its hTR RNA template. Although these are potent catalytic inhibitors of telomerase, they are challenging to implement in the clinic due to their delayed effectiveness. Drugs that directly bind to the telomeres, the complex structures that are associated at the telomeric ends, and stabilize secondary DNA structures such as G-quadruplexes are also potent inhibitors of telomerase. Special focus is given here to the telomeres, the biological machinery that works in tandem with telomerase to elongate telomeres, the causes of telomere disruption or dysfunction, and the consequences of disruption/dysfunction on the activity and design of anticancer agents.
• Shammas, M. A., J., R., Akiyama, M., Koley, H., Chauhan, D., Hideshima, T., Goyal, R. K., Hurley, L. H., Anderson, K. C., & Munshi, N. C. (2003). Telomerase inhibition and cell growth arrest by G-quadruplex interactive agent in multiple myeloma. Molecular Cancer Therapeutics, 2(9), 825-833.
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  PMID: 14555701;Abstract: Objective: The aim of this study was to test the efficacy of telomerase inhibitor (TMPyP4 [tetra( N-methyl-4-pyridyl)-porphyrin chloride]; a G-quadruplex-intercalating porphyrin) as a potential therapeutic agent for multiple myeloma. Materials and Methods: We studied telomere length, telomerase activity, and effect of telomerase inhibition in multiple myeloma cells. Several myeloma cell lines were analyzed for telomerase activity, telomere length, and gene expression. Three myeloma cell lines (U266, ARH77, and ARD) were treated with TMPyP4 for 3-4 weeks. Viable cell number was assessed by trypan blue exclusion, and nature of cell death was determined by annexin labeling and/or DNA fragmentation. In situ oligo ligation technique was used to identify specific DNase I-type DNA cleavage. Results: We report high telomerase activity and shortened telomeres in myeloma cells compared to normal B cells. We have also observed inhibition of telomerase activity, reduction in telomere length, and decline of myeloma cell growth, as measured by trypan blue dye exclusion, following exposure to TMPyP4. Exposure to porphyrin reduced telomerase activity of U266, ARH77, and ARD myeloma cells by 98%, 92%, and 99%, respectively. Exposure to porphyrin had no effect on viability for the first 14 days, followed by death of 75-90% of cells over the next 2 weeks. The nature of cell death was apoptotic, as determined by annexin and DNA nick labeling. Majority of cells showed DNA fragmentation specific to caspase-3-activated DNase I. Conclusions: These results demonstrate antiproliferative activity of G-quadruplex- intercalating agents, and suggest telomerase as an important therapeutic target for myeloma therapy. © 2003 American Association for Cancer Research.
• Tao, L. u., Shi, D., Sun, D., Han, H., & Hurley, L. H. (2003). Preparation and bioactivity of cationic porphyrins bearing mixed 3-quinolyl and 3-pyridyl meso groups. Journal of China Pharmaceutical University, 34(2), 109-115.
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  Abstract: AIM: To search for the potent telomerase inhibitors with structures of cationic porphyrins to improve the interactions between G-quadruplex and porphyrins by systematically varying the meso substituents. METHOD: Porphyrins bearing mixed 3-quinolyl/3-pyridyl meso groups were synthesized using the Adler-Longo method by condensation of aldehydes with pyrrole, followed by methylation and ion-exchange. The compounds were tested for the telomerase inhibitory activity and c-Myc inhibitory activity. RESULT: All compounds were found to be potent and approximately equivalent in terms of their ability to inhibit the action of telomerase in a cell-free assay. Compound 4 had the best inhibitory activity on c-Myc.
• Weisman-Shomer, P., Cohen, E., Hershco, I., Khateb, S., Wolfovitz-Barchad, O., Hurley, L. H., & Fry, M. (2003). The cationic porphyrin TMPyP4 destabilizes the tetraplex form of the fragile X syndrome expanded sequence d(CGG)_n. Nucleic Acids Research, 31(14), 3963-3970.
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  PMID: 12853612;PMCID: PMC165968;Abstract: Fragile X syndrome, the most common cause of inherited mental retardation, is instigated by dynamic expansion of a d(CGG) trinucleotide repeat in the 5′-untranslated region of the first exon of the FMR1 gene, resulting in its silencing. The expanded d(CGG)n tract readily folds into hairpin and tetraplex structures which may contribute to the blocking of FMR1 transcription. In this work, we report that the cationic porphyrin 5,10,15,20-tetra(N-methyl-4-pyridyl)porphin (TMPyP4) effectively destabilizes in vitro the G′2 bimolecular tetraplex structure of d(CGG)n while it stabilizes the G′2 tetraplex: form of the telomeric sequence d(TTAGGG)2. Similarly to TMPyP4, the hnRNP-related protein CBF-A also destabilizes G′2 tetrahelical d(CGG)n while binding and stabilizing tetraplex telomeric DNA. We report that relative to each agent individually, successive incubation of G′2 d(CGG)n with TMPyP4 followed by exposure to CBF-A results in a nearly additive extent of disruption of this tetraplex form of the repeat sequence. Our observations open up the prospect of unfolding secondary structures of the expanded FMR1 d(CGG)n tract of fragile X cells by their exposure to low molecular size drugs or to proteins such as TMPyP4 or CBF-A.
• Grand, C. L., Han, H., Mũnoz, R. M., Weitman, S., D., D., Hurley, L. H., & Bearss, D. J. (2002). The cationic porphyrin TMPyP4 down-regulates c-MYC and human telomerase reverse transcriptase expression and inhibits tumor growth in vivo. Molecular Cancer Therapeutics, 1(8), 565-573.
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  PMID: 12479216;Abstract: Cationic porphyrins are being studied as possible anticancer agents because of their ability to bind to and stabilize DNA guanine quadruplexes (G-quadruplexes). We have shown previously that the cationic porphyrin TMPyP4 is able to bind to and stabilize G-quadruplexes in human telomere sequences, resulting in inhibition of telomerase activity. To better understand the mechanism of action behind telomerase inhibition by TMPyP4, we performed a cDNA microarray analysis on cells treated with TMPyP4 and TMPyP2, a positional isomer of TMPyP4 that has low affinity for G-quadruplexes. Analysis of time course data from the microarray experiments revealed that TMPyP4 and TMPyP2 treatment altered the expression of several gene clusters. We found that c-MYC, an oncogene nearly ubiquitous in human tumors that bears the potential in its promoter to form a G-quadruplex, was among the genes specifically down-regulated by TMPyP4, but not by TMPyP2. The hTERT gene, which encodes the catalytic subunit of telomerase, is transcriptionally regulated by c-MYC, and we have found that TMPyP4 also causes a decrease in human telomerase reverse transcriptase transcripts, suggesting two possible mechanisms for the effect of TMPyP4 on telomerase activity. We also show that TMPyP4, but not TMPyP2, is able to prolong survival and decrease tumor growth rates in two xenograft tumor models. We believe that, because of the actions of TMPyP4 in decreasing both c-MYC protein levels and telomerase activity, as well as its anticancer effects in vivo, it is a worthwhile agent to pursue and develop further. © 2002 American Association for Cancer Research.
• Herzig, M. C., Rodriguez, K. A., Trevino, A. V., Dziegielewski, J., Arnett, B., Hurley, L., & Woynarowski, J. M. (2002). The genome factor in region-specific DNA damage: The DNA-reactive drug U-78779 prefers mixed A/T-G/C sequences at the nucleotide level but is region-specific for long pure AT islands at the genomic level. Biochemistry, 41(5), 1545-1555.
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  PMID: 11814348;Abstract: Bizelesin is the first anticancer drug capable of damaging specific regions of the genome with clusters of its binding sites T(A/T)4A. This study characterized the sequence- and region-specificity of a bizelesin analogue, U-78779, designed to interact with mixed A/T-G/C motifs. At the nucleotide level, U-78779 was found to prefer runs of A/Ts interspersed with 1 or 2 G/C pairs, although 25% of the identified sites corresponded to pure AT motifs similar to bizelesin sites. The in silico computational analysis showed that the preferred mixed A/T-G/C motifs distribute uniformly at the genomic level. In contrast, the secondary, pure AT motifs (A/T)6A were found densely clustered in the same long islands of AT-rich DNA that bizelesin targets. Mapping the sites and quantitating the frequencies of U-78779 adducts in model AT island and non-AT island naked DNAs demonstrated that clusters of pure AT motifs outcompete isolated mixed A/T-G/C sites in attracting drug binding. Regional preference of U-78779 for AT island domains was verified also in DNA from drug-treated cells. Thus, while the primary sequence preference gives rise to non-region-specific scattered lesions, the clustering of the minor pure AT binding motifs seems to determine region-specificity of U-78779 in the human genome. The closely correlated cytotoxic activities of U-78779 and bizelesin in several cell lines further imply that both drugs may share common cellular targets. This study underscores the significance of the genome factor in a drug's potential for region-specific DNA damage, by showing that it can take precedence over drug binding preferences at the nucleotide level.
• Hurley, L. H. (2002). DNA and its associated processes as targets for cancer therapy. Nature Reviews Cancer, 2(3), 188-200.
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  PMID: 11990855;Abstract: DNA is the molecular target for many of the drugs that are used in cancer therapeutics, and is viewed as a non-specific target of cytotoxic agents. Although this is true for traditional chemotherapeutics, other agents that were discovered more recently have shown enhanced efficacy. Furthermore, a new generation of agents that target DNA-associated processes are anticipated to be far more specific and effective. How have these agents evolved, and what are their molecular targets?
• Hurley, L., & Hurley, L. -. (2002). DNA and its associated processes as targets for cancer therapy. Nature reviews. Cancer, 2(3).
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  DNA is the molecular target for many of the drugs that are used in cancer therapeutics, and is viewed as a non-specific target of cytotoxic agents. Although this is true for traditional chemotherapeutics, other agents that were discovered more recently have shown enhanced efficacy. Furthermore, a new generation of agents that target DNA-associated processes are anticipated to be far more specific and effective. How have these agents evolved, and what are their molecular targets?
• Hurley, L., Kim, M., Vankayalapati, H., Shin-Ya, K., Wierzba, K., & Hurley, L. -. (2002). Telomestatin, a potent telomerase inhibitor that interacts quite specifically with the human telomeric intramolecular g-quadruplex. Journal of the American Chemical Society, 124(10).
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  Telomestatin is a natural product isolated from Streptomyces anulatus 3533-SV4 and has been shown to be a very potent telomerase inhibitor. The structural similarity between telomestatin and a G-tetrad suggested to us that the telomerase inhibition might be due to its ability either to facilitate the formation of or trap out preformed G-quadruplex structures, and thereby sequester single-stranded d[T(2)AG(3)](n) primer molecules required for telomerase activity. Significantly, telomestatin appears to be a more potent inhibitor of telomerase (5 nM) than any of the previously described G-quadruplex-interactive molecules. In this communication we provide the first experimental evidence that telomestatin selectively facilitates the formation of or stabilizes intramolecular G-quadruplexes, in particular, that produced from the human telomeric sequence d[T(2)AG(3)](4). A simulated annealing (SA) docking approach was used to study the binding interactions of telomestatin with the intramolecular antiparallel G-quadruplex structure. Each intramolecular G-quadruplex molecule was found to bind two telomestatin molecules (unpublished results). A 2:1 model for the telomestatin bound in the external stacking mode in an energy minimized complex with the human telomeric basket-type G-quadruplex was constructed. Our observation that a G-quadruplex-interactive molecule without significant groove interactions is able to reorient in a G-quadruplex structure proints to the importance of core interaction with an asymmetric G-quadruplex structure in producing selective binding. Furthermore, the G-quadruplex interactions of telomestatin are more selective for the intramolecular structure in contrast to other G-quadruplex-interactive agents, such as TMPyP4.
• Hurley, L., Rezler, E. M., Bearss, D. J., & Hurley, L. -. (2002). Telomeres and telomerases as drug targets. Current opinion in pharmacology, 2(4).
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  Recent advances in telomerase inhibition have been achieved by using antisense oligonucleotides and ribozymes to target the telomerase mRNA or the telomerase RNA template. Also, small molecules are potent catalytic inhibitors of telomerase. However, therapeutic regimes incorporating these agents will be challenging to implement in the clinic because of their delayed effectiveness. Drugs that directly bind to the telomeres and stabilize secondary DNA structures such as G-quadruplexes are also potent inhibitors of telomerase and disrupt telomere structure. These G-quadruplex-interactive drugs could feasibly be used in synergy with more conventional cytotoxic agents to bring about more immediate responses in cancer cells that are less dependent upon telomere length. Recently, an emerging possible novel use of G-quadruplex-interactive drugs employs their ability to target G-quadruplexes in promoter regions of genes (such as c-MYC), which then serves to repress the production of the human telomerase reverse transcriptase protein.
• Hurley, L., Siddiqui-Jain, A., Grand, C. L., Bearss, D. J., & Hurley, L. -. (2002). Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proceedings of the National Academy of Sciences of the United States of America, 99(18).
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  The nuclease hypersensitivity element III(1) upstream of the P1 promoter of c-MYC controls 85-90% of the transcriptional activation of this gene. We have demonstrated that the purine-rich strand of the DNA in this region can form two different intramolecular G-quadruplex structures, only one of which seems to be biologically relevant. This biologically relevant structure is the kinetically favored chair-form G-quadruplex, which is destabilized when mutated with a single G --> A transition, resulting in a 3-fold increase in basal transcriptional activity of the c-MYC promoter. The cationic porphyrin TMPyP4, which has been shown to stabilize this G-quadruplex structure, is able to suppress further c-MYC transcriptional activation. These results provide compelling evidence that a specific G-quadruplex structure formed in the c-MYC promoter region functions as a transcriptional repressor element. Furthermore, we establish the principle that c-MYC transcription can be controlled by ligand-mediated G-quadruplex stabilization.
• Kim, M., Vankayalapati, H., Shin-Ya, K., Wierzba, K., & Hurley, L. H. (2002). Telomestatin, a potent telomerase inhibitor that interacts quite specifically with the human telomeric intramolecular G-quadruplex. Journal of the American Chemical Society, 124(10), 2098-2099.
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  PMID: 11878947;Abstract: Telomestatin is a natural product isolated from Streptomyces anulatus 3533-SV4 and has been shown to be a very potent telomerase inhibitor. The structural similarity between telomestatin and a G-tetrad suggested to us that the telomerase inhibition might be due to its ability either to facilitate the formation of or trap out preformed G-quadruplex structures, and thereby sequester single-stranded d[T2AG3]n primer molecules required for telomerase activity. Significantly, telomestatin appears to be a more potent inhibitor of telomerase (5 nM) than any of the previously described G-quadruplex-interactive molecules. In this communication we provide the first experimental evidence that telomestatin selectively facilitates the formation of or stabilizes intramolecular G-quadruplexes, in particular, that produced from the human telomeric sequence d[T2AG3]4. A simulated annealing (SA) docking approach was used to study the binding interactions of telomestatin with the intramolecular antiparallel G-quadruplex structure. Each intramolecular G-quadruplex molecule was found to bind two telomestatin molecules (unpublished results). A 2:1 model for the telomestatin bound in the external stacking mode in an energy minimized complex with the human telomeric basket-type G-quadruplex was constructed. Our observation that a G-quadruplex-interactive molecule without significant groove interactions is able to reorient in a G-quadruplex structure proints to the importance of core interaction with an asymmetric G-quadruplex structure in producing selective binding. Furthermore, the G-quadruplex interactions of telomestatin are more selective for the intramolecular structure in contrast to other G-quadruplex-interactive agents, such as TMPyP4. Copyright © 2002 American Chemical Society.
• Rezler, E. M., Bearss, D. J., & Hurley, L. H. (2002). Telomeres and telomerases as drug targets. Current Opinion in Pharmacology, 2(4), 415-423.
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  PMID: 12127874;Abstract: Recent advances in telomerase inhibition have been achieved by using antisense oligonucleotides and ribozymes to target the telomerase mRNA or the telomerase RNA template. Also, small molecules are potent catalytic inhibitors of telomerase. However, therapeutic regimes incorporating these agents will be challenging to implement in the clinic because of their delayed effectiveness. Drugs that directly bind to the telomeres and stabilize secondary DNA structures such as G-quadruplexes are also potent inhibitors of telomerase and disrupt telomere structure. These G-quadruplex-interactive drugs could feasibly be used in synergy with more conventional cytotoxic agents to bring about more immediate responses in cancer cells that are less dependent upon telomere length. Recently, an emerging possible novel use of G-quadruplex-interactive drugs employs their ability to target G-quadruplexes in promoter regions of genes (such as c-MYC), which then serves to repress the production of the human telomerase reverse transcriptase protein.
• Siddiqui-Jain, A., Grand, C. L., Bearss, D. J., & Hurley, L. H. (2002). Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proceedings of the National Academy of Sciences of the United States of America, 99(18), 11593-11598.
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  PMID: 12195017;PMCID: PMC129314;Abstract: The nuclease hypersensitivity element III1 upstream of the P1 promoter of c-MYC controls 85-90% of the transcriptional activation of this gene. We have demonstrated that the purine-rich strand of the DNA in this region can form two different intramolecular G-quadruplex structures, only one of which seems to be biologically relevant. This biologically relevant structure is the kinetically favored chair-form G-quadruplex, which is destabilized when mutated with a single G → A transition, resulting in a 3-fold increase in basal transcriptional activity of the c-MYC promoter. The cationic porphyrin TMPyP4, which has been shown to stabilize this G-quadruplex structure, is able to suppress further c-MYC transcriptional activation. These results provide compelling evidence that a specific G-quadruplex structure formed in the c-MYC promoter region functions as a transcriptional repressor element. Furthermore, we establish the principle that c-MYC transcription can be controlled by ligand-mediated G-quadruplex stabilization.
• Windsor, J. B., Thomas, C., Hurley, L., Roux, S. J., & Lloyd, A. M. (2002). Automated colorimetric screen for apyrase inhibitors. BioTechniques, 33(5), 1024-1030.
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  PMID: 12449379;Abstract: Apyrases are enzymes that efficiently hydrolyze ATP and ADP and may operate both inside and outside the cell. Although apyrases are important to a variety of cellular mechanisms and uses in industry, there are no available apyrase-specific inhibitors. Colorimetric assays based on the Fiske-Subbarow method for measuring inorganic phosphate are able to detect the release of inorganic phosphate, from ATP and other nucleotides. We found that this type of assay could be automated and used to screen, for apyrase-inhibiting compounds by assaying for a reduction in released phosphate in the presence of potential inhibitors. The automation of this assay allowed for the successful screening of a commercially available compound library. Several low molecular weight compounds were identified that, when used at micromolar concentrations, effectively inhibited apyrase activity.
• Duan, W., Rangan, A., Vankayalapati, H., Kim, M., Zeng, Q., Sun, D., Han, H., Fedoroff, O. Y., Nishioka, D., Rha, S. Y., Izbicka, E., D., D., & Hurley, L. H. (2001). Design and synthesis of fluoroquinophenoxazines that interact with human telomeric g-quadruplexes and their biological effects. Molecular Cancer Therapeutics, 1(2), 103-120.
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  PMID: 12467228;Abstract: In this study we have identified a new structural motif for a ligand with G-quadruplex interaction that results in biological effects associated with G-quadruplex-interactive compounds. Fluoroquinolones have been reported to possess weak telomerase inhibitory activity in addition to their better known bacterial gyrase poisoning. Starting with a fluoroquinobenzoxazine, which has modest potency in a human topoisomerase II assay, we have designed a more potent inhibitor of telomerase that has lost its topoisomerase II poisoning activity. This fluoroquinophenoxazine (FQP) interacts with G-quadruplex structures to inhibit the progression of Taq polymerase in a G-quadruplex polymerase stop assay. In addition, we demonstrate by 1H NMR studies that this compound interacts with telomeric G-quadruplex structures by external stacking to the G-tetrad with both the unimolecular fold-over and the parallel G-quadruplex structures. A photocleavage assay confirms the FQP interaction site, which is located off center of the external tetrad but within the loop region. Molecular modeling using simulated annealing was performed on the FQP-parallel G-quadruplex complex to determine the optimum FQP orientation and key molecular interactions with the telomeric G-quadruplex structure. On the basis of the results of these studies, two additional FQP analogues were synthesized, which were designed to test the importance of these key interactions. These analogues were evaluated in the Taq polymerase stop assay for G-quadruplex interaction. The data from this study and the biological evaluation of these three FQPs, using cytotoxicity and a sea urchin embryo system, were in accord with the predicted more potent telomeric G-quadruplex interactions of the initial lead compound and one of the analogues. On the basis of these structural and biological studies, the design of more potent and selective telomeric G-quadruplex-interactive compounds can be envisaged. © 2001 American Association for Cancer Research.
• Han, H., Langley, D. R., Rangan, A., & Hurley, L. H. (2001). Selective interactions of cationic porphyrins with G-quadruplex structures. Journal of the American Chemical Society, 123(37), 8902-8913.
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  PMID: 11552797;Abstract: G-quadruplex DNA presents a potential target for the design and development of novel anticancer drugs. Because G-quadruplex DNA exhibits structural polymorphism, different G-quadruplex typologies may be associated with different cellular processes. Therefore, to achieve therapeutic selectivity using G-quadruplexes as targets for drug design, it will be necessary to differentiate between different types of G-quadruplexes using G-quadruplex-interactive agents. In this study, we compare the interactions of three cationic porphyrins, TMPyP2, TMPyP3, and TMPyP4, with parallel and antiparallel types of G-quadruplexes using gel mobility shift experiments and a helicase assay. Gel mobility shift experiments indicate that TMPyP3 specifically promotes the formation of parallel G-quadruplex structures. A G-quadruplex helicase unwinding assay reveals that the three porphyrins vary dramatically in their abilities to prevent the unwinding of both the parallel tetrameric G-quadruplex and the antiparallel hairpin dimer G-quadruplex DNA by yeast Sgsl helicase (Sgs1p). For the parallel G-quadruplex, TMPyP3 has the strongest inhibitory effect on Sgslp, followed by TMPyP4, but the reverse is true for the antiparallel G-quadruplex. TMPyP2 does not appear to have any effect on the helicasecatalyzed unwinding of either type of G-quadruplex. Photocleavage experiments were carried out to investigate the binding modes of all three porphyrins with parallel G-quadruplexes. The results reveal that TMPyP3 and TMPyP4 appear to bind to parallel G-quadruplex structures through external stacking at the ends rather than through intercalation between the G-tetrads. Since intercalation between G-tetrads has been previously proposed as an alternative binding mode for TMPyP4 to G-quadruplexes, this mode of binding, versus that determined by a photocleavage assay described here (external stacking), was subjected to molecular dynamics calculations to identify the relative stabilities of the complexes and the factors that contribute to these differences. The △G° for the external binding mode was found to be driven by △H° with a small unfavorable T△S° term. The △G° for the intercalation binding model was driven by a large T△S° term and complemented by a small △H° term. One of the main stabilizing components of the external binding model is the energy of solvation, which favors the external model over the intercalation model by -67.94 kcal/mol. Finally, we propose that intercalative binding, although less favored than external binding, may occur, but because of the nature of the intercalative binding, it is invisible to the photocleavage assay. This study provides the first experimental insight into how selectivity might be achieved for different G-quadruplexes by using structural variants within a single group of G-quadruplex-interactive drugs.
• Hurley, L. H. (2001). Secondary DNA structures as molecular targets for cancer therapeutics. Biochemical Society Transactions, 29(6), 692-696.
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  PMID: 11709056;Abstract: DNA sequence information is pivotal to transcription, replication and recombination. DNA structure is dependent upon intracellular conditions such as ion concentration and the presence of proteins that may bind to DNA to facilitate the interconversion between different forms and to stabilize specific secondary structures. Dependent upon the primary DNA sequence, purine- and pyrimidine-rich strands of DNA can adopt four-stranded structures known as G-quadruplexes and i-motifs, respectively. These structures have been proposed to exist in biologically important regions of DNA, e.g. at the end of chromosomes and in the regulatory regions of oncogenes such as c-myc. Proteins such as topoisomerase I and Rap1 can facilitate the formation of G-quadruplex structures, and for transcriptional activation of c-myc, proteins such as NM23-H2 and hnRNP K are required. These proteins bind to the non-duplex forms of the nuclease hypersensitivity element III1 of c-myc. The design and synthesis of small molecules that target these secondary DNA structures and the biochemical and biological effects of these compounds are of potential importance in cancer chemotherapy.
• Hurley, L. H., & Zewail-Foote, M. (2001). The antitumor agent ecteinascidin 743: Characterization of its covalent DNA adducts and chemical stability. Advances in Experimental Medicine and Biology, 500, 289-299.
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  PMID: 11764956;Abstract: Ecteinascidin 743 (Et 743), a natural product derived from the Caribbean tunicate Eteinascidia turbinata, is a potent antitumor agent currently in phase II clinical trials. Et 743 binds in the minor groove of DNA, forming covalent adducts by reacting with N2 of guanine. Although DNA is considered to be the macromolecular receptor for Et 743, the precise mechanism by which Et 743 exerts its remarkable antitumor activity has not yet been elucidated. The aim of this study is to provide a rationale for the antitumor activity of Et 743 by studying its fundamental interactions with DNA at the molecular level. First, DNA structural distortions induced by Et 743 were characterized using gel electrophoresis. Surprisingly, Et 743 bends DNA toward the major groove, a unique feature among DNA-interactive agents that occupy the minor groove. Second, in order to gain further insight into the molecular basis behind the apparent sequence selectivity of Et 743, the stability and structure of Et 743 adducts at different target sequences were determined. On the basis of this data, the overall stability of the Et 743-DNA adducts was found to be governed by the DNA target sequence, where the inability of Et 743 to form optimum bonding networks with its optimum recognition sites leads to the formation of an unstable adduct. Consequently, the reaction of Et 743 with DNA is reversible, and the rate of the reverse reaction is a function of the target and flanking sequences. The results from this study demonstrate that Et 743 differs from other DNA alkylating agents by its effects on DNA structure and sequence-dependent chemical stability. This information provides important insight into the underlying mechanisms for its unique profile of antitumor activity.
• Rangan, A., Fedoroff, O. Y., & Hurley, L. H. (2001). Induction of Duplex to G-quadruplex Transition in the c-myc Promoter Region by a Small Molecule. Journal of Biological Chemistry, 276(7), 4640-4646.
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  PMID: 11035006;Abstract: A major control element of the human c-myc oncogene is the nuclease-hypersensitive purine/pyrimidine-rich sequence. This double-stranded DNA fragment, corresponding to the 27-base pair segment in the nuclease-hypersensitive element of the c-myc promoter region, forms a stable Watson-Crick double helix under physiological conditions. However, this duplex DNA can be effectively converted to G-quadruplex DNA by a small molecular weight ligand. Both intermolecular and intramolecular G-quadruplex forms can be induced by this ligand. Similar transitional changes are also observed with the duplex telomeric sequence from the Oxytricha species. These results provide additional support to the idea that G-quadruplex structures may play structural roles in vivo and also provide insight into novel methodologies for rational drug design. These structurally altered DNA elements might serve as regulatory signals in gene expression or in telomere dynamics and hence are promising targets for drug action.
• Shi, D. -., Wheelhouse, R. T., Sun, D., & Hurley, L. H. (2001). Quadruplex-interactive agents as telomerase inhibitors: Synthesis of porphyrins and structure-activity relationship for the inhibition of telomerase. Journal of Medicinal Chemistry, 44(26), 4509-4523.
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  PMID: 11741471;Abstract: The cationic porphyrin 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphyrin (TMPyP4) binds to quadruplex DNA and is thereby an inhibitor of human telomerase (Wheelhouse et al. J. Am. Chem. Soc. 1998, 120, 3261-3262). Herein the synthesis and telomerase-inhibiting activity of a wide range of analogues of TMPyP4 are reported, from which rules for a structure-activity relationship (SAR) have been discerned: (1) stacking interactions are critical for telomerase inhibition, (2) positively charged substituents are important but may be interchanged and combined with hydrogen-bonding groups, and (3) substitution is tolerated only on the meso positions of the porphyrin ring, and the bulk of the substituents should be matched to the width of the grooves in which they putatively lie. This SAR is consistent with a model presented for the complexation of TMPyP4 with human telomeric quadruplex DNA.
• Sun, D., & Hurley, L. H. (2001). Targeting telomeres and telomerase. Methods in Enzymology, 340, 573-592.
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  PMID: 11494871;
• Zewail-Foote, M., & Hurley, L. H. (2001). Differential rates of reversibility of ecteinascidin 743-DNA covalent adducts from different sequences lead to migration to favored bonding sites. Journal of the American Chemical Society, 123(27), 6485-6495.
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  PMID: 11439034;Abstract: Ecteinascidin 743 (Et 743), one of a series of structurally related antitumor antibiotics isolated from a marine tunicate, is currently in phase II clinical trials. Et 743 alkylates guanine N2 through the minor groove of DNA. Hydrogen-bonding networks that associate the drug with a three base pair DNA recognition site have been proposed to contribute to both the reactivity and the stability of the Et 743-DNA adduct. Here, we report that the reaction of Et 743 with DNA is reversible under nondenaturing conditions and that the rate of this reverse reaction depends critically upon the DNA-modified sequence. Quite unexpectedly, it was found that although the rates of alkylation are similar for the 5′-AGT and 5′-AGC sequences, reversal from the 5′-AGT sequence occurs faster than from the 5′-AGC sequence. Consequently, it is the differences in the rate of the reverse reaction that dictate the sequence selectivity of Et 743 toward its favored target sequence. As a direct consequence of the reversible nature of Et 743 with DNA, Et 743 can migrate from the nonfavored bonding sequence (e.g., 5′-AGT) to the favored DNA target site (e.g., 5′-AGC). The data suggest that the observed differences in the rate of reversibility arise from differences in the stability of the Et 743-DNA adduct at the 5′-AGT and 5′-AGC target sequences. On the basis of gel electrophoresis and IH NMR experiments, the Et 743-AGT adduct is less stable, has more dynamic motion, and produces different conformational changes in the DNA than the more stable Et 743-AGC adduct. The shuffling of Et 743-DNA adducts to the more stable alkylation sites has important implications for understanding the underlying relationship between the structural modification of DNA by Et 743 and its biological potency and efficacy in tumor cells.
• Zewail-Foote, M., Li, V., Kohn, H., Bearss, D., Guzman, M., & Hurley, L. H. (2001). The inefficiency of incisions of ecteinascidin 743-DNA adducts by the UvrABC nuclease and the unique structural feature of the DNA adducts can be used to explain the repair-dependent toxicities of this antitumor agent. Chemistry and Biology, 8(11), 1033-1049.
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  PMID: 11731295;Abstract: Background: Ecteinascidin 743 (Et 743), a natural product derived from a marine tunicate, is a potent antitumor agent presently in phase II clinical trials. Et 743 binds in the minor groove of DNA and alkylates N2 of guanine via a unique mechanism involving catalytic activation. The sequence selectivity of Et 743 is governed by different patterns of hydrogen-bonding to DNA, which results in differential reversibility of the covalent adducts. As determined by nuclear magnetic resonance spectroscopy, the preferred sequences 5′-PuGC and 5′-PyGG are stabilized by a hydrogen-bonding network, while the non-preferred sequences 5′-NG(A/T) are much less stabilized due to the lack of a key hydrogen bond to the GC base pair on the 3′-side of the alkylated guanine. Results: Mammalian cell lines (XPB, XPD, XPF, XPG, and ERCC1) deficient in the nucleotide excision repair (NER) gene products show resistance to Et 743. The recognition and subsequent incision of Et 743-DNA adducts by the bacterial multisubunit endonuclease UvrABC were used to evaluate DNA repair-mediated toxicity as a rationale for the resistance of NER-defective cell lines and the antitumor activity of Et 743. The Et 743-DNA adducts are indeed recognized and incised by the UvrABC repair proteins; however, the pattern of incision indicated that the non-preferred, and less stable, sequences (i.e. 5′-NG(A/T)) modified with Et 743 are generally incised at a much higher efficiency than the preferred, more stable sequences (i.e. 5′-PuGC or 5′-PyGG). In addition, within the same Et 743 recognition sequence, the level of incision varies, indicating that flanking regions also contribute to the differential incision frequency. Conclusions: The inefficient repair incision by the UvrABC nuclease of Et 743-DNA adducts provides a basis for rationalizing the observed repair-dependent cytotoxicities of these DNA adducts, if other associated structural properties of Et 743-DNA adducts are taken into account. In particular, the wedge-shaped Et 743, which forces open the minor groove of DNA, introducing a major groove bend, and the extrahelical protrusion of the C-subunit of Et 743 provide unique characteristics alongside the hydrogen-bonding stabilization of a covalent DNA adduct, which we propose traps an intermediate in NER processing of Et 743-DNA adducts. This trapped intermediate protein-Et 743-DNA adduct complex can be considered analogous to a poisoned topoisomerase I- or topoisomerase II-DNA complex. In the absence of an intact NER nuclease complex, this toxic lesion is unable to form, and the Et 743-DNA adducts, although not repaired by the NER pathway, are less toxic to cells. Conversely, elevated levels of either of these nucleases should lead to enhanced Et 743 toxicity. © 2001 Elsevier Science Ltd. All right reserved.
• Zhou, Q., Duan, W., Simmons, D., Shayo, Y., Raymond, M. A., Dorr, R. T., & Hurley, L. H. (2001). Design and synthesis of a novel DNA-DNA interstrand adenine-guanine cross-linking agent [1]. Journal of the American Chemical Society, 123(20), 4865-4866.
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  PMID: 11457309;
• Bearss, D. J., Hurley, L. H., & D., D. (2000). Telomere maintenance mechanisms as a target for drug development. Oncogene, 19(56), 6632-6641.
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  PMID: 11426649;Abstract: The shortening of the telomeric DNA sequences at the ends of chromosomes is thought to play a critical role in regulating the lifespan of human cells. Since all dividing cells are subject to the loss of telomeric sequences, cells with long proliferative lifespans need mechanisms to maintain telomere integrity. It appears that the activation of the enzyme telomerase is the major mechanism by which these cells maintain their telomeres. The proposal that a critical step in the process of the malignant transformation of cells is the upregulation of expression of telomerase has made this enzyme a potentially useful prognostic and diagnostic marker for cancer, as well as a new target for therapeutic intervention for the treatment of patients with cancer. It is now clear that simply inhibiting telomerase may not result in the anticancer effects that were originally hypothesized. While telomerase may not be the universal target for cancer therapy, we certainly believe that targeting the telomere maintenance mechanisms will be important in future research aimed toward a successful strategy for curing cancer.
• Fedoroff, O., Rangan, A., Chemeris, V. V., & Hurley, L. H. (2000). Cationic porphyrins promote the formation of i-motif DNA and bind peripherally by a nonintercalative mechanism. Biochemistry, 39(49), 15083-15090.
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  PMID: 11106486;Abstract: Telomeric C-rich strands can form a noncanonical intercalated DNA structure known as an i-motif. We have studied the interactions of the cationic porphyrin 5,10,15,20-tetra-(N-methyl-4-pyridyl)porphine (TMPyP4) with the i-motif forms of several oligonucleotides containing telomeric sequences. TMPyP4 was found to promote the formation of the i-motif DNA structure. On the basis of 1H NMR studies, we have created a model of the i-motif-TMPyP4 complex that is consistent with all the available experimental data. Two-dimensional NOESY data prompted us to conclude that TMPyP4 binds specifically to the edge of the intercalated DNA core by a nonintercalative mechanism. Since we have shown that TMPyP4 binds to and stabilizes the G-quadruplex form of the complementary G-rich telomeric strand, this study raises the intriguing possibility that TMPyP4 can trigger the formation of unusual DNA structures in both strands of the telomeres, which may in turn explain the recently documented biological effects of TMPyP4 in cancer cells.
• Han, H., & Hurley, L. H. (2000). G-quadruplex DNA: A potential target for anti-cancer drug design. Trends in Pharmacological Sciences, 21(4), 136-142.
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  PMID: 10740289;Abstract: In addition to the familiar duplex DNA, certain DNA sequences can fold into secondary structures that are four-stranded; because they are made up of guanine (G) bases, such structures are called G-quadruplexes. Considerable circumstantial evidence suggests that these structures can exist in vivo in specific regions of the genome including the telomeric ends of chromosomes and oncogene regulatory regions. Recent studies have demonstrated that small molecules can facilitate the formation of, and stabilize, G-quadruplexes. The possible role of G-quadruplex-interactive compounds as pharmacologically important molecules is explored in this article. Copyright (C) 2000 Elsevier Science Ltd.
• Han, H., Bennett, R. J., & Hurley, L. H. (2000). Inhibition of unwinding of G-quadruplex structures by Sgs1 helicase in the presence of N,N'-Bis[2-(1-piperidino)ethyl]-3,4,9,10-perylenetetracarboxylic diimide, a G-quadruplex-interactive ligand. Biochemistry, 39(31), 9311-9316.
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  PMID: 10924124;Abstract: N,N'-Bis[2-(1-piperidino)ethyl]-3,4,9,10-perylenetetracarboxylic diimide (PIPER), a perylene derivative, is a very potent and selective G-quadruplex DNA-interactive agent. It has been shown to inhibit DNA polymerase and telomerase by stacking externally to the G-tetrads in the G-quadruplex structures. Recently, we have demonstrated that this small molecule greatly accelerates the assembly of G-quadruplex structures in a cell-free system. In this report, we present data demonstrating that PIPER prevents the unwinding of G-quadruplex structures by yeast Sgsl helicase. Sgsl belongs to the RecQ DNA helicase family whose members include other G-quadruplex DNA unwinding helicases, such as human Bloom's syndrome and human Werner's syndrome helicases. PIPER specifically prevents the unwinding of G-quadruplex DNA but not duplex DNA by Sgsl. Competition experiments indicate that this inhibitory activity is due to the interaction of PIPER with G-quadruplex structures rather than the helicase itself. These results combined with previous studies suggest a possible mechanism of action for these G-quadruplex-interactive agents inside cells: they might induce G-quadruplex formation in G-rich regions on genomic DNA, stabilize these structures, and prevent them from being cleared by enzymes such as helicases. The G-quadruplex structures may, in turn, disrupt some critical cellular events such as DNA replication, transcription regulation, and telomere maintenance.
• Hurley, L. H., Wheelhouse, R. T., Sun, D., Kerwin, S. M., Salazar, M., Fedoroff, O. Y., Han, F. X., Han, H., Izbicka, E., & D., D. (2000). G-quadruplexes as targets for drug design. Pharmacology and Therapeutics, 85(3), 141-158.
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  PMID: 10739869;Abstract: G-quadruplexes are a family of secondary DNA structures formed in the presence of monovalent cations that consist of four-stranded structures in which Hoogsteen base-pairing stabilizes G-tetrad structures. These structures are proposed to exist in vivo, although direct confirmatory evidence is lacking. Guanine-rich regions of DNA capable of forming G-quadruplex structures are found in a variety of chromosomal regions, including telomeres and promoter regions of DNA. In this review, we describe the design of three separate groups of G-quadruplex-interactive compounds and their interaction with G-quadruplex DNA. Using the first group of compounds (anthraquinones), we describe experiments that provide the proof of concept that a G-quadruplex is required for inhibition of telomerase. Using the second group of compounds (perylenes), we describe the structure of a G-quadruplex-ligand complex and its effect on the dynamics of formation and enzymatic unwinding of the quadruplex. For the third group of compounds (porphyrins), we describe the experiments that relate the biological effects to their interactions with G-quadruplexes. Copyright (C) 2000 Elsevier Science Inc.
• Raymond, E., Soria, J., Izbicka, E., Boussin, F., Hurley, L., & D., D. (2000). DNA G-quadruplexes, telomere-specific proteins and telomere-associated enzymes as potential targets for new anticancer drugs. Investigational New Drugs, 18(2), 123-137.
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  PMID: 10857992;Abstract: Telomeres and telomerase have been subjects to a tremendous attention from scientists and oncologists during the past 5 years. This interest has been motivated by the potential of telomerase as a tumor marker for the diagnosis and the prognosis of cancer. The possible use of telomerase or telomeres as new targets for anticancer drugs also triggered investigations. The expression of telomerase was found in overall 85% of cancers. Telomerase is early expressed during oncogenesis with a gradient indicating that a high level of telomerase expression could be associated with a bad prognosis. Therefore, drugs targeting telomerase and telomeres might be useful in many human tumors with little restrictions regarding the tumor type or on the stage of the disease. Moreover, since telomerase is not or slightly expressed in normal cells, it has been postulated that drugs targeting telomerase would induce low toxicity. The race for the discovery of telomerase inhibitors has started while the identification of the components controlling telomerase, telomeres, cell survival, senescence, and apoptosis was still in progress. The recent identification of components regulating telomere length and telomerase expression (TRF1, TRF2, and tankyrase) opened a variety of new opportunities to control telomerase/telomere interactions. Meanwhile, a proof of principle was provided that changing telomere interactions with telomere binding proteins by chemical or biological means can induce cancer cell death. Interestingly, recent data challenge the old paradigm which suggested that a long exposure to telomerase and telomere inhibitors is necessary to induce anticancer effects. In this paper, we review the most recent information concerning the regulation of telomere length and telomerase expression, with emphasis on mechanisms that might translate into new drug discovery.
• Rha, S. Y., Izbicka, E., Lawrence, R., Davidson, K., Sun, D., Moyer, M. P., Roodman, G. D., Hurley, L., & Hoff, D. V. (2000). Effect of telomere and telomerase interactive agents on human tumor and normal cell lines. Clinical Cancer Research, 6(3), 987-993.
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  PMID: 10741725;Abstract: Shortening of telomeres along with an up-regulation of telomerase is implicated in the immortality of tumor cells. Targeting either telomeres or telomerase with specific compounds has been proposed as an anticancer strategy. Because telomerase activity and telomeres are found in normal cells, telomere or telomerase targeting agents could induce side effects in normal tissues. We evaluated the effects of telomere and telomerase interactive agents in human tumor and normal cell lines to try to determine the potential side effects those agents might induce in patients. Toxicity of the G-quadruplex interactive porphyrins (TMPyP4, TMPyP2) and azidothymidine (AZT) were tested using a cell-counting technique against normal human cell lines (CRL-2115 and CRL-2120, fibroblasts; NHEK-Ad, adult keratinocytes; CCL- 241, small intestinal cells; NCM 460, colonic mucosal epithelial cells) and human tumor cell lines (MDA-MB 231 and Hs 578T, breast cancer; SK-N-FI, neuroblastoma; HeLa, cervix cancer; MIA PaCa-2, pancreatic cancer; HT-29 and HCT-116, colon cancer; DU 145, prostatic cancer cell line). Telomerase activity of these cell lines was measured by a non-PCR-based conventional assay. The effects of TMPyP2, TMPyP4, and AZT were also evaluated against normal human bone marrow specimens, using a granulocyte-macrophage colony- forming assay (CFU-GM). AZT showed very low cytotoxic effects against normal and tumor cell lines, with the IC50 values above 200 μM. The IC50 values for TMPyP2 and TMPyP4 in normal human cell lines were in the range of 2.9- 48.3 μM and 1.7-15.5 μM, respectively, whereas in tumor cell lines the IC50 values were 11.4-53 μM and 9.0-28.2 μM, respectively. Within the tissue types, keratinocytes were more sensitive to TMPyP4 than fibroblasts, and small intestinal cells were more sensitive than colonic mucosal epithelial cells. The IC50 for TMPyP2 and TMPyP4 in the normal marrow colony-forming assays were 19.3 ± 5.1 μM and 47.9 ± 1.0 μM, respectively. In conclusion, the in vitro cytotoxicity of the telomere interactive agent TMPyP4 is comparable in human tumor and normal cell lines, which indicates that TMPyP4 could have effects on normal tissues.
• Yu, H., Kwok, Y., Hurley, L. H., & Kerwin, S. M. (2000). Efficient, Mg²⁺-dependent photochemical DNA cleavage by the antitumor quinobenzoxazine (S)-A-62176. Biochemistry, 39(33), 10236-10246.
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  PMID: 10956013;Abstract: The quinobenzoxazines, a group of structural analogues of the antibacterial fluoroquinolones, are topoisomerase II inhibitors that have demonstrated promising anticancer activity in mice. It has been proposed that the quinobenzoxazines form a 2:2 drug-Mg2+ self-assembly complex on DNA. The quinobenzoxazine (S)-A-62176 is photochemically unstable and undergoes a DNA-accelerated photochemical reaction to afford a highly fluorescent photoproduct. Here we report that the irradiation of both supercoiled DNA and DNA oligonucleotides in the presence of (S)-A-62176 results in photochemical cleavage of the DNA. The (S)-A-62176-mediated DNA photocleavage reaction requires Mg2+. Photochemical cleavage of supercoiled DNA by (S)-A-62176 is much more efficient that the DNA photocleavage reactions of the fluoroquinolones norfloxacin, ciprofloxacin, and enoxacin. The photocleavage of supercoiled DNA by (S)-A-62176 is unaffected by the presence of SOD, catalase, or other reactive oxygen scavengers, but is inhibited by deoxygenation. The photochemical cleavage of supercoiled DNA is also inhibited by 1 mM KI. Photochemical cleavage of DNA oligonucleotides by (S)-A-62176 occurs most extensively at DNA sites bound by drug, as determined by DNase I footprinting, and especially at certain G and T residues. The nature of the DNA photoproducts, and inhibition studies, indicate that the photocleavage reaction occurs by a free radical mechanism initiated by abstraction of the 4'- and 1'-hydrogens from the DNA minor groove. These results lend further support for the proposed DNA binding model for the quinobenzoxazine 2:2 drug-Mg2+ complex and serve to define the position of this complex on the minor groove of DNA.
• Cathers, B. E., Sun, D., & Hurley, L. H. (1999). Accurate determination of quadruplex binding affinity and potency of G-quadruplex-interactive telomerase inhibitors by use of a telomerase extension assay requires varying the primer concentration. Anti-Cancer Drug Design, 14(4), 367-372.
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  PMID: 10625929;
• Haiyong, H., Cliff, C. L., & Hurley, L. H. (1999). Accelerated assembly of G-quadruplex structures by a small molecule. Biochemistry, 38(22), 6981-6986.
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  PMID: 10353809;Abstract: In the presence of alkali cations, notably potassium and sodium, DNA oligomers that possess two G-rich repeats associate into either a tetrameric parallel G-quadruplex or a variety of dimeric antiparallel G-quadruplexes. The formation of such structures is normally a very slow process. Some proteins, such as the β-subunit of the Oxytricha telomere-binding protein, promote the formation of G-quadruplex structures in a chaperone-like manner. In this report, we present data concerning the role of a perylene derivative, PIPER, in the assembly of G-quadruplex structures as the first example of a small ligand behaving as a driver in the assembly of polynucleotide secondary structures. Gel-shift experiments demonstrate that PIPER can dramatically accelerate the association of a DNA oligomer containing two tandem repeats of the human telomeric sequence (TTAGGG) into di- and tetrameric G-quadruplexes. In so doing, PIPER alters the oligomer dimerization kinetics from second to first order. The presence of 10 μM PIPER accelerates the assembly of varied dimeric G-quadruplexes an estimated 100-fold from 2 μM oligomer. These results imply that some biological effects elicited by G-quadruplex- interactive agents, such as the induction of anaphase bridges, may stem from the propensity such compounds have for assembling G-quadruplexes.
• Han, F. X., Wheelhouse, R. T., & Hurley, L. H. (1999). Interactions of TMPyP4 and TMPyP2 with quadruplex DNA. Structural basis for the differential effects on telomerase inhibition. Journal of the American Chemical Society, 121(15), 3561-3570.
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  Abstract: The cationic porphyrins TMPyP4 and TMPyP2 possess similar structures but have strikingly different potencies for telomerase inhibition. To rationalize this difference, the interactions of TMPyP4 and TMPyP2 with an antiparallel quadruplex DNA were investigated. A single-stranded DNA oligonucleotide (G4A) containing four human telomere repeats of GGGTTA has been designed to form an intramolecular quadruplex DNA and was confirmed to form such a structure under 100 mM KCl by a DNA ligase assay, DMS footprinting, and CD spectrum analysis. By carrying out UV spectroscopic studies of the thermal melting profiles of G4A-porphyrin complexes, we provide evidence that TMPyP4 and TMPyP2 both stabilized quadruplex DNA to about the same extent. A photocleavage assay was used to determine the precise location for TMPyP4 and TMPyP2 in their interactions with quadruplex DNA. The results show that TMPyP4 binds to the intramolecular quadruplex DNA by stacking externally to the guanine tetrad at the GT step, while TMPyP2 binds predominantly to the same G4 DNA structure via external binding to the TTA loop. We propose that the inability of TMPyP2 to bind to the G4A by stacking externally to the guanine tetrad accounts for the differential effects on telomerase inhibition by TMPyP4 and TMPyP2.
• Han, H., Cliff, C. L., & Hurley, L. H. (1999). Accelerated assembly of G-quadruplex structures by a small molecule. Biochemistry, 38(22), X-6986.
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  Abstract: In the presence of alkali cations, notably potassium and sodium, DNA oligomers that possess two G-rich repeats associate into either a tetrameric parallel G-quadruplex or a variety of dimeric antiparallel G-quadruplexes. The formation of such structures is normally a very slow process. Some proteins, such as the β-subunit of the Oxytricha telomere-binding protein, promote the formation of G-quadruplex structures in a chaperone-like manner. In this report, we present data concerning the role of a perylene derivative, PIPER, in the assembly of G-quadruplex structures as the first example of a small ligand behaving as a driver in the assembly of polynucleotide secondary structures. Gel-shift experiments demonstrate that PIPER can dramatically accelerate the association of a DNA oligomer containing two tandem repeats of the human telomeric sequence (TTAGGG) into di- and tetrameric G-quadruplexes. In so doing, PIPER alters the oligomer dimerization kinetics from second to first order. The presence of 10 μM PIPER accelerates the assembly of varied dimeric G-quadruplexes an estimated 100-fold from 2 μM oligomer. These results imply that some biological effects elicited by G-quadruplex-interactive agents, such as the induction of anaphase bridges, may stem from the propensity such compounds have for assembling G-quadruplexes.
• Han, H., Hurley, L. H., & Salazar, M. (1999). A DNA polymerase stop assay for G-quadruplex-interactive compounds. Nucleic Acids Research, 27(2), 537-542.
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  PMID: 9862977;PMCID: PMC148212;Abstract: We have developed and characterized an assay for G-quadruplex-interactive compounds that makes use of the fact that G-rich DNA templates present obstacles to DNA synthesis by DNA polymerases. Using Taq DNA polymerase and the G-quadruplex binding 2,6-diamidoanthraquinone BSU-1051, we find that BSU-1051 leads to enhanced arrest of DNA synthesis in the presence of K+ by stabilizing an intramolecular G-quadruplex structure formed by four repeats of either TTGGGG or TTAGGG in the template strand. The data provide additional evidence that BSU-1051 modulates telomerase activity by stabilization of telomeric G-quadruplex DNA and point to a polymerase arrest assay as a sensitive method for screening for G-quadruplex-interactive agents with potential clinical utility.
• Izbicka, E., Nishioka, D., Marcell, V., Raymond, E., Davidson, K. K., Lawrence, R. A., Wheelhouse, R. T., Hurley, L. H., Wu, R. S., & D., D. (1999). Telomere-interactive agents affect proliferation rates and induce chromosomal destabilization in sea urchin embryos. Anti-Cancer Drug Design, 14(4), 355-365.
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  PMID: 10625928;Abstract: Cationic porphyrins, which interact with guanine quadruplex (G4) telomeric folds, inhibit telomerase activity in human tumor cells. In this study, we have further examined effects of porphyrins and other telomere- and telomerase-interactive agents on proliferation rates and chromosome stability in a novel in viveo model, developing sea urchin embryos. We studied two porphyrins: (i) TMPyP4, a potent telomerase inhibitor; and (ii) TMPyP2, an isomer of TMPyP4 and an inefficient telomerase inhibitor, azidothymine (AZT), the reverse transcriptase inhibitor, antisense phosphorothioate oligonucleotide to telomerase RNA (TAG6) and a control scrambled sequence (ODN). TMPyP4, AZT and TAG6 (but not TMPyP2 or ODN) decreased the rates of cell proliferation and increased the percentage of cells trapped in mitosis. Nuclear localization of TAG6, but not of ODN, was demonstrated with 5'-fluoresceinated analogs of TAG6 and ODN. Formation of elongated chromosomes incapable of separating in anaphase, induced by TMPyP4, AZT and TAG6, closely resembled phenotypes resulting from telomerase template mutation or dominant negative TRF2 allele. Our data suggest that G4-interactive agents exert their antiproliferative effects via chromosomal destabilization and warrant their further development as valuable anticancer tools.
• Izbicka, E., Wheelhouse, R. T., Raymond, E., Davidson, K. K., Lawrence, R. A., Sun, D., Windle, B. E., Hurley, L. H., & D., D. (1999). Effects of cationic porphyrins as G-quadruplex interactive agents in human tumor cells. Cancer Research, 59(3), 639-644.
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  PMID: 9973212;Abstract: A series of cationic porphyrins has been identified as G-quadruplex interactive agents (QIAs) that stabilize telomeric G-quadruplex DNA and thereby inhibit human telomerase; 50% inhibition of telomerase activity was achieved in HeLa cell-free extract at porphyrin concentrations in the range ≤50 μM. Cytotoxicity of the porphyrins in vitro was assessed in normal human cells (fibroblast and breast) and human tumor cells representing models selected for high telomerase activity and short telomeres (breast carcinoma, prostate, and lymphoma). In general, the cytotoxicity (EC50, effective concentration for 50% inhibition of cell proliferation) against normal and tumor cells was >50 μM. The porphyrins were readily absorbed into tumor cell nuclei in culture. Inhibition of telomerase activity in MCF7 cells by subcytotoxic concentrations of TMPyP4 showed time and concentration dependence at 1-100 μM TMPyP4 over 15 days in culture (10 population doubling times). The inhibition of telomerase activity was paralleled by a cell growth arrest in G2-M. These results suggest that relevant biological effects of porphyrins can be achieved at concentrations that do not have general cytotoxic effects on cells. Moreover, the data support the concept that a rational, structure-based approach is possible to design novel telomere-interactive agents with application to a selective and specific anticancer therapy.
• Kshirsagar, T. A., & Hurley, L. H. (1999). Mechanistic insight into the aromatization of cyclic p-quinonemethides to indoles. Heterocycles, 51(1), 185-189.
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  Abstract: Two mechanisms have been previously proposed for the aromatization of cyclic p-quinonemethides to indoles. A novel synthetic route to indoles via an unstable cyclic p-quinonemethide has provided additional insight into the mechanism of cyclization. Since this key intermediate lacks the functional groups required for one of the mechanistic pathways (Pathway B), it appears that cyclization occurs via Pathway A.
• Kwok, Y., Sun, D., Clement, J. J., & Hurley, L. H. (1999). The quinobenzoxazines: Relationship between DNA binding and biological activity. Anti-Cancer Drug Design, 14(5), 443-450.
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  PMID: 10766299;Abstract: The quinobenzoxazine compounds, derived from antibacterial quinolones, is active in vitro and in vivo against murine and human tumors. In this contribution, we show that the relative DNA binding affinity of the quinobenzoxazine compounds correlates with their cytotoxicity, their ability to inhibit gyrase-DNA complex formation, and the decatenation of kinetoplast DNA by human topoisomerase II. DNA binding studies with the descarboxy-A-62176 analogue indicate that the β-keto acid moiety of the quinobenzoxazine compounds plays an important role in their interaction with DNA.
• Kwok, Y., Zeng, Q., & Hurley, L. H. (1999). Structural insight into a quinolone-topoisomerase II-DNA complex. Further evidence for a 2:2 quinobenzoxazine-Mg²⁺ self-assembly model formed in the presence of topoisomerase II. Journal of Biological Chemistry, 274(24), 17226-17235.
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  PMID: 10358081;Abstract: Quinobenzoxazine A-62176, developed from the anti-bacterial fluoroquinolones, is active in vitro and in vivo against murine and human tumors. It has been previously claimed that A-62176 is a catalytic inhibitor of mammalian topoisomerase II that does not stabilize the cleaved complex. However, at low drug concentrations and pH 6-7, we have found that A-62176 can enhance the formation of the cleaved complex at certain sites. Using a photocleavage assay, mismatched sequences, and competition experiments between psorospermin and A-62176, we pinpointed the drug binding site on the DNA base pairs between positions +1 and +2 relative to the cleaved phosphodiester bonds. A 2:2 quinobenzoxazine-Mg2+ self-assembly model was previously proposed, in which one drug molecule intercalates into the DNA helix and the second drug molecule is externally bound, held to the first molecule and DNA by two Mg2+ bridges. The results of competition experiments between psorospermin and A-62176, as well as between psorospermin and A-62176 and norfloxacin, are consistent with this model and provide the first evidence that this 2:2 quinobenzoxazine-Mg2+ complex is assembled in the presence of topoisomerase II. These results also have parallel implications for the mode of binding of the quinolone antibiotics to the bacterial gyrase-DNA complex.
• Lee, S., & Hurley, L. H. (1999). A thymine: Thymine mismatch enhances the pluramycin alkylation site downstream of the TBP - TATA box complex. Journal of the American Chemical Society, 121(39), X.
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  Abstract: The DNA groove interactions of the pluramycins determine the base-pair specificity to the 5′-side of the covalently modified guanine. The DNA reactivity of these drugs at defined sites can be further increased by structural and dynamic DNA distortion induced by TATA binding protein (TBP) binding to the TATA box. This enhanced drug reactivity has led to the proposal that protein-induced DNA conformational dynamics might be responsible for the more selective biological consequences of the pluramycins. To identify the structural and/or dynamic determinants that account for the enhanced drug reactivity, DNA heteroduplexes that contain base mismatches were examined for enhanced alkylation by altromycin B. The results demonstrate that base mismatches located at the 5′-side of the target guanine enhance drug reactivity. An analysis of the structural and dynamic properties of the base mismatches demonstrates that the pluramycin reactivities are not only determined by dynamic conformation of the base mismatch, which improves the accessibility of the drug to the DNA helix, but also by the specific groove interactions between the DNA and the drug, which results in stabilization of the precovalent drug - DNA complex. Having established that the highest pluramycin reactivity with heteroduplex DNA is produced by a thymine:thymine (T:T) mismatch, we next addressed how the same mismatch affects pluramycin reactivity in the flanking region to the TBP - TATA box complex. When this mismatch is introduced into the downstream flanking sequence of the TATA box, TBP binding cooperatively enhances the drug alkylation on a downstream guanine adjacent to the inserted mismatch. While an obvious structural similarity between the T:T mismatch and the TBP-induced effects at a downstream site of the TATA box does not exist, we propose that the base pair destabilizing effects of the T:T mismatch may resemble the dynamically accessible intercalation site on the downstream side of the TATA box induced by binding of TBP to the TATA box.
• Lee, S., & Hurley, L. H. (1999). A thymine:thymine mismatch enhances the pluramycin alkylation site downstream of the TBP-TATA box complex. Journal of the American Chemical Society, 121(39), 8971-8977.
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  Abstract: The DNA groove interactions of the pluramycins determine the base-pair specificity to the 5'-side of the covalently modified guanine. The DNA reactivity of these drugs at defined sites can be further increased by structural and dynamic DNA distortion induced by TATA binding protein (TBP) binding to the TATA box. This enhanced drug reactivity has led to the proposal that protein-induced DNA conformational dynamics might be responsible for the more selective biological consequences of the pluramycins. To identify the structural and/or dynamic determinants that account for the enhanced drug reactivity, DNA heteroduplexes that contain base mismatches were examined for enhanced alkylation by altromycin B. The results demonstrate that base mismatches located at the 5'-side of the target guanine enhance drug reactivity. An analysis of the structural and dynamic properties of the base mismatches demonstrates that the pluramycin reactivities are not only determined by dynamic conformation of the base mismatch, which improves the accessibility of the drug to the DNA helix, but also by the specific groove interactions between the DNA and the drug, which results in stabilization of the precovalent drug-DNA complex. Having established that the highest pluramycin reactivity with heteroduplex DNA is produced by a thymine:thymine (T:T) mismatch, we next addressed how the same mismatch affects pluramycin reactivity in the flanking region to the TBP-TATA box complex. When this mismatch is introduced into the downstream flanking sequence of the TATA box, TBP binding cooperatively enhances the drug alkylation on a downstream guanine adjacent to the inserted mismatch. While an obvious structural similarity between the T:T mismatch and the TBP-induced effects at a downstream site of the TATA box does not exist, we propose that the base pair destabilizing effects of the T:T mismatch may resemble the dynamically accessible intercalation site on the downstream side of the TATA box induced by binding of TBP to the TATA box.
• Seaman, F. C., & Hurley, L. H. (1999). ³¹P-Nmr as a probe for drug-nucleic acid interactions. Phosphorus, Sulfur and Silicon and Related Elements, 144-146, 297-300.
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  Abstract: The structural impact of covalent and noncovalent interactions of drugs with DNA is an important component for understanding the biochemical and biological consequences of DNA damage. Work in this laboratory has focused on a number of potentially therapeutically important drugs that distort DNA by unwinding, bending DNA into the major or minor groove. These lead to enhanced recognition of DNA by proteins involved in transcription and replication. In this paper, we will present the structures of one of these complexes and show how 31P-NMR can be used to monitor these distortive effects.
• Sun, D., Lopez-Guajardo, C. C., Quada, J., Hurley, L. H., & D., D. (1999). Regulation of catalytic activity and processivity of human telomerase. Biochemistry, 38(13), 4037-4044.
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  PMID: 10194316;Abstract: The ends of eukaryotic chromosomes are specialized sequences, called telomeres comprising tandem repeats of simple DNA sequences. Those sequences are essential for preventing aberrant recombination and protecting genomic DNA against exonucleolytic DNA degradation. Telomeres are maintained at a stable length by telomerase, an RNA-dependent DNA polymerase. Recently, human telomerase has been recognized as a unique diagnostic marker for human tumors and is potentially a highly selective target for antitumor drugs. In this study, we have examined the major factors affecting the catalytic activity and processivity of human telomerase. Specifically, both the catalytic activity and processivity of human telomerase were modulated by temperature, substrate (dNTP and primer) concentration, and the concentration of K+. The catalytic activity of telomerase increased as temperature (up to 37°C), concentrations of dGTP, primer, and K+ were increased. However, the processivity of human telomerase decreased as temperature, primer concentration, and K+ were increased. Our results support the current model for human telomerase reaction and strengthen the hypothesis that a G- quadruplex structure of telomere DNA plays an important role in the regulation of the telomerase reaction.
• Zewail-Foote, M., & Hurley, L. H. (1999). Ecteinascidin 743: A minor groove alkylator that bends DNA toward the major groove. Journal of Medicinal Chemistry, 42(14), 2493-2497.
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  PMID: 10411470;Abstract: The ecteinascidins (Ets), which are natural products derived from marine tunicates, exhibit potent antitumor activity. Of the numerous Ets isolated, Et 743 is presently being evaluated in phase II clinical trials. Et 743 binds in the minor groove of DNA and alkylates N2 of guanine. Although structurally similar to saframycin, which exhibits poor activity in cellular assays, Et 743 has shown good efficacy as an antitumor agent. In this study, DNA structural distortions induced by Et 743 were examined to provide insight into the molecular basis for the antitumor activity of Et 743. Electrophoretic mobility shifts of ligated oligomers containing site-directed adducts were used to examine the extent and direction of the Et 743-induced bend. Surprisingly, we find that Et 743 bends DNA toward the major groove, which is a unique feature among DNA-interactive agents that occupy the minor groove.
• Zewail-Foote, M., & Hurley, L. H. (1999). Molecular approaches to achieving control of gene expression by drug intervention at the transcriptional level. Anti-Cancer Drug Design, 14(1), 1-9.
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  PMID: 10363023;Abstract: In this article we first very briefly review current approaches to the design of drugs that have specificity for the modulation of gene expression and selectivity for target cells at the transcription level by targeting DNA. We focus this review on our approaches to gaining selectivity by drug-induced architectural alteration in DNA structure, selectivity achieved by protein-induced changes in DNA structure or dynamics, and hijacking of nuclear receptors.
• Fedoroff, O. Y., Salazar, M., Han, H., Chemeris, V. V., Kerwin, S. M., & Hurley, L. H. (1998). NMR-based model of a telomerase-inhibiting compound bound to G- quadruplex DNA. Biochemistry, 37(36), 12367-12374.
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  PMID: 9730808;Abstract: The single-stranded (TTAGGG)(n) tail of human telomeric DNA is known to form stable G-quadruplex structures. Optimal telomerase activity requires the nonfolded single-stranded form of the primer, and stabilization of the G- quadruplex form is known to interfere with telomerase binding. We have identified 3,4,9,10-perylenetetracarboxylic diimide-based ligands as potent inhibitors of human telomerase by using a primer extension assay that does not use PCR-based amplification of the telomerase primer extension products. A set of NMR titrations of the ligand into solutions of G-quadruplexes using various oligonucleotides related to human telomeric DNA showed strong and specific binding of the ligand to the G-quadruplex. The exchange rate between bound and free DNA forms is slow on the NMR time scale and allows the unequivocal determination of the binding site and mode of binding. In the case of the 5'-TTAGGG sequence, the ligand-DNA complex consists of two quadruplexes oriented in a tail-to-tail manner with the ligand sandwiched between terminal G4 planes. Longer telomeric sequences, such as TTAGGGTT, TTAGGGTTA, and TAGGGTTA, form 1:1 ligand-quadruplex complexes with the ligand bound at the GT step by a threading intercalation mode. On the basis of 2D NOESY data, a model of the latter complex has been derived that is consistent with the available experimental data. The determination of the solution structure of this telomerase inhibitor bound to telomeric quadruplex DNA should help in the design of new anticancer agents with a unique and novel mechanism of action.
• Fletcher, T. M., Sun, D., Salazar, M., & Hurley, L. H. (1998). Effect of DNA secondary structure on human telomerase activity. Biochemistry, 37(16), 5536-5541.
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  PMID: 9548937;Abstract: Telomeres are specialized DNA-protein complexes located at the chromosome ends. The guanine-rich telomeric sequences have the ability to form G-quadruplex structures under physiological ionic conditions in vitro. Human telomeres are maintained through addition of TTAGGG repeats by the enzyme telomerase. To determine a correlation between DNA secondary structure and human telomerase, telomerase activity in the presence of various metal cations was monitored. Telomerase synthesized a larger proportion of products corresponding to four, five, eight, and nine full repeats of TTAGGG in 100 mM K+ and to a lesser extent in 100 mM Na+ when a d(TTAGGG)3 input primer was used. A more even product distribution was observed when the reaction mixture contained no added Na+ or K+. Increasing concentrations of Cs+ resulted in a loss of processivity but not in the distinct manner observed in K+. When the input primer contained 7-deaza-dG, the product distribution resembled that of reactions without K+ even in the presence of 100 mM K+. Native polyacrylamide gel electrophoresis indicated that d(TTAGGG)4, d(TTAGGG)5, d(TTAGGG)8, and d(TTAGGG)9 formed compact structures in the presence of K+. The oligonucleotide d(TTAGGG)4 had a UV spectrum characteristic of that of the G-quadruplex only in the presence of K+ and Na+. A reasonable explanation for these results is that four, five, eight, and nine repeats of TTAGGG form DNA secondary structures which promote dissociation of the primer from telomerase. This suggests that telomerase activity in cells can be modulated by the secondary structure of the DNA template. These findings are of probable relevance to the concept of telomerase as a therapeutic target for drug design.
• II, B. M., Seaman, F. C., Wheelhouse, R. T., & Hurley, L. H. (1998). Erratum: Mechanism for the catalytic activation of ecteinascidin 743 and its subsequent alkylation of guanine N2 (Journal of the American Chemical Society (1998) 120 (2490-91)). Journal of the American Chemical Society, 120(38), 9975-.
• II, B. M., Seaman, F. C., Wheelhouse, R. T., & Hurley, L. H. (1998). Mechanism for the catalytic activation of ecteinascidin 743 and its subsequent alkylation of guanine N2. Journal of the American Chemical Society, 120(10), 2490-2491.
• Kshirsagar, T. A., & Hurley, L. H. (1998). A facile synthesis of 5-mesyl-3-benzylbenz[e]indole: Implications for the involvement of a p-quinone methide intermediate. Journal of Organic Chemistry, 63(16), 5722-5724.
• Kshirsagar, T. A., & Hurley, L. H. (1998). Erratum: A facile synthesis of 5-mesyl-3-benzylbenze[e]indole: Implications for the involvement of a p-quinone methide intermediate (Journal of Organic Chemistry (1998) 63 (5724)). Journal of Organic Chemistry, 63(25), 9604-.
• Kwok, Y., & Hurley, L. H. (1998). Topoisomerase II site-directed alkylation of DNA by psorospermin and its effect on topoisomerase II-mediated DNA cleavage. Journal of Biological Chemistry, 273(49), 33020-33026.
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  PMID: 9830055;Abstract: Psorospermin, a plant-derived antitumor agent, has been shown to selectively alkylate a guanine at the topoisomerase II cleavage site to trap the topoisomerase II-DNA cleaved complex. The results of this study provide further important insight into the mechanism of the topoisomerase II site- directed alkylation of DNA by psorospermin and its subsequent effects on the topoisomerase II-induced DNA cleavage. First, we demonstrate that the topoisomerase II-induced alkylation of DNA by psorospermin occurs at a time preceding the topoisomerase II-mediated strand cleavage event, because it occurs in the absence of Mg2+. We confirm that the alkylation of DNA by psorospermin takes place at N-7 of guanine in the presence of topoisomerase II, because substitution of the target guanine by 7-deazaguanine prevents alkylation. Because the stimulation of the topoisomerase II-induced DNA cleavage by psorospermin can be slowly reversed by the addition of excess salt, this indicates that alkylation of DNA by psorospermin traps a reversible topoisomerase II-DNA complex. Both the DNA alkylation by psorospermin in the presence of topoisomerase II and the enzyme-mediated DNA cleavage elevated by psorospermin are more enhanced at acidic pH values, in accordance with the increased stability of the topoisomerase II-DNA complex at acidic pH values. Finally, our results suggest that it is the psorospermin-DNA adducts, not the abasic sites resulting from depurination, that are responsible for the stimulation of the topoisomerase II-mediated cleavage. Because the precise location of the psorospermin within the topoisomerase II cleavage site is known, together with the covalent DNA linkage chemistry and the conformation of the psorospermin-DNA adduct, this structural insight provides an excellent opportunity for the design and synthesis of new, more effective topoisomerase II poisons.
• Kwok, Y., Zeng, Q., & Hurley, L. H. (1998). Topoisomerase II-mediated site-directed alkylation of DNA by psorospermin and its use in mapping other topoisomerase II poison binding sites. Proceedings of the National Academy of Sciences of the United States of America, 95(23), 13531-13536.
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  PMID: 9811834;PMCID: PMC24853;Abstract: Psorospermin is a plant natural product that shows significant in vivo activity against P388 mouse leukemia. The molecular basis for this selectivity is unknown, although psorospermin has been demonstrated to intercalate into DNA and alkylate N7 of guanine. Significantly, the alkylation reactivity of psorospermin at specific sites on DNA increased 25- fold in the presence of topoisomerase II. In addition, psorospermin trapped the topoisomerase II-cleaved complex formation at the same site. These results imply that the efficacy of psorospermin is related to its interaction with the topoisomerase H-DNA complex. Because thermal treatment of (N7 guanine)-DNA adducts leads to DNA strand breakage, we were able to determine the site of alkylation of psorospermin within the topoisomerase II gate site and infer that intercalation takes place at the gate site between base pairs at the +1 and +2 positions. These results provide not only additional mechanistic information on the mode of action of the anticancer agent psorospermin but also structural insights into the design of an additional class of topoisomerase II poisons. Because the alkylation site for psorospermin in the presence of topoisomerase II can be assigned unambiguously and the intercalation site inferred, this drug is a useful probe for other topoisomerase poisons where the sites for interaction are less well defined.
• Seaman, F. C., & Hurley, L. H. (1998). Molecular basis for the DNA sequence selectivity of ecteinascidin 736 and 743: Evidence for the dominant role of direct readout via hydrogen bonding. Journal of the American Chemical Society, 120(50), 13028-13041.
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  Abstract: The marine natural product ecteinascidin 743 (Et 743) is currently in phase II clinical trials. We have undertaken parallel structural and modeling studies of an Et 743-(N2-guanine) 12-mer DNA adduct and an adduct involving the structurally related Et 736 of the same sequence in order to ascertain the structural basis for the ecteinascidin-DNA sequence selectivity. In contrast to the C-subunit differences found in Et 736 and Et 743, they have identical A-B-subunit scaffolds, which are the principal sites of interaction with DNA bases. These identical scaffolds generate parallel networks of drug- DNA hydrogen bonds that associate the drugs with the three base pairs at the recognition site. We propose that these parallel hydrogen bonding networks stabilize the Et 736 and Et 743 A- and B-subunit prealkylation binding complex with the three base pairs and are the major factors governing sequence recognition and reactivity. The possibility that a unique hydrogen- bonding network directs the course of sequence recognition was examined by first characterizing the hydrogen-bonding substituents using 1H NMR properties of the exchangeable protons attached to the hydrogen-bond donor and other protons near the proposed acceptor. Using these experimental findings as indicators of hydrogen bonding, Et 736-12-mer duplex adduct models (binding and covalent forms) containing the favored sequences 5'-AGC and 5'-CGG were examined by molecular dynamics (MD) in order to evaluate the stability of the hydrogen bonds in the resulting conformations. The MD- generated models of these favored sequences display optimal donor/acceptor positions for maximizing the number of drug-DNA hydrogen bonds prior to covalent reaction. The results of MD analysis of the carbinolamine (binding) forms of the sequences 5'-GGG (moderately reactive) and 5'-AGT (poorly reactive) suggested reasons for their diminished hydrogen-bonding capability. These experimental and modeling results provide the structural basis for the following sequence specificity rules: For the target sequence 5'-XGY, the favored base to the 3'-side, Y, is either G or C. When Y is G, then a pyrimidine base (T or C) is favored for X. When Y is C, a purine (A or G) is favored for X.
• Sun, D., Hurley, L. H., & D., D. (1998). Telomerase assay using biotinylated-primer extension and magnetic separation of the products. BioTechniques, 25(6), 1046-1051.
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  PMID: 9863060;Abstract: Human telomerase, a ribonucleoprotein enzyme, is known to be associated with immortalized cancer cells but is absent in most normal tissues. Thus, telomerase appears to be an attractive new target for anticancer agents and an important diagnostic marker of human cancers. Here, we describe an improved telomerase assay method based on the Dynabead® biomagnetic separation theory. In this method, 5'-biotinylated (TTAGGG)3 was used as a primer for the telomerase reaction. Telomerase reaction products were then immobilized on streptavidin-coated Dynabeads and washed intensively to eliminate excess [α32P]dGTP. Using this method, without the amplification of telomerase reaction products by the PCR, we were able to quantitatively detect telomerase activity in human HeLa cell extracts equivalent to between 200-500 cells. This method is anticipated to be useful for the measurement of telomerase activity in various tumor cells, for assessing potential telomerase and for understanding the biochemical aspects of the telomerase reaction.
• Wheelhouse, R. T., Sun, D., Han, H., Han, F. X., & Hurley, L. H. (1998). Cationic porphyrins as telomerase inhibitors: The interaction of tetra- (N-methyl-4-pyridyl)porphine with quadruplex DNA [1]. Journal of the American Chemical Society, 120(13), 3261-3262.
• Zeng, Q., Kwok, Y., Kerwin, S. M., Mangold, G., & Hurley, L. H. (1998). Design of new topoisomerase II inhibitors based upon a quinobenzoxazine self-assembly model. Journal of Medicinal Chemistry, 41(22), 4273-4278.
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  PMID: 9784102;Abstract: A new class of pyridobenzophenoxazine compounds has been developed as topoisomerase II inhibitors for anticancer chemotherapy. These compounds were designed based on a proposed model of a quinobenzoxazine self-assembly complex on DNA. They showed excellent inhibitory effects on several tumor cell lines with nanomolar IC50 values. Their cytotoxic potency correlates with theft ability to unwind DNA and inhibit topoisomerase II.
• II, B. M., Seaman, F. C., & Hurley, L. H. (1997). NMR-based model of an ecteinascidin 743-DNA adduct [16]. Journal of the American Chemical Society, 119(23), 5475-5476.
• Lee, S., & Hurley, L. H. (1997). DNA conformation selective intercalation of pluramycins into TBP-TATA box complex. FASEB Journal, 11(9), A1324.
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  Abstract: TDP (TATA binding protein), the primary transcription factor tliat recruits subsequent transcription machinery, binds io the TATA box through the extensive minor groove contacts and locally bends ihe DNA. This protein-induced distortion transiently creates an unv-ound site on the downstream side of the TATA box. and is favorably seized by piuramyrins, a group of novel threading intercalating arititumor antibiotics. To understand the detail of the dynamics of Ihe TATA box upon TBP binding, we have investigated the TUP-1 ATA box complexes using D Nase I arid piuramyrins. D Nase I foot print ing ex periinpnts revealed overdigested pattern rather than protection on both the if half A tract of the TATA box and the downstream flanking sequences at low i oncen t rations of protein, implying the unusual déformai ion of t he minor groove. A downstream ba.se-pair step in the protein-DNA complex showed the enhanced modification by a.11 pluramycins. However, pluramycins that have distinct, sugar substituants alkylated different guanines in the same base-pair step (CG : GC), indicating t hat the sugar substituents modulated orientations of ihe drugs. Taken together, it is proposed that the asymmetric recognition of the TBP-TATA complex originates trom the preferred distortion on the 3′ half A tract of the TATA box and that the propagated distortion results in the un winding of the specific downstream base-pair step. Those results also suggest the potential of pluramycins as molecular probes that detect uniquely deformed DNA duplexes through the specifii positioning of sugar substituents.
• Lee, S., Seaman, F. C., Sun, D., Xiong, H., Kelly, R. C., & Hurley, L. H. (1997). Replacement of the Bizelesin ureadiyl linkage by a guanidinium moiety retards translocation from monoalkylation to cross-linking sites on DNA. Journal of the American Chemical Society, 119(15), 3434-3442.
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  Abstract: In this contribution we demonstrate that Bizelesin can translocate along the minor groove of DNA from a kinetically favored monoalkylation site to a thermodynamically favored cross-linking site. This translocation is prevented in compounds that have a charged guanidino linkage substituting for the ureadiyl linkage. Furthermore, the manipulative interplay of Bizelesin and the target sequence 5'-TAATTA (Seaman, F.C.; Chu, J.; Hurley, L.H. J. Am. Chem. Soc. 1996, 118, 5383-5395) that is required to produce a suitably rearranged product for cross-linking is prevented by the substitution of a guanidino for the ureadiyl linkage. A structural basis involving hydrogen bonding of the guanidino linkage with phosphates on the backbone of DNA is proposed to account for the absence of translocation, the slow conversion of monoalkylated to cross-linked species, and the non-rearranged cross-linked product.
• Lokey, R. S., Kwok, Y., Guelev, V., Pursell, C. J., Hurley, L. H., & Iverson, B. L. (1997). A new class of polyintercalating molecules. Journal of the American Chemical Society, 119(31), 7202-7210.
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  Abstract: We have synthesized a series of polyintercalating compounds, including the first known tetraintercalator, based on the 1,4,5,8-naphthalenetetracarboxylic diimide chromophore. The chromophores are attached in a head-to-tail arrangement by peptide linkers and are synthesized by standard solid phase peptide synthesis methods. We report evidence, based on UV-visible spectroscopy and viscometry, that the compounds are fully intercalated upon binding to double-stranded DNA. Using DNAse I footprinting experiments, the bisintercalator 2 was found to bind to DNA in a cooperative manner. The footprinting results as well as association and dissociation kinetics data reveal that the compounds exhibit a tremendous preference for GC over AT sequences. A mode of binding is proposed in which the compounds intercalate completely from the major groove, and not in a threading manner as may be suggested by their structures. A kinetic scheme is proposed that takes into account the observed cooperativity and fits the data for the dissociations of the polyintercalators from poly(dAdT), although a similar scheme could not adequately model their dissociations from poly(dGdC) or from calf thymus DNA.
• Park, H. -., & Hurley, L. H. (1997). Covalent modification of N3 of guanine by (+)-CC-1065 results in protonation of the cross-strand cytosine. Journal of the American Chemical Society, 119(3), 629-630.
• Sun, D., Thompson, B., Cathers, B. E., Salazar, M., Kerwin, S. M., Trent, J. O., Jenkins, T. C., Neidle, S., & Hurley, L. H. (1997). Inhibition of human telomerase by a G-Quadruplex-Interactive compound. Journal of Medicinal Chemistry, 40(14), 2113-2116.
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  PMID: 9216827;
• Han, F. X., & Hurley, L. H. (1996). A model for the T-antigen-induced structural alteration of the SV40 replication origin based upon experiments with specific probes for bent, straight, and unwound DNA. Biochemistry, 35(24), 7993-8001.
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  PMID: 8672503;Abstract: The T-antigen-induced structural changes of the SV40 replication origin were probed with three DNA-reactive antitumor agents: (+)-CC-1065, bizelesin, and pluramycin, (+)-CC-1065 is an N3 adenine minor groove alkylating agent that selectively reacts with AT-rich DNA sequences with a bent conformation; bizelesin also reacts with the minor groove of AT-rich sequences but is selective for a straight DNA conformation. Pluramycin is an intercalative guanine alkylator whose reactivity is increased by unwinding and decreased by compression of the minor and/or major grooves of DNA. We show that while binding of T-antigen reduced the ability of (+)-CC-1065 to alkylate the AT tract in the SV40 replication origin, it did not interfere with bizelesin modification of the same sequence. These unexpected results suggest that when T-antigen binds to the SV40 origin the AT tract is in a straight DNA conformation. High-resolution DNase I footprinting experiments indicate that at least three helically in-phase T-antigen binding sites exist in the GC box region located immediately downstream of the AT tract. The binding of T- antigen enhances the reactivity of (+)-CC-1065 to the two 5'-AGTTA* (the asterisk indicates the covalent bonding site) drug modification sites in the GC box region, demonstrating that these sites are in a bent conformation. In contrast, T-antigen inhibited the reactivity of pluramycin at sequences within the GC box region that are known not to bind T-antigen. These data, in combination with the DNase I footprinting results, suggest that T-antigen binding induces a conformational change in the DNA that no longer favors pluramycin intercalation. Based on our results, we propose that T-antigen binds tightly to the upstream region of the AT tract of SV40 replication origin forming double hexamers. In the downstream region, binding of T- antigen to the helically in-phase sites in the GC box region induces DNA bending in the opposite direction of the natural AT tract bending, while simultaneously transforming the naturally bent AT tract DNA into a straight conformation.
• Hansen, M., Lee, S., Cassady, J. M., & Hurley, L. H. (1996). Molecular details of the structure of a psorospermin - DNA covalent/intercalation complex and associated DNA sequence selectivity. Journal of the American Chemical Society, 118(24), 5553-5561.
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  Abstract: Psorospermin is a DNA-reactive natural product isolated from the roots of the tropical African plant Psorospermum febrifugum that shows significant promise as an antileukemic agent. Incubation of this antineoplastic agent with DNA results in the production of sequence selective abasic sites on the DNA. Using high-field NMR and gel electrophoresis, the mechanism of covalent modification of DNA and the mode of interaction with DNA are determined. Psorospermin intercalates the DNA molecule, positioning the tricyclic xanthone chromophore in an orientation parallel to the adjacent base pairs. This places the epoxide in the major groove, resulting in site-directed electrophilic addition of the epoxide to N7 of guanine located to the 3' side of the site of DNA intercalation. It is proposed in this study that the subsequent depurination of the psorospermin - N7-guanine adduct is the source of the previously observed in vivo formation of abasic sites on the DNA. Significantly, although the chemical structure and mechanism of covalent modification of DNA are very similar to those of the pluramycin class of agents, there are distinct differences in the relative reactivities and sequence selectivity between psorospermin and pluramycin-like compounds that may give rise to observed variances in biological activity. Specifically, while psorospermin is much less reactive than most of the pluramycins, it shows unique selectivity for 5'GG* sequences (the asterisk designates the site of covalent modification), which is the least reactive site for all of the pluramycins investigated thus far. On the basis of the solution NMR structure of the psorospermin - DNA covalent adduct, the underlying structural differences that give rise to this lower reactivity and different sequence specificities are proposed.
• Henderson, D., & Hurley, L. H. (1996). Specific targeting of protein-DNA complexes by DNA-reactive drugs (+)- CC-1065 and pluramycins. Journal of Molecular Recognition, 9(2), 75-87.
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  PMID: 8877797;Abstract: To gain insight into the interactions between transcriptional factor proteins and DNA, the DNA-reactive drugs (+)-CC-1065 and pluramycin were used to target specific protein-DNA complexes. The structural features of the complex between the transcriptional activator Sp1 and the 21-base-pair repeat of the early promoter region of SV40 DNA were examined using hydroxyl-radical footprinting; (+)-CC-1065, a sequence-specific minor groove bending probe; and circularization experiments. The results show that the 21-base-pair repeat region has an intrinsically in-phase bent structure that is stabilized upon saturation Sp1 binding by protein-DNA and protein-protein interactions to produce a looping structure. The intercalating drug pluramycin was used to probe the structural details of the interaction between the TATA binding protein (TBP) and the TATA box DNA sequence. TBP, which directs initiation of RNA transcription, exhibits two-fold symmetry and apparently interacts with the TATA box in a symmetrical fashion. However, the interaction results in an asymmetric effect, in that transcription is initiated only in the downstream direction. Using pluramycin as a probe, it was determined that TBP binding to the human myoglobin TATA sequences enhances pluramycin reactivity at a site immediately downstream of the TATA box. The implications on transcriptional control of ternary complexes comprised of transcriptional factors, DNA, and DNA-reactive compounds will be presented.
• Hongtao, Y. u., Hurley, L. H., & Kerwin, S. M. (1996). Evidence for the formation of 2:2 drug-Mg²⁺ dimers in solution and for the formation of dimeric drug complexes on DNA from the DNA-accelerated photochemical reaction of antineoplastic quinobenzoxazines. Journal of the American Chemical Society, 118(30), 7040-7048.
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  Abstract: The quinobenzoxazines are a group of topoisomerase II catalytic inhibitors that have demonstrated promising anticancer activity in mice. They have been proposed to form an unprecedented 2:2 drug-Mg2+ self-assembly complex on DNA. We have exploited the photochemical decomposition of the quinobenzoxazines to gain further support and insights into the nature of 2:2 quinobenzoxazine-Mg2+ dimers and the 2:2 drug-Mg2+ complex on duplex DNA. The quinobenzoxazine A-62176 undergoes photodecomposition to highly fluorescent products. Methyl viologen (MV2+) accelerates this photoreaction almost 500-fold. The formation of 2:2 drug-Mg2+ dimers in solution is deduced from the Mg2+-dependent difference in the MV2+-facilitated photoreaction rates of racemic and scalemic A-62176. However, both racemic and scalemic A-62176 have identical MV2+-facilitated photoreaction rates in the presence of Mg2+ and the achiral fluoroquinolone norfloxacin, due to heterochemical norfloxacin/A-62176 dimer complex formation. DNA also accelerates the photochemical decomposition of A-62176 up to 80-fold. This DNA-acceleration requires Mg2+, duplex DNA, molecular oxygen, and intercalation of the drug into the DNA duplex. In the proposed model for drug-DNA complexation, only one drug molecule of each 2:2 drug-Mg2+ dimer intercalates into the DNA duplex, the other molecule binds externally to the DNA. Norfloxacin, which can only play the external binding role, was able to modulate the photochemical reaction of the quinobenzoxazines on DNA. Furthermore, it appears that the precise positioning of the intercalated molecule, which is modulated by the structure and stereochemistry of the externally bound molecule, plays an important role in determining the rate of photoreaction on DNA. The implications of the observed photochemical reaction of the quinobenzoxazines are described for human phototoxicity, photodynamic therapy, mechanism of action studies, and improved drug design for both topoisomerase and gyrase inhibitors.
• Hurley, L. H., & Chaires, J. B. (1996). Preface. Advances in DNA Sequence-Specific Agents, 2(C), ix-x.
• Park, H., Kelly, R. C., & Hurley, L. H. (1996). The chemical evolution of DNA-DNA interstrand cross-linkers that recognize defined mixed AT and GC sequences. Journal of the American Chemical Society, 118(42), 10041-10051.
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  Abstract: The monoalkylation and cross-linking reactivities of a group of four structurally related DNA-DNA interstrand cross-linkers have been determined on restriction enzyme fragments and select oligomers. These highly potent cytotoxic DNA-DNA cross-linkers consist of two cyclopropa[c]pyrrolo[3,4-3]indol-4(5H)-ones indoles [(+)-CPI-I] joined by a urea (Bizelesin) or a bisamido furan, bisamido pyrrole, or bisamido N-methylpyrrole linker. Using a thermal cleavage assay in combination with radio-labeled restriction enzyme fragments, we have shown that these compounds cross-link duplex DNA six or seven base pairs apart on opposite strands, but they differ among themselves for both alkylation reactivity and DNA sequence selectivity. Bizelesin and the [(+)-CPI-I]2 bisamido furan and [(+)-CPI-I]2 bisamido N-methyl pyrrole compounds prefer purely AT-rich sequences (e.g., 5'-T(A/T)(4 or 5)A*-3', where T represents the cross-strand adenine alkylation and A* represents an adenine alkylation), while the [(+)-CPI-I]2 bisamido pyrrole requires a centrally positioned GC base pair for high cross-linking reactivity (i.e., 5'-T(A/T)2G(A/T)2A*-3'). By comparison of the cross-linking reactivity of the four compounds in 21-mer duplex oligomers containing strategically placed GC or IC base pairs, the sequence and linker requirements for high reactivity of the six- and seven-base-pair cross-linkers in 5'-T(N)(4 or 5)5A*-3' sequences were determined. In the duplex, to attain highest reactivity, a centrally placed GC base pair and the exocyclic 2-amino group were required, while for the linker in the bisamido pyrrole compound, an unsubstituted amine in the pyrrole ring was necessary. On the basis of the known requirements for monoalkylation of duplex DNA by (+)-CPI-derived compounds and the structural consequences of monoalkylation, together with the information gleaned from this study, we are able to provide a rationale for the structural requirements for the specific sequence cross-linked with high reactivity by the pyrrole compound. We propose that, because monoalkylation of the duplex produced a bent DNA duplex that is unsuitable for cross-linking, the duplex has to first undergo a ligand-induced rearrangement involving two hydrogen-bonding donor-acceptor pairs, which reinstates the requirements necessary for the second alkylation reaction.
• Salazar, M., Thompson, B. D., Kerwin, S. M., & Hurley, L. H. (1996). Thermally induced DNA·RNA hybrid to G-quadruplex transitions: Possible implications for telomere synthesis by telomerase. Biochemistry, 35(50), 16110-16115.
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  PMID: 8973182;Abstract: Telomerase is a specialized reverse transcriptase that contains its own RNA template for synthesis of telomeric DNA [Greider, C. W., and Blackburn, E. H. (1989) Nature 337, 331-337; Shippen-Lentz, D., and Blackburn, E. H. (1990) Science 247, 546-552]. The activity of this ribonucleoprotein enzyme has been associated with cancer cells [Kim et al. (1994) Science 266, 2011- 2015] and is thus a potential target for anticancer chemotherapy. Telomeric DNA·RNA hybrids are important intermediates in telomerase function and form after extension of the growing telomere on the telomerase RNA template. Translocation is a critical step in telomerase function and consists of unwinding of the telomeric DNA·telomerase RNA hybrid followed by repositioning of the 3'-end of the extended telomere. A central question in telomerase function is how translocation of the extended telomere occurs in the absence of ATP or GTP. It has been hypothesized that unwinding of the telomeric hybrid may be facilitated by the formation of stable hairpins or G-quadruplexes by the telomere product (i.e., a hybrid to G-quadruplex transition) and that this may provide at least part of the driving force for translocation [Shippen-Lentz and Blackburn. 1990; Zahler et al. (1991) Nature 350, 718-720]. However, so far there has been no effort aimed at examining the possibility that a hybrid/G-quadruplex equilibrium can occur and to what extent this equilibrium depends on buffer and concentration conditions. Examination of these transitions may provide insight into telomerase function and may also provide clues for the development of anti- telomerase agents. Using a model system consisting of the DNA·RNA hybrid d(GGTTAAGGGTTAG)·r(cuaacccuaacc), we present evidence that a thermally induced transition of telomeric DNA·RNA hybrid to G-quadruplex can occur under certain conditions. These results provide support for the hypothesis that G-quadruplex formation by the telomere product may in fact regulate telomerase function at the translocation step (Zahler et al., 1991) and suggest an Achilles' heel for indirectly targeting telomerase. Thus, on the basis of the insight gained from the present studies and the result of Zahler et al. (1991), we propose that ligands that selectively bind or cleave G-quadruplex structures may modulate telomerase processivity.
• Seaman, F. C., & Hurley, L. H. (1996). Manipulative interplay of the interstrand cross-linker Bizelesin with d(TAATTA)₂ to achieve sequence recognition of DNA. Journal of the American Chemical Society, 118(42), 10052-10064.
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  Abstract: As expected, Bizelesin, which is a biscyclopropa[c]pyrrolo[3,2-e]indol-4(5H)-one [(+)-CPI]-derived DNA-DNA cross-linker, has a high interstrand cross-linking reactivity with the palindromic sequence 5'-d(CGTAATTȦCG)2. Contrary to expectations, the target duplex is rearranged to yield two products: one (major product) contains an AT step wherein both adenines are syn-oriented and hydrogen bonded to thymines forming a stable Hoogsteen base-paired region flanked by Watson-Crick base-paired regions (5HG); the other (minor product) contains anti-oriented AT-step adenines that show no evidence of hydrogen bonding with pairing thymines (5OP) in an otherwise normally base-paired duplex. In another unexpected outcome, the reaction of two 'uncoupled' monoalkylating (+)-CPI 'halves' of Bizelesin with the same duplex alkylates same-strand adenines three base pairs apart [5'-d(CGTAȦTTȦCG)2] rather than the anticipated opposite-strand adenines six base pairs apart (which would mimic Bizelesin). To probe the molecular mechanism that leads to Bizelesin's unusual DNA rearrangement, which appears to be a requirement for DNA-DNA interstrand cross-linking, we have carried out conformational exchange analyses (NOESY and ROESY) and restrained molecular dynamics simulations of these adducts. These studies suggest that Bizelesin controls the rearrangement of the six-base-pair target prior to cross-linkage and restricts the cross-linked DNA adduct's range of motion, freezing-out adduct conformers defined by alternative drug-DNA hydrogen-bond regimes. The two competing cross-linkage pathways share a common first step, the opening of the central AT-step base pairs, an event that is facilitated by the energetics of monoadduct-induced DNA bending distortion. One pathway (to 5HG) stabilizes these open bases by reorganizing the AT-step region into two Hoogsteen base pairs, the thymine bases of which also hydrogen bond with Bizelesin's ureadiyl subunit. A second pathway (to 5OP) directly stabilizes the open bases by forming a hydrogen-bonding complex between the AT-step thymines and Bizelesin's ureadiyl subunit. Cross-linked DNA motion drives both of the 5HG and 5OP adducts from one ephemeral hydrogen-bonding regime to another, a process documented in the NOESY conformational exchange data and simulated in restrained molecular dynamics trajectories. These results, together with the analysis of other six-base-pair Bizelesin cross-linked species, suggest a novel mechanism for sequence recognition by this cross-linker where monoalkylation distortive stress associated with a bent DNA conformation must be dissipated by a cooperative interaction between drug and duplex to produce a straight B-form-like structure before cross-linking can proceed. This example provides a new mechanism for DNA sequence recognition involving a 'drug-induced rearrangement' of DNA that critically depends upon the interplay of drug and sequence recognition elements.
• Seaman, F. C., Chu, J., & Hurley, L. (1996). Cross-linkage by "intact" Bizelesin and bisalkylation by the "separated halves" of the Bizelesin dimer: Contrasting drug manipulation of DNA conformation (5′-TAATTA-3′) directs alkylation toward different adenine targets. Journal of the American Chemical Society, 118(23), 5383-5395.
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  Abstract: Gel electrophoresis analysis of CPI-I bisalkylation of a 21-mer duplex containing 5′-TAA2TTA1-3′ (the palindromic preferred cross-linking sequence of the (+)-CC-1065 analog Bizelesin) shows same-strand (strand one) alkylation of first A1 and then A2 instead of the anticipated symmetrical A1 alkylation of strands one and two. Two-dimensional NMR analyses (NOESY and COSY) confirm the head-to-tail minor groove orientation of the same-strand-bound drugs. CPI-I contrasts sharply with Bizelesin (two CPI-I units linked tail-to-tail by a ureadiyl "linker"), which symmetrically cross-links this sequence at A1 (strands one and two), but only by first rearranging the duplex structure and consequently removing the duplex distortion stemming from monoadduct formation. CPI-I induces no such major DNA rearrangement prior to or during bisalkylation. Why does CPI-I react with the adenines of only a single strand? Two possible causes for the unexpected strand one A2 alkylation are, first, retardation of strand two A' site's reactivity by focusing of monoadduct conformational distortion on this site and, second, elevation of A2 reactivity above other competing adenine sites due to unusual monoadduct strand one A2T-step conformational properties. The relative importance of these two nonmutually exclusive factors was investigated using gel electrophoresis experiments: Time-course CPI-I bisalkylation studies were conducted on the AT-step sequence 5′-TAA2TTA1-3′ and an A-tract sequence, 5′-TAA2AAA1-3′, to see if the former sequence's AT-step flexibility, high base-pair opening rate, and unwinding capability (traits not shared by the latter sequence) controlled selection of the second target site. The observed parallel AT-step and A-tract sequence A1 and A2 bisalkylation patterns suggest that AT-step properties play at best a secondary role (compared to 5′-end TA-step behavior) in directing the second alkylation reaction to the AT-step site. rMD (solvated) simulations of the AT-step and A-tract monoadducts display distortion that is focused on this 5′-end TA-step site. While two-dimensional 1H NMR spectra of the final bisadduct reveal no significant TA-step conformational distortion, they demonstrate that conformational adjustment at the A2 ligand attachment site diminishes head-to-tail steric clash of the two drugs. These results suggest that the CPI-I monoadduct propagates bending distortion (to the 5′-side) through five base pairs toward the TA-step junction site. In the AT-step and A-tract sequences, neither adenine straddling this TA-step junction site is alkylated by CPI-I, suggesting that the base pairs forming the junction site are distorted away from a suitable orientation or are unable to assume a conformation suitable for alkylation. Consequently, the second alkylation occurs at a site (AT-step) that requires a modest displacement of the second ligand away from the already attached drug. The results and analysis of the data included in this paper provide important lessons for the design of inter- and intrastrand DNA-DNA cross-linkers.
• Seaman, F. C., Chu, J., & Hurley, L. (1996). Erratum: Cross-linkage by 'intact' bizelesin and bisalkylation by the 'separated halves' of the bizelesin dimer: Contrasting drug manipulation of DNA conformation (5'-TAATTA-3') directs alkylation toward different adenine targets (J. Am. Chem. Soc. 1996, 118, 5383-5395). Journal of the American Chemical Society, 118(33), 7871-.
• Sun, D., Hurley, L. H., & Harshey, R. M. (1996). Structural distortions induced by integration host factor (IHF) at the H' site of phage λ probed by (+)-CC-1065, pluramycin, and KMnO ₄ and by DNA cyclization studies. Biochemistry, 35(33), 10815-10827.
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  PMID: 8718873;Abstract: Integration Host Factor (IHF) is a sequence-specific DNA-bending protein that is proposed to interact with DNA primarily through the minor groove. We have used various chemical probes [(+)-CC-1065, a minor-groove-specific agent that alkylates N3 of adenine and traps bends into the minor groove; pluramycin, a minor-major-groove threading intercalator that alkylates N7 of guanine; KMnO 4, which reacts more strongly with bases in denatured DNA] to gain more information on the interaction of IHF with the H' site of phage λ. In addition to the 13-bp core consensus recognition element present at all IHF binding sites, the H' site also has an upstream AT-rich element that increases the affinity of IHF for this site. Our results reveal new details of IHF-DNA interaction at this site. Results with (+)-CC-1065 modification suggest that IHF interacts with the adenines on the 3'-side of the AT-rich element and likely induces a minor-groove bend in its vicinity, which in turn stabilizes the interaction. Pluramycin modification experiments suggest the presence of both short- and long-range structural perturbations (possible DNA unwinding events) on either side of the IHF contact region. Although IHF is known to induce a large bend in DNA at the H' site, no separation of base pairs was detected when the bent DNA was probed with KMnO 4. DNA cyclization studies indicate a large magnitude (approximately 180°) for the IHF-induced bend at the H' site, consistent with > 140° bend estimated by gel electrophoresis methods. These studies suggest that IHF-induced DNA bending is accompanied by the introduction of a DNA node, DNA unwinding, and/or by some other DNA distortion. An enhanced binding and stability of IHF was observed on small circular DNA.
• Thurston, D. E., Bose, D. S., Thompson, A. S., Howard, P. W., Leoni, A., Croker, S. J., Jenkins, T. C., Neidle, S., Hartley, J. A., & Hurley, L. H. (1996). Synthesis of sequence-selective C8-linked pyrrolo[2,1-c][1,4]benzodiazepine DNA interstrand cross-linking agents. Journal of Organic Chemistry, 61(23), 8141-8147.
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  Abstract: An efficient convergent synthesis of a homologous series of C8-linked pyrrolobenzodiazepine dimers with remarkable DNA interstrand cross-linking activity and potent in vitro cytotoxicity is reported. The 'amino thioacetal' cyclization procedure was used to produce the electrophilic DNA-interactive N10-C11 imine moiety during the final synthetic step. In order to construct the key A-ring fragments (9a-d), a versatile convergent approach has been developed to join two units of vanillic acid with α,ω-dihaloalkanes of varying length to provide the required bis(4-carboxy-2-methoxyphenoxy)alkanes while avoiding the formation of mixtures of monoalkylated and bisalkylated products.
• Fan, J., Sun, D., Hongtao, Y. u., Kerwin, S. M., & Hurley, L. H. (1995). Self-assembly of a quinobenzoxazine-Mg²⁺ complex on DNA: A new paradigm for the structure of a drug-DNA complex and implications for the structure of the quinolone bacterial gyrase-DNA complex. Journal of Medicinal Chemistry, 38(3), 408-424.
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  PMID: 7853333;Abstract: The quinobenzoxazine compounds A-62176 and A-85226 belong to a novel class of antineoplastic agents that are catalytic inhibitors of topoisomerase II and also structural analogs of the antibacterial DNA gyrase inhibitor Norfloxacin. In vitro studies have shown that their antineoplastic activity is dependent upon the presence of divalent metal ions such as Mg2+ and Mn2+, although the precise role of these ions in the mechanism of action is unknown. In this study we have investigated the structures of the binary complex between the quinobenzoxazines and Mg2+ and the ternary complex between quinobenzoxazine-Mg2+ and DNA. The stoichiometry of the binary and ternary complexes and the biophysical studies suggest that a 2:2 drug:Mg2+ complex forms a "heterodimer complex" with respect to DNA in which one drug molecule is intercalated into DNA and the second drug molecule is externally bound, held to the first molecule by two Mg2+ bridges, which themselves are chelated to phosphates on DNA. There is a cooperativity in binding of the quinobenzoxazines to DNA, and a 4:4 drug:Mg2+ complex is proposed in which the two externally bound molecules from two different 2:2 dimers interact via π-π interactions. The externally bound quinobenzoxazine molecules can be replaced by the quinolone antibacterial compound Norfloxacin to form mixed-structure dimers on DNA. Based upon the proposed model for the 2:2 quinobenzoxazine:Mg2+ complex on DNA, a parallel model for the antibacterial quinolone-Mg2+-DNA gyrase complex is proposed that relies upon the ATP-fueled unwinding of DNA by gyrase downstream of the cleavable complex site. These models, which have analogies to leucine zippers, represent a new paradigm for the structure of drug-DNA complexes. In addition, these models have important implications for the design of new gyrase and topoisomerase II inhibitors, in that optimization for structure-activity relationships should be carried out on two different quinolone molecules rather than a single molecule. © 1995 American Chemical Society.
• Hansen, M., & Hurley, L. (1995). Altromycin B threads the DNA helix interacting with both the major and the minor grooves to position itself for site-directed alkylation of guanine N7. Journal of the American Chemical Society, 117(9), 2421-2429.
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  Abstract: The pluramycins are a class of antitumor antibiotics for which a detailed structural investigation of their interaction with DNA is lacking. Using altromycin B as a prototypical pluramycin, we have characterized the drug's interaction with the self-complementary DNA duplex [d(GAAG*TACTTC)]2 diadduct (*denotes the site of covalent modification) by two-dimensional NMR and have gained considerable insight into the role played by the drug's glycosidic substituents in sequence selectivity. The drug intercalates into the DNA molecule and stacks to the 5' side of the modified guanine, thereby placing a disaccharide into the minor groove and a monosaccharide into the major groove. As a result of these interactions, the epoxide is positioned in the major groove of the DNA to perform electrophilic attack on N7 of guanine.
• Hansen, M., Yun, S., & Hurley, L. (1995). Hedamycin intercalates the DNA helix and, through carbohydrate-mediated recognition in the minor groove, directs N7-alkylation of guanine in the major groove in a sequence-specific manner. Chemistry and Biology, 2(4), 229-240.
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  PMID: 9383425;Abstract: Background: The pluramycins are a class of antitumor antibiotics that exert their biological activity through interaction with DNA. Recent studies with the analog altromycin B have determined that these agents intercalate into the DNA molecule, position carbohydrate substituents into both major and minor grooves, and alkylate the DNA molecule by epoxide-mediated electrophilic attack on N7 of guanine located to the 3′ side of the drug molecule. Alkylation is sequence dependent and appears to be modulated by glycoside substituents attached at the corners of a planar chromophore. The altromycin B-like analogs preferentially alkylate 5′AG sequences; hedamycin-like analogs prefer 5′TG and 5′CG sequences. Although the mechanism of guanine modification by altromycin B has been extensively studied, the mechanism of action of hedamycin has not been previously determined. Results: Using high-field NMR, we have shown that hedamycin stacks to the 5′ side of the guanine nucleotide at the site of intercalation in a DNA decamer, positioning both aminosaccharides into the minor groove to direct alkylation by the epoxide moiety on N7 of guanine.The C10 linked N,N-dimethylvancosamine sugar moiety interacts to the 5′ side of the intercalation site, while the C8 linked anglosamine moiety interacts to the 3′ side.The binding interactions of the two aminosugars steer the C2 double epoxide located in the major groove into the proximity of N7 of guanine. Unexpectedly, it is not the first epoxide that undergoes electrophilic addition to N7 of guanine, which would correspond to altromycin B, but the second, terminal epoxide. Conclusions: We have used two-dimensional NMR to elucidate the sequence-selective recognition of DNA by hedamycin and the mechanism of covalent modification of guanine by this antibiotic. Characterization of the intermolecular interactions between both hedamycin and altromycin B and their targeted DNA sequences has yielded a better understanding of the reasons for variations in sequence selectivity and alkylation reactivity among the pluramycin compounds.
• Henderson, D., & Hurley, L. H. (1995). Molecular struggle for transcriptional control. Nature Medicine, 1(6), 525-527.
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  PMID: 7585116;
• Mountzouris, J. A., Wang, J. -., Thurston, D., & Hurley, L. H. (1995). Comparison of a DSB-120 DNA interstrand cross-linked adduct with the corresponding bis-tomaymycin adduct: An example of a successful template- directed approach to drug design based upon the monoalkylating compound tomaymycin (Journal of Medicinal Chemistry (1994) 37 (3138)). Journal of Medicinal Chemistry, 38(6), 1052-.
• Sun, D., & Hurley, L. H. (1995). TBP binding to the TATA box induces a specific downstream unwinding site that is targeted by pluramycin. Chemistry and Biology, 2(7), 457-469.
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  PMID: 9383448;Abstract: Background: The TATA-binding protein (TBP) is one of the major components of the human TFIID multiprotein complex. It is important in directing the initiation of RNA transcription at a site immediately downstream of the TATA sequence (TATA box) found in many eukaryotic promoters. The crystal structure of TBP complexed with an oligonucleotide containing the TATA box revealed a protein with an approximate two-fold symmetry which apparently has symmetrical interactions with DNA. It is not known how an asymmetric effect involving downstream activation can be produced by an apparent symmetric complex. We set out to examine the state of DNA in the TBP-DNA complex using pluramycin, a small molecular weight probe of DNA accessibility. Results: Binding of TBP to the TATA box facilitates intercalation of pluramycin at a defined site immediately downstream of the TATA sequence through an apparent transient unwinding of the DNA. Pluramycin adducts are detected by the production of DNA strand breakage products upon heating. Incubation of pluramycin with the TBP-DNA complex facilitates the trapping of the specific complex by intercalation. Gel mobility shift and circularization assays reveal that the binding of pluramycin on the 3′-side of the TATA box region considerably stabilizes the TBP-DNA complex. Conclusions: We propose that the TBP-DNA-pluramycin ternary complex is a 'specific' binding mode in which TBP and pluramycin make compensatory alterations in DNA, accounting for the improved stability of the ternary complex. We also propose a model of the ternary complex that explains the observed asymmetric effect of TBP binding to the TATA box.
• Sun, D., Hansen, M., & Hurley, L. (1995). Molecular basis for the DNA sequence specificity of the pluramycins. A novel mechanism involving groove interactions transmitted through the helix via intercalation to achieve sequence selectivity at the covalent bonding step. Journal of the American Chemical Society, 117(9), 2430-2440.
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  Abstract: The pluramycin antitumor antibiotics, which include the altromycins, pluramycin, hedamycin, and rubiflavin, are a group of highly evolved DNA-reactive compounds that have structural features reminiscent of both nogalamycin and the aflatoxins. As such, they are characterized as "threading intercalators" with the added ability to alkylate N7 of guanine (see preceding article in this issue). In this article we have demonstrated that different members of this group of antibiotics have sequence specificities that differ for the base pair to the 5′ side of the alkylated guanine and also have a range of reactivities with susceptible sequences. Subsequent experiments were designed to determine the molecular origin for both these observed contrasting sequence specificities and covalent reactivities. First, neopluramycin, an analog of pluramycin that lacks the epoxide, and thus is unable to covalently modify DNA, but is in other respects structurally similar, exhibits no discernible sequence selectivity. This suggests that the sequence selectivity of the pluramycins is determined at the covalent bonding step rather than the precovalent binding interactions. Second, using An tracts of varying length (n = 1-5) to modulate the minor groove geometry to the 5′ side of the covalent alkylation site, this structural parameter has been shown to have a major effect on both sequence specificity and alkylation reactivity. Last, the electronegativity of the N7 position of the alkylated base can also affect reactivity and, to a lesser extent, sequence specificity. In order to determine the molecular details of the interactions in the minor and major grooves, which could give rise to the different sequence specificities of the nonclassical (typified by altromycin B) and classical (typified by hedamycin) pluramycins, we have used molecular models of the altromycin B and hedamycin-DNA adducts that are derived from high-field NMR data of their 10-mer duplex diadducts. These studies demonstrate that it is likely that the sequence-dependent reactivities of the epoxide of the pluramycin to N7 of guanine are dependent upon the relative extent of a "proximity effect". The magnitude of the proximity effect is determined by a "steering reaction", which takes place in the minor and major grooves due to the different placement of the carbohydrate substituents on the pluramycins and their hydrogen bonding and van der Waals interactions with the base pairs to the 5′ side of the alkylation site. This is proposed to be a novel mechanism for sequence recognition, where cooperative interactions in the minor and major grooves transmitted via the intercalation moiety dictate the positioning of the epoxide in the major groove and, thus, sequence reactivity. Finally, we propose that the increased reactivity of the classical pluramycins in contrast to the altromycins is at least partially determined by the "reach" of the reactive epoxide in the major groove, which varies from one group to another. The molecular mechanisms for sequence recognition described here provide a new paradigm for sequence recognition by minor and major groove interactions mediated by intercalactive binding but achieved at the covalent bonding step. * Address correspondence to this author.
• Thompson, A. S., & Hurley, L. H. (1995). Solution conformation of a bizelesin A-tract duplex adduct: DNA-DNA cross-linking of an A-tract straightens out bent DNA. Journal of Molecular Biology, 252(1), 86-101.
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  PMID: 7666436;Abstract: The DNA cross-linker bizelesin has been previously shown to form interhelical interstrand cross-links with adenine residues six base-pairs apart (including the modified adenine residues). Sequence specificity studies have shown that the ligand has a high affinity for the intrinsically bent A-tract sequence [d(CGTTTTTACG):d(CGTAAAAACG)]. However, gel retardation studies have shown that the cross-linked duplex retains none of the characteristic A-tract bending observed within the unmodified duplex. Two-dimensional 1H-NMR experiments have not only confirmed the sites of cross-linking into the duplex, but have also shown the loss of inherent A-tract characteristics, including reduced crosspeak intensities between the H2s of the central adenine residues and the cross-strand H1' of the base one base removed to the 3' side. This observation suggests loss of propeller twisting within these central adenine residues and provides insight into the controversial origin of A-tract bending. This study is important because it validates the use of bizelesin as a probe for determining the importance of A-tract-induced bending in transcriptional and replicational elements.
• Thompson, A. S., Fan, J. -., Sun, D., Hansen, M., & Hurley, L. H. (1995). Determination of the structural role of the internal guanine-cytosine base pair in recognition of a seven-base-pair sequence cross-linked by Bizelesin. Biochemistry, 34(35), 11005-11016.
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  PMID: 7669758;Abstract: Bizelesin (formerly U77,779, The Upjohn Co.) is a bifunctional DNA cross- linking antitumor antibiotic consisting of two open-ring homologs of the (+)- CC-1065 cyclopropa[c]pyrrolo[3,2-e]indol-4(5H)-one (CPI) subunits connected by a rigid linking moiety. Previous studies have shown that Bizelesin most often forms an interstrand cross-link through the N3 of two adenines 6 base pairs (bp) apart (inclusive of the modified adenines). However, gel electrophoresis studies have also indicated that Bizelesin forms 7-bp cross- links in specific sequences. In most of these sequences the cross-linked adenines represent the only possible cross-link site (i.e., no 6-bp site is available); however, in several sequences, a 7-bp sequence is selected in overwhelming preference to a possible 6-bp sequence. In this study, we demonstrate the unique requirement for a G·C base pair within this sequence and the critical presence of the exocyclic 2-amino group of guanine. In a subsequent two-dimensional 1H-NMR study that concentrates on the 7-bp cross- link formed with the sequence 5'-TTAGTTA-3', the role of the central G·C base pairs in the formation of a 7-bp cross-link is probed. 1H-NMR analysis coupled with restrained molecular dynamics (rMD) provides evidence for distortion around the covalently modified adenines. Because of this distortion, the modified bases are twisted toward the center of the duplex adduct, effectively reducing the cross-linked distance. The rMD study also indicates that a hydrogen bond is formed between the exocyclic amine of the central guanine and the carbonyl of the ureylene linker. On the basis of the observation of the distortion in the duplex and the hydrogen bonding between the drag and DNA, it is possible to speculate on the role of the central G·C bases in this sequence preference and propose a mechanism by which Bizelesin forms a 7-bp rather than a 6-bp cross-link with this sequence.
• Thompson, A. S., Sun, D., & Hurley, L. H. (1995). Monoalkylation and cross-linking of DNA by cyclopropapyrroloindoles entraps bent and straight forms of A-tracts. Journal of the American Chemical Society, 117(8), 2371-2372.
• Ding, Z. -., Harshey, R. M., & Hurley, L. H. (1994). Erratum: (+)-CC-1065 as a structural probe of Mu transposase-induced bending of DNA: Overcoming limitations of hydroxyl-radical footprinting (Nucleic Acids Research (1993) 21 (4281-4287)). Nucleic Acids Research, 22(2), 256-.
• Hurley, L. H. (1994). The minor groove covalent reactive drugs anthramycin and (+)-CC-1065 and their interstrand cross-linking derivatives.. IARC scientific publications, 295-312.
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  PMID: 7806319;
• Hurley, L. H., & Daekyu, S. (1994). (+)-CC-1065 as a probe for intrinsic and protein-induced bending of DNA. Journal of Molecular Recognition, 7(2), 123-132.
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  PMID: 7826672;
• Jenkins, T. C., Hurley, L. H., Neidle, S., & Thurston, D. E. (1994). Structure of a covalent DNA minor groove adduct with a pyrrolobenzodiazepine dimer: Evidence for sequence-specific interstrand cross-linking. Journal of Medicinal Chemistry®, 37(26), 4529-4537.
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  PMID: 7799403;Abstract: The structure of the interstrand cross-linked adduct formed between a C8-C8′-linked pyrrolobenzodiazepine (PBD) dimer (DSB-120; 1,1′-(propane-1,3-diyldioxy)bis[(11aS)-7-methoxy-1,2,3,11a-tetrahydro-5H- pyrrolo[2,1-c][1,4]benzodiazepin-5-one]) and a self-complementary d(CICGATCICG)2 duplex has been determined from high-field 1D- and 2D-NMR data using a simulated annealing procedure. The refined structure supports earlier observations from solution NMR experiments and indicates that the covalently bound molecule spans six DNA base pairs in the minor groove, forming a symmetric cross-link between the spatially separated internal guanines and with active recognition of an embedded 5′-GATC bonding site. This result confirms that template-directed approaches are useful for the design of linked DNA-interactive PBD dimers with viable DNA cross-linking potential. Further, head-to-head connection of the PBD moieties results in an overall retention of 5′-GA bonding site preference for each alkylating PBD subunit. Structural analysis indicates that cross-link formation results in a localized perturbation of the DNA duplex, attributable in part to a mutual reduction in dynamic mobility or "covalent clamping" within the Gua4-Cyt7 base tract. However, ligand-induced distortion is confined to the Cyt7 and Ino8 residues on each strand. The Gua(N2)-Gua(N2) cross-link is stabilized by two directed H-bonds from the formed aminal residues to N3 acceptor atoms of adenine bases on the 3′-side of each covalently modified guanine. Evidence for sequence-specific cross-linking with DSB-120 is provided by extended modeling studies which suggest that recognition of the favored d(̇GATĊ) motif is dominated by van der Waals steric factors, although electrostatic and H-bonded interaction terms also play a key role. This conclusion supports recent covalent footprinting studies revealing that this PBD dimer shows a selectivity for embedded base sequences of the type 5′-(pu/py)GATC(py/pu). © 1994 American Chemical Society.
• Mountzouris, J. A., Wang, J., Thurston, D., & Hurley, L. H. (1994). Comparison of a DSB-120 DNA interstrand cross-linked adduct with the corresponding bis-tomaymycin adduct: An example of a successful template-directed approach to drug design based upon the monoalkylating compound tomaymycin. Journal of Medicinal Chemistry, 37(19), 3132-3140.
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  PMID: 7932537;Abstract: The interstrand cross-linked DSB-120-d(CICG*ATCICG)2 DNA adduct (* indicates covalently modified guanine) was examined by two-dimensional NMR and compared to the bis-tomaymycin adduct on the same oligomer. Tomaymycin and DSB-120 form self-complementary adducts with the d(CICGATCICG)2 duplex sequence in which the covalent linkage sites occur between C11 of either drug and the exocyclic 2-amino group of the single reactive guanine on each strand of d(CICGATCICG)2. In the case of DSB-120, this is evidence for the formation of a guanine-guanine DNA interstrand cross-link. Both drugs show formation of an S stereochemistry at the covalent linkage site with an associated 3′ orientation. While the majority of DNA in these adducts appears to be B-form, DSB-120 interstrand cross-linking induces atypical properties in the 81 nucleotide, indicated by broadening of the 8IH2 proton resonance, non-C2′ endo sugar geometry, and unusually weak internucleotide NOE connectivity to the 7C nucleotide. Tomaymycin does not produce this regional dislocation. For tomaymycin, while there are strong NOE connectivities from protons on the five-membered ring to the 8IH2 proton on the floor of the minor groove, the equivalent internucleotide connectivities in DSB-120 are weaker. This indicates that the tomaymycin tail is close to the floor of the minor groove, while the five-membered ring of DSB-120 is more shallowly immersed, perhaps due to strain from cross-linking with a very short linker unit. Last, the conformational stresses induced on the duplex by DSB-120 appear to make the region of covalent attachment more accessible to solvent than is the case for tomaymycin. The 4GN2Hb resonance appears in 100% D2O on the tomaymycin adduct but is only observed in 90% H2O/10% D2O for the DSB-120 adduct. On the basis of these results, the strategies for template-directed DNA cross-linker design are assessed. © 1994 American Chemical Society.
• Sun, D., & Hurley, L. H. (1994). Binding of Sp1 to the 21-bp repeat region of SV40 DNA: effect of intrinsic and drug-induced DNA bending between GC boxes. Gene, 149(1), 165-172.
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  PMID: 7958981;Abstract: The effect of the antitumor antibiotic (+)-CC-1065 on the binding of Spl to the 21-bp repeats of SV40 DNA has been investigated. (+)-CC-1065 alkylates N3 of adenine in DNA and resides in the minor groove. As a consequence of alkylation of the two 5prime;-AGTTA* sequences (* indicates covalent modification site), which reside between GC boxes III and IV, and boxes V and VI, protein binding to the 3prime; sites is completely abolished and there is a significant decrease in Sp1 binding to the other regions. The effect of substituting A5 tracts for the (+)-CC-1065-bonding sequence was intermediate between the unmodified 5prime;-AGTTA* and the drug-modified sequences. It is proposed that a structural distortion of DNA associated with stiffening of the helix induced by the drug-adduct formation is primarily responsible for the inhibition of binding of Spl molecules to 21-bp repeats, rather than steric hindrance due to the occupancy by drug molecules of the minor groove within that region. © 1994.
• Sun, D., & Hurley, L. H. (1994). Cooperative bending of the 21-base-pair repeats of the SV40 viral early promoter by human Sp1. Biochemistry, 33(32), 9578-9587.
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  PMID: 8068633;Abstract: The overall structural features of the multimeric complex between Sp1 and the 21-base-pair repeat of the early promoter region of SV40 DNA have been determined using hydroxyl-radical footprinting; (+)-CC-1065, a sequence-specific minor groove bending probe; and circularization experiments. The results show that the 21-base-pair repeat region has an intrinsically in-phase bent structure that is stabilized upon saturation Sp1 binding by protein-DNA and protein-protein interactions to produce a looping structure. The direction of the Sp1-stabilized bending of DNA occurs into the minor groove and is localized between each of the Sp1 binding sites. These results are used as the basis to propose a looping structure for the multimeric Sp1 21-base-pair repeat region of SV40 DNA. Last, these results provide a rationale for the recently observed inhibition of basal transcriptional levels by site-specific triple-helical DNA complexes. © 1994 American Chemical Society.
• Aristoff, P. A., Johnson, P. D., Sun, D., & Hurley, L. H. (1993). Synthesis and biochemical evaluation of the CBI-PDE-I-dimer, a benzannelated analog of (+)-CC-1065 that also produces delayed toxicity in mice. Journal of Medicinal Chemistry, 36(14), 1956-1963.
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  PMID: 8336335;Abstract: A practical synthesis of CBI (2) was developed and applied to the synthesis of benzannelated analogs of CC-1065, including CBI-PDE-I-dimer (13) and CBI-bis-indole [(+)-A'BC]. The CBI-PDE-I-dimer was shown to have similar DNA sequence selectivity and structural effects on DNA as (+)-CC-1065. Of particular importance was the observed duplex winding effect that has been associated with the pyrrolidine ring of the nonalkylated subunits of (+)-CC-1065 and possibly correlated with its delayed toxicity effects. The effect of CBI-PDE-I-dimer was also compared to (+)-CC-1065 in the inhibition of duplex unwinding by helicase II and nick sealing by T4 ligase and found to be quantitatively similar. The in vitro and in vivo potencies of the CBI compounds corresponded very closely to the corresponding CPI derivatives. Finally, CBI-PDE-I-dimer was like (+)-CC-1065 in causing delayed toxicity in mice. © 1993 American Chemical Society.
• Ding, Z., Harshey, R. M., & Hurley, L. H. (1993). (+)-CC-1065 as a structural probe of Mu transposase-induced bending of DNA: Overcoming limitations of hydroxyl-radical footprinting. Nucleic Acids Research, 21(18), 4281-4287.
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  PMID: 8414983;PMCID: PMC310062;Abstract: Phage Mu transposase (A-protein) is primarily responsible for transposition of the Mu genome. The protein binds to six att sites, three at each end of Mu DNA. At most att sites interaction of a protein monomer with DNA is seen to occur over three minor and two consecutive major grooves and to result in bending up to about 90°. To probe the directionality and locus of these A-protein-induced bends, we have used the antitumor antibiotic (+)-CC-1065 as a structural probe. As a consequence of binding within the minor groove, (+)-CC-1065 is able to alkylate N3 of adenine in a sequence selective manner. This selectivity is partially determined by conformational flexibility of the DNA sequence, and the covalent adduct has a bent DNA structure in which narrowing of the minor groove has occurred. Using this drug in experiments in which either gel retardation or DNA strand breakage are used to monitor the stability of the A-protein - DNA complex or the (+)-CC-1065 alkylation sites on DNA (att site L3), we have demonstrated that of the three minor grooves implicated in the interaction with A-protein, the peripheral two are 'open' or accessible to drug bonding following protein binding. These drug-bonding sites very likely represent binding at at least two A-protein-induced bending sites. Significantly, the locus of bending at these sites is spaced approximately two helical turns apart, and the bending is proposed to occur by narrowing of the minor groove of DNA. The intervening minor groove between these two peripheral sites is protected from (+)-CC-1065 alkylation. The results are discussed in reference to a proposed model for overall DNA bending in the A-protein att L3 site complex. This study illustrates the utility of (+)-CC-1065 as a probe for protein-induced bending of DNA, as well as for interactions of minor groove DNA bending proteins with DNA which may be masked in hydroxyl radical footprinting experiments.
• Hurley, L. H. (1993). Structure of the altromycin B (N7-Guanine)-DNA adduct. A proposed prototypic DNA adduct structure for the pluramycin antitumor antibiotics. Biochemistry, 32(32), 8068-8074.
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  PMID: 8347608;Abstract: Altromycin B belongs to the pluramycin family of antitumor antibiotics, which also includes kidamycin, hedamycin, pluramycin, neopluramycin, DC92-B, and rubiflavin A. These potent antitumor compounds react with DNA in as yet imprecisely determined ways. In the present investigation, we have used gel electrophoresis methods in combination with nuclear magnetic resonance and mass spectrometry to determine the structure of the altromycin B-DNA adduct. High-resolution gel electrophoresis demonstrated that guanine was the reactive base, and N7 was implicated from experiments in which N7-deazaguanine was used in place of guanine in a strand breakage assay. Experiments using supercoiled DNA demonstrated that altromycin B and related drugs intercalated into DNA, which implicated this as a common mechanism for binding of the pluramycin antibiotics to DNA. The altromycin B-guanine adduct was isolated from calf thymus DNA after thermal depurination of the alkylated DNA. Mass spectrometry confirmed that altromycin alkylated DNA through guanine, and 1H- and 13C-NMR was used to confirm the covalent linkage sites between altromycin B and guanine. On the basis of these results, we propose that altromycin B first intercalates into DNA via a threading mechanism, reminiscent of nogalamycin, to insert the disaccharide into the minor groove and position the epoxide in the major groove in proximity to N7 of guanine. Nucleophilic attack from N7 of guanine leads to an acid-catalyzed opening of the epoxide, resulting in the altromycin B-DNA adduct. On the basis of these results, a general mechanism for the interaction of the pluramycin family of antibiotics with DNA is proposed. © 1993 American Chemical Society.
• Sun, D., & Hurley, L. H. (1993). Analysis of the monoalkylation and cross-linking sequence specificity of bizelesin, a bifunctional alkylation agent related to (+)-CC-1065. Journal of the American Chemical Society, 115(14), 5925-5933.
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  Abstract: The sequence specificity of bizelesin, an interstrand DNA-DNA cross-linker related to the monoalkylating compound (+)-CC-1065, was studied using restriction enzyme fragments. Bizelesin, like (+)-CC-1065, forms monoalkylation adducts through N3 of adenine but can also form DNA-DNA cross-links six base pairs apart on opposite strands. Compared to many other minor groove cross-linking compounds, bizelesin is very efficient at cross-linking DNA. There is a higher than expected proportion of cross-linked adducts based upon the relative number of cross-linked vs monoalkylated adducts. This is rationalized based upon the relative thermodynamic stability of the cross-linked vs monoalkylated species. Where bizelesin monoalkylation occurs, the sequence specificity is significantly higher than those of (+)-CC-1065 and other monoalkylating (+)-CPI analogs. The bizelesin GC tolerance at cross-linking sites is twice as high as for the monoalkylation sites. This increased GC tolerance can be largely explained by the covalent immobilization of the second alkylation arm at sequences that are not normally reactive toward CPI monoalkylation compounds but are made reactive due to a proximity effect. This same rationale can be used to explain the reactivity of the second alkylation arm of bizelesin with guanine, cytosine, and thymine on some sequence. There are some sequences that appear to be unusual in their reactivity with bizelesin in that bizelesin formed cross-linking spanning seven base pairs, and bizelesin forms monoakylation adducts on guanine. In these cases, it is proposed that bizelesin may trap out rare conformational forms during the second alkylation step, or bizelesin may alkylate unusual sites due to the strong precovalent affinity of bizelesin for those sites.
• Sun, D., Lin, C. H., & Hurley, L. H. (1993). A-tract and (+)-CC-1065-induced bending of DNA. Comparison of structural features using non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR. Biochemistry, 32(17), 4487-4495.
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  PMID: 8387334;Abstract: (+)-CC-1065 is a biologically potent DNA-reactive antitumor antibiotic produced by Streptomyces zelensis. In a previous study we have reported that (+)-CC-1065 produces bending of DNA that has similarities to that intrinsically associated with A-tracts [Lin, C. H., Sun, D., & Hurley, L. H. (1991) Chem. Res. Toxicol. 4, 21-26]. In this article we provide evidence using a combination of non-denaturing gel analysis, hydroxyl-radical footprinting, and high-field NMR for both distinctions between the two types of bends and the importance of junctions in both types of bends. For A-tracts we demonstrate that the locus of bending is at the center of an A-tract and that upon modification of the 3′ adenine with (+)-CC-1065 this locus is moved less than 1 base pair to the 3′ side, and the bending magnitude is significantly increased. For drug bonding sequences such as 5′-AGTTA* or 5′-GATTA* (where * denotes the drug bonding site), the locus of bending is found to be between the two thymines, and the bending is focused over a 2-base-pair sequence rather than a 5-base-pair sequence, as is the case for the A-tract. An important distinction between an A-tract intrinsic bend and a (+)-CC-1065-induced bend is the effect of temperature. While, as shown previously, the magnitude of A-tract bending increases with decrease in temperature, for drug-induced bending of 5′-AGTTA* the bending magnitude increases with increased temperature. Hydroxyl-radical footprinting of the drug-modified 5′-AGTTA* sequence shows a decrease in cleavage centered around the TT sequence, which is presumably associated with a decrease in minor groove width. In a parallel study, the non-self-complementary 12-mer duplex (5′-GGCGGAGTTA*GG-3′)·(5′-CCTAACTC-CGCC-3′) (Figure 2B) and the corresponding (+)-CC-1065-modified duplex adduct were examined thoroughly by one- and two-dimensional 1H NMR and NOESY restrained molecular mechanics and dynamics calculations. Both the 12-mer duplex and the (+)-CC-1065-12-mer duplex adduct maintain an overall B-form DNA with the anti base orientation throughout in aqueous solution at room temperature. The 18C nucleotide of both the 12-mer duplex and its drug-modified adduct has an average C3′-endo sugar pucker The 12-mer duplex exhibits a unique internal motion at the 16A nucleotide, which is located to the 3′ side of the complementary partner of the covalently modified adenine, and a major kink at the 18C-19T step Following covalent bonding with (+)-CC-1065, the discontinuity around 18C is entrapped and further exaggerated. In addition, the 12-mer duplex adduct displays a compression of the minor groove at the 8T to 9T step and widening on both sides, but especially abruptly at the covalent modification site. Structurally the 12-mer duplex adduct bears many similarities to a bent DNA structure, which is intrinsically associated with A-tracts. The major drug-induced distortion on DNA is localized at the 9T and 10A step of the covalently modified strand. A truncated junction model for the drug-entrapped/induced bending of DNA is proposed, and a comparison to intrinsic A-tract bending is made. © 1993 American Chemical Society.
• Sun, D., Park, H., & Hurley, L. H. (1993). Alkylation of guanine and cytosine in DNA by bizelesin. Evidence for a covalent immobilization leading to a proximity-driven alkylation of normally unreactive bases by a (+)-CC-1065 cross-linking compound. Chemical Research in Toxicology, 6(6), 889-894.
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  PMID: 8117929;Abstract: Bizelesin, an intrahelical DNA-DNA interstrand cross-linker related to (+)-CC-1065, has been shown to alkylate DNA through guanine in restriction enzyme sequences in which there is a suitably positioned adenine contained in a highly reactive monoalkylation sequence on the opposite strand. Oligomers containing the sequence 5′-TTTTTN*, in which "N" was either G, C, or T, were synthesized to evaluate the cross-linking potential of bizelesin at nonadenine bases. Kinetic analysis of monoalkylation and cross-linking events demonstrates that it is the reaction at "N" (guanine or cytosine) that results in the cross-link which is the slow step. On the basis of this analysis and the normal unreactivity of guanine and cytosine to alkylation by the cyclopropapyrroloindole alkylating moiety of (+)-CC-1065, we propose that the molecular mechanism for this type of cross-linking reaction most likely involves a covalent immobilization of the second alkylating arm, resulting in a "proximity-driven" reaction. © 1993 American Chemical Society.
• Bose, D. S., Thompson, A. S., Ching, J., Hartley, J. A., Berardini, M. D., Jenkins, T. C., Neidle, S., Hurley, L. H., & Thurston, D. E. (1992). Rational design of a highly efficient irreversible DNA interstrand cross-linking agent based on the pyrrolobenzodiazepine ring system. Journal of the American Chemical Society, 114(12), 4939-4941.
• Lin, C. H., Hill, G. C., & Hurley, L. H. (1992). Characterization of a 12-mer duplex d(GGCGGAGTTAGG)·d(CCTAACTCCGCC) containing a highly reactive (+)-CC-1065 sequence by ¹H and ³¹P NMR, hydroxyl-radical footprinting, and NOESY restrained molecular dynamics calculations. Chemical Research in Toxicology, 5(2), 167-182.
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  PMID: 1322736;Abstract: The solution structure of the GC-rich non-self-complementary DNA 12-mer duplex (I), which contains a (+)-CC-1065 highly reactive bonding sequence 5′AGTTA* (where * denotes the (Matrix Presented) covalent modification site), has been examined thoroughly by one- and two-dimensional proton and phosphorus NMR spectroscopy, hydroxyl-radical footprinting, and NOESY restrained molecular mechanics and dynamics calculations. The assignments of the nonexchangeable proton resonances (except some of the H5′ and H5″ protons due to severe resonance overlap), phosphorus resonances, and the exchangeable resonances (except amino protons of adenosine and guanosine) of this 12-mer duplex have been made. The results show that this 12-mer duplex maintains an overall B-form DNA with all anti base orientation throughout in aqueous solution at room temperature. Hydroxyl-radical footprinting experiments on a 21-mer sequence that contains this 12-mer duplex used for NMR studies showed that the minor groove is somewhat narrowed at the 7G-8T and 17A-18C steps, as indicated by the inhibition of cleavage at these locations. Although both high-field NMR and hydroxyl-radical footprinting experiments supported a bent-like structure for this 12-mer duplex, nondenaturing gel electrophoresis on the ligated 21-mer sequence that contains this 12-mer duplex did not show the abnormally slow migration characteristic of a bent DNA duplex. Analysis of the NMR data sets reveals several local structural perturbations similar to those found on an (A)n tract DNA duplex. For example, the existence of a propeller twist was detected within the A·T-rich region for both the 12-mer and the (A)n tract DNA duplexes. The 18CH5 aromatic resonance that is directly adjacent to the 3′ side of the 5′TAA segment was significantly shifted upfield with a chemical shift of 5.10 ppm, which is almost within the region normally associated with sugar H3′ protons. The sugar geometries for 18C and 7G, which are located to the 3′ side of the 5′TAA segment, are proposed to be in the neighborhood of C3′-endo and O1′-endo ⇔ C3′-endo, respectively. We propose that this unusually upfield-shifted resonance signal for 18CH5 and the average C3′-endo sugar geometry for 18C nucleotide on the 12-mer duplex is connected with the peculiar conformation, possibly a transient kink, within the 5′AC/GT step. The results of the NOESY restrained molecular mechanics and dynamics calculations on the 12-mer sequence reveal two kinks, which are located on either side of the 18C nucleotide that has an average C3′-endo sugar geometry. The two phosphorus resonance signals that are located at the 7G-8T and the 18C-19T steps, where the minor groove is narrowed as indicated by the hydroxyl-radical footprinting experiments, displayed unusual upfield chemical shifts. Also identified were two unusually broadened base protons of the 16A nucleotide and one imino proton belonging to the 9T·16A base pair within the A·T-rich segment. We proposed that this broadening phenomenon is most likely due to a unique internal motion characterized by a rapid local conformational equilibrium between microstates of the 12-mer duplex in aqueous solution at room temperature. This local conformational flexibility, a transient kink, and the bent-like structure are proposed to play a critical role in the sequence-specific recognition of the DNA duplex by (+)-CC-1065. © 1992 American Chemical Society.
• Maine, I. P., Sun, D., Hurley, L. H., & Kodadek, T. (1992). Erratum: The antitumor agent CC-1065 inhibits helicase-catalyzed unwinding of duplex DNA (Biochemistry (April 28, 1992) 31:16 (3968-3975)). Biochemistry, 31(43), 10642-.
• Maine, I. P., Sun, D., Hurley, L. H., & Kodadek, T. (1992). The antitumor agent CC-1065 inhibits helicase-catalyzed unwinding of duplex DNA. Biochemistry, 31(16), 3968-3975.
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  PMID: 1314652;Abstract: The antitumor drug CC-1065 is thought to exert its effects by covalent bonding to N3 of adenine in DNA and interfering with some aspect of DNA metabolism. Therefore, it is of interest to determine what effect this drug has on enzymes involved in various aspects of DNA metabolism. In this report, we examine the ability of two DNA helicases, the dda protein of phage T4 and helicase II of Escherichia coli, to unwind CC-1065-adducted, tailed, oligonucleotides. It is shown that the presence of the drug on DNA strongly inhibits unwinding catalyzed by the T4 and E. coli proteins. A significant difference between the results obtained with the two helicases is that DNAs containing drug on either the tailed or the completely duplex strands are poor substrates for helicase II but dda protein-mediated unwinding is inhibited only when the drug is on the tailed strand. The drug-modified, helicase-released, strands migrate abnormally through a native gel, suggesting that the drug traps an unusual secondary structure generated in the course of protein-mediated unwinding. A kinetic analysis of the drug-inhibited reactions reveals that the helicases are trapped by the DNA-drug complex. This is evidenced by a decrease in the rate of helicase exchange between drug-bound substrate and drug-free duplex. The implications of these results with respect to the mechanism of action of CC-1065 in vivo are discussed. © 1992 American Chemical Society.
• Sun, D., & Hurley, L. H. (1992). Effect of the (+)-CC-1065-(N3-adenine)DNA adduct on in vitro DNA synthesis mediated by Escherichia coli DNA polymerase. Biochemistry, 31(10), 2822-2829.
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  PMID: 1547223;Abstract: (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have shown that the potent cytotoxic and antitumor activities of (+)-CC-1065 are due to the ability of this compound to covalently modify DNA. (+)-CC-1065 reacts with duplex DNA to form an N3- adenine DNA adduct which lies in the minor groove of the DNA helix overlapping with a 5-base-pair region. As a consequence of covalent modification with (+)-CC-1065, the DNA helix bends into the minor groove and also undergoes winding and stiffening [Lee, C.-S., Sun, D., Kizu, R., and Hurley, L. H. (1991) Chem. Res. Toxicol. 4, 203-213]. In the studies described here, in which we have constructed site-directed DNA adducts on single-stranded DNA templates, we have shown that (+)-CC-1065 and select synthetic analogues, which have different levels of cytotoxicity, all show strong blocks against progression of Klenow fragment, E. coli DNA polymerase, and T4 DNA polymerase. The inhibition of bypass of drug lesions by polymerase could be partially alleviated by increasing the concentration of dNTPs and, to a small extent, by increasing polymerase levels. Klenow fragment binds equally well to a DNA template adjacent to a drug modification site and to unmodified DNA. These results taken together lead us to suspect that it is primarily inhibition of base pairing around the drug modification site and not prevention of polymerase binding that leads to blockage of DNA synthesis. Unexpectedly, the exact termination site of the in vitro DNA synthesis by Klenow fragment is not dependent on the species of covalently bound drug molecule but on the sequence to the 5' side of the drug-modified adenine. Misincorporation of dA for dG by Klenow fragment occurred at the secondary pausing site specifically for (+)-CC-1065 contained within the covalently modified sequence 5'-GATTA-3'. Although (+)-CC-1065 and its analogues evaluated in this study did not produce dramatically different effects on DNA polymerases when the drugs were bound to a single-stranded template, polymerization from a primer site containing a drug lesion in the duplex region did show a selective inhibitory effect with (+)-CC-1065 and (+)-AB'C'. When this observation is considered alongside results of experiments showing selective inhibition by these same compounds of T4 ligase and helicase II, the winding phenomena uniquely found with these compounds may be associated with the potent biological effect known as delayed lethality.
• Sun, D., & Hurley, L. H. (1992). Inhibition of T4 DNA ligase activity (+)-CC-1065: Demonstration of the importance of the stiffening and winding effects of (+)-CC-1065 on DNA. Anti-Cancer Drug Design, 7(1), 15-36.
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  PMID: 1543525;Abstract: Non-denaturing gel electrophoresis analysis demonstrates that the stiffening and winding effects of (+)-CC-1065 produce unusual proximal and distal inhibition of T4 DNA ligase-catalysed ligation of covalently modified DNA. (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. This drug selectively bonds through N3 of adenine in DNA and lies in the minor groove of DNA, reacting in a highly sequence-selective manner. Previous studies (Lee et al., 1991) have shown that (+)-CC-1065 produces bending and winding of DNA. The DNA bending and sequence specificity is mediated by the alkylating 'A' subunit of (+)-CC-1065, while the close van der Waals contacts between the non-alkylating B and C subunits of (+)-CC-1065 and the floor of the minor groove of DNA are responsible for the winding of DNA. Covalent modification of oligomers with (+)-CC-1065 and structurally related drugs leads to preferential inhibition of T4 DNA ligase on the non-covalently modified strand to the 5' side of the covalent adduct site, but enhanced ligation of the covalently modified strand. We speculate that the differential effect on proximal strand ligation is due to a drug-induced winding and helix-stabilizing effect which occurs predominantly to the 5' side of the adduct. In addition to the proximal inhibition of ligation, we also describe a distal inhibition of T4 DNA ligase activity which occurs exclusively with drug-modified oligomers and that, if successful, would result in 180° out-of-phase bent DNA following ligation. In this case, two 25 mers or a 21 plus a 29 mer are inhibited from ligation when modified with (+)-CC-1065. This distal ligation is unique to (+)-CC-1065 and its analogs that cause stiffening of the DNA helix. The (+)-CC-1065-induced stiffening effect was demonstrated using a circularization assay and was found to be associated with the close van der Waals contacts between the inside edge of (+)-CC-1065 and the floor of the minor groove, and also to the benzofuran moiety of (+)-ABC' (Adozelesin), a (+)-CC-1065 analog. We conclude from these studies that the DNA-winding and helix-stabilizing effects of these drug molecules can dramatically affect the efficiency of proximal ligation mediated by T4 DNA ligase, and the unusual helix-stiffening effect of (+)-CC-1065, (+)-AB'C' and (+)-ABC' can stabilize the structure of bent DNA formed by drug modification, which results in distal ligase inhibition.
• Sun, D., & Hurley, L. H. (1992). Structure-activity relationships of (+)-CC-1065 analogues in the inhibition of helicase-catalyzed unwinding of duplex DNA. Journal of Medicinal Chemistry, 35(10), 1773-1782.
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  PMID: 1588557;Abstract: (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have shown that the potent cytotoxic and antitumor activities of (+)-CC-1065 are due to the ability of this compound to covalently modify DNA. (+)-CC-1065 reacts with duplex DNA to form a (NS-adenine)-DNA adduct which lies in the minor groove of DNA overlapping with a five base-pair region. As a consequence of covalent modification with (+)-CC-1065, the helix bends into the minor groove and also undergoes winding and stiffening. In the studies described here, we have constructed templates for helicase-catalyzed unwinding of DNA that contain site-directed (+)-CC-1065 and analogue DNA adducts. Using these templates we have shown that (+)-CC-1065 and select synthetic analogues, which have different levels of cytotoxicity, all produce a significant inhibition of unwinding of a 3′-tailed oligomer duplex by helicase II when the displaced strand is covalently modified. However, the extent of helicase II inhibition is much more significant for (+)-CC-1065 and an analogue which also produced DNA winding when the winding effects are transmitted in the opposite direction to the helicase unwinding activity. This observed pattern of inhibition of helicase-catalyzed unwinding of drug-modified templates was the same for a 3′-T-tail, for different duplex region sequences, and with the Escherichia coli rep protein. Unexpectedly, the gel mobility of the displaced drug-modified single strand was dependent on the species of drug attached to the DNA. Last, strand displacement by helicase II coupled to primer extension by E. coli DNA polymerase I showed the same pattern of inhibition when the lagging strand was covalently modified. In addition, the presence of helicase II on single-stranded regions of templates caused the premature termination of primer extension by DNA polymerase. These results are discussed from the perspective that (+)-CC-1065 and its analogues have different effects on DNA structure, and these resulting structural changes in DNA molecules are related to the different in vivo biological consequences caused by these drug molecules. © 1992 American Chemical Society.
• Wang, J., Hill, G. C., & Hurley, L. H. (1992). Template-directed design of a DNA-DNA cross-linker based upon a bis-tomaymycin-duplex adduct. Journal of Medicinal Chemistry, 35(16), 2995-3002.
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  PMID: 1501225;Abstract: A template-directed approach to the design of a DNA-DNA interstrand cross-linker based upon the structure of a bis-tomaymycin-duplex adduct has been carried out. Tomaymycin is a member of the pyrrolo[1,4]benzodiazepines antitumor antibiotics. In a previous study (F. L. Boyd et al., Biochemistry 1990, 29, 2387-2403), we have shown that two tomaymycin molecules can be covalently bound to a 12-mer duplex molecule, where the drug molecules are on opposite strands six base-pairs apart, and the stereochemistry at the drug bonding site, and orientation in the minor groove, was defined by high-field NMR. This bis-tomaymycin 12-mer duplex adduct maintains the self-complementarity of the duplex and a B-type structure. In the present study we have shown using high-field NMR that this same 12-mer sequence can be truncated by two base pairs so that the two tomaymycin-modified guanines are now only four base-pairs apart, the two species of tomaymycin molecules are still bound with the same stereochemistry and orientation, and the 10-mer duplex adduct maintains its self-complementarity. In a second 10-mer duplex we have shown that changing the bonding sequence from 5′CGA to 5′AGC does not significantly affect the structure of the bis-tomaymycin-duplex adduct. However, when the sequence is rearranged so that the drugs point in a tail-to-tail orientation rather than in the previous head-to-head configuration, there are more than one species of tomaymycin bound to DNA, and, as a consequence, the bis-tomaymycin 10-mer duplex adduct loses its self-complementarity. Last, we have used the 10-mer duplex containing the 5′CGA sequence, in which the tomaymycin molecules are oriented head to head, to design an interstrand cross-linking species in which the two drug molecules are linked together with a flexible linker molecule. © 1992 American Chemical Society.
• Ding, Z. -., & Hurley, L. H. (1991). DNA interstrand cross-linking, DNA sequence specificity, and induced conformational changes produced by a dimeric analog of (+)-CC-1065. Anti-Cancer Drug Design, 6(5), 427-452.
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  PMID: 1662513;Abstract: U-77779 is a symmetrical dimer of the spirocyclopropyl allkylating subunit of (+)-CC-1065 in which the linker consists of two indole subunits separated by a ureido group. This compound was synthesized by scientists of the Upjohn Company and was found to be more active in both anti-tumor efficacy and cytotoxicity than its mono-alkylating analogs. Using three different 21-base pair DNA duplexes containing U-77779 reactive sequences, we have shown that U-77779 produces a stable interstrand cross-linked species that loses its internal self complementarity. A comparison of U-77779 with the mono-alkylating analogs of (+)-CC-1065 shows that it appears to have an increased sequence selectivity such that, while monoalkylating compounds like (+)-CC-1065 react at more than one site, U-77779 reacts only at sites where there are two suitably positioned alkylation sites. Chemical footprinting with 1,10-phenanthroline-copper complex revealed a six base pair cross-linked region between the two covalently modified adenines with modulated cleavage outside this region. In the case of hydroxyl radical footprinting, considerable variability of the extent of cleavage within the cross-linked sequence was found. These results are discussed in terms of likely induced conformational changes in DNA. In contrast to (+)-CC-1065, non-denaturing gel electrophoresis did not reveal any net bending of DNA due to U-77779, which we believe is due to the 180 out-of-phase bending produced on opposite strands of DNA by the cross-linker.
• Lee, C., Sun, D., Kizu, R., & Hurley, L. H. (1991). Determination of the structural features of (+)-CC-1065 that are responsible for bending and winding of DNA. Chemical Research in Toxicology, 4(2), 203-213.
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  PMID: 1782349;Abstract: Analysis of the anomalous migration in electrophoretic mobilities of (+)-CC-1065-modified oligomers following ligation reveals that (+)-CC-1065 induces DNA bending and winding of the helix. (+)-CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. This drug selectively bonds covalently to N3 of adenine and lies in the minor groove of DNA, reacting in a highly sequence-selective manner. Structurally, (+)-CC-1065 consists of three subunits: two identical pyrroloindole units (subunits B and C) and a third subunit containing the DNA-reactive cyclopropane ring (subunit A). While the bonding reaction is the main determinant of DNA sequence selectivity of (+)-CC-1065, binding interactions between the inside edge substituents of the B and C subunits and the floor of the minor groove of DNA can modulate or fine tune this sequence selectivity [Hurley, L. H., Lee, C.-S., McGovren, J. P., Mitchell, M. A., Warpehoski, M. A., Kelly, R. C., & Aristoff, P. A. (1988) Biochemistry 27, 3886-3892]. The A subunit of (+)-CC-1065 is responsible for the bending of DNA, and close van der Waals contacts between the inside edge of (+)-CC-1065 and the floor of the minor groove of DNA cause winding equivalent to about 1 base pair per alkylation site and stiffening of DNA. The magnitude of DNA bending induced by (+)-CC-1065 and related compounds is about 14-19°, which is equivalent to that produced by an adenine-thymine tract of about 5-6 base pairs in length. Experiments using oligomers containing both an adenine tract and a unique (+)-CC-1065 bonding site approximately one helix turn apart demonstrate that the directionality of drug-induced bending is in toward the minor groove and the locus of bending is about 2-3 base pairs to the 5′-side of the covalently modified adenine. A circularization efficiency assay shows that the optimum size of circles produced by (+)-CC-1065 and related drugs is between 168 and 180 base pairs. These results are discussed in relation to the molecular basis of the DNA sequence selectivity of (+)-CC-1065, and the (+)-CC-1065-induced DNA bending is compared with the intrinsic bending associated with adenine tracts. Since (+)-CC-1065 induces effects on local DNA structure that appear similar to those produced naturally by adenine tracts and certain DNA binding proteins, the relevance of this phenomenon to biological effects of (+)-CC-1065 and related drugs is considered. © 1991 American Chemical Society.
• Lin, C. H., Beale, J. M., & Hurley, L. H. (1991). Structure of the (+)-CC-1065-DNA adduct: Critical role of ordered water molecules and implications for involvement of phosphate catalysis in the covalent reaction. Biochemistry, 30(15), 3597-3602.
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  PMID: 2015216;Abstract: (+)-CC-1065 is an extremely potent antitumor agent produced by Streptomyces zelensis. The potent effects of (+)-CC-1065 and its alkylating analogues are thought to be due to the formation of a covalent adduct through N3 of adenine in DNA. It has been previously postulated, on the basis of modeling studies, that a phosphate may be involved in stabilization of the adduct and in acid catalysis of this reaction. In this study, using 1H NMR in combination with 17O-labeled water and phosphate, we demonstrate the involvement of a bridging water molecule between a phenolic proton on the alkylating subunit of (+)-CC-1065 and an anionic oxygen in the phosphate on the noncovalently modified strand of DNA. In addition, a second ordered water molecule associated with one of the protons on N6 of the covalently modified adenine is also identified. This structure has important implications for catalytic activation of the covalent reaction between (+)-CC-1065 and DNA and, consequently, the molecular basis for sequence-selective recognition of DNA by the alkylating subunit of (+)-CC-1065. On the basis of the example described here, the use of 1H NMR in 17O-labeled water may be a powerful probe to examine other structures and catalytic processes for water-mediated hydrogen-bonded bridges that occur between small molecules and DNA or enzymes. © 1991 American Chemical Society.
• Lin, C. H., Sun, D., & Hurley, L. H. (1991). (+)-CC-1065 produces bending of DNA that appears to resemble adenine/thymine tracts [2]. Chemical Research in Toxicology, 4(1), 21-26.
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  PMID: 1655087;
• Tang, M., Nazimiec, M. E., Doisy, R. P., Pierce, J. R., Hurley, L. H., & Alderete, B. E. (1991). Repair of Helix-stabilizing anthramycin-N2 guanine DNA adducts by UVRA and UVRB proteins. Journal of Molecular Biology, 220(4), 855-866.
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  PMID: 1831859;Abstract: The transfectivity of anthramycin (Atm)-modified φX174 replicative form (RF) DNA in Escherichia coli is lower in uvrA and uvrB mutant cells but much higher in uvrC mutant cells compared to wild-type cells. Pretreatment of the Atm-modified phage DNA with purified UVRA and UVRB significantly increases the transfectivity of the DNA in uvrA or uvrB mutant cells. This pretreatment greatly reduces the UVRABC nuclease-sensitive sites (UNSS) and Atm-induced absorbance at 343 nm in the Atm-modified DNA without producing apurinic sites. The reduction of UNSS is proportional to the concentrations of UVRA and UVRB and the enzyme-DNA incubation time and requires ATP. We conclude that there are two different mechanisms for repairing Atm-N2 guanine adducts by UVR proteins: (1) UVRA and UVRB bind to the Atm-N2 guanine double-stranded DNA region and consequently release the Atm from the adducted guanine; (2) UVRABC makes an incision at both sides of the Atm-DNA adduct. The latter mechanism produces potentially lethal double-strand DNA breaks in Atm-modified φX174 RF DNA in vitro. © 1991 Academic Press Limited.
• Boyd, F. L., Cheatham, S. F., Remers, W., Hill, G. C., & Hurley, L. H. (1990). Characterization of the structure of the anthramycin-d(ATGCAT)₂ adduct by NMR and molecular modeling studies. Determination of the stereochemistry of the covalent linkage site, orientation in the minor groove of DNA, and effect on local DNA structure. Journal of the American Chemical Society, 112(9), 3279-3289.
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  Abstract: Anthramycin is a member of the pyrrolo[1,4]benzodiazepine group of antitumor antibiotics. Previous studies have demonstrated that anthramycin binds covalently through N-2 of guanine within the minor groove of DNA, resulting in a relatively nondistortive DNA adduct. From the nuclear Overhauser effect spectroscopy (NOESY) proton NMR spectra of the anthramycin-d(ATGCAT)2 adduct, we have obtained results that unambiguously assign the orientation of the drug molecule in the minor groove of DNA. Four sets of NOE cross-peaks between anthramycin protons and nucleotide protons on either the covalently or the noncovalently modified strands reveal that the drug is specifically oriented with the aromatic ring to the 3′-side of the covalently modified guanine. Unequivocal assignment of the geometry at the site of attachment of anthramycin to d(ATGCAT)2 cannot be made by J-correlated spectroscopy (COSY). However, when combined with the results of modeling with the molecular mechanics program AMBER, an 11S stereochemistry at this site can be confidently predicted. 31P NMR studies show that two of the resonance signals in the anthramycin-d(ATGCAT)2 adduct have moved significantly downfield. Both downfield 31P NMR signals have been assigned by 17O isotopic labeling and 1H-31P two-dimensional J-correlation experiments and shown to correspond to the phosphates on the 5′-sides of the covalently modified deoxyguanine and the deoxycytosine on the opposite strand. Assignment of resonance signals of nonexchangeable base and sugar protons of the anthramycin-d(ATGCAT)2 has been made with two-dimensional Fourier transform NMR methods (COSY and NOESY). Conformational details about the sugar puckers, the glycosidic dihedral angles, and the effect of anthramycin bonding on secondary structure of the duplex have been obtained from the relative intensities of cross-peaks in the two-dimensional NMR spectra in aqueous solution. All of the sugars that are amenable to this analysis possess a conformation consistent with B-type DNA. Molecular mechanics calculations with AMBER are predictive of the orientation and stereochemistry of anthramycin bound to d(ATGCAT)2. The species having an 11S stereochemistry at the covalent bonding site and oriented with the aromatic ring of anthramycin to the 3′-side of the covalently modified guanine of anthramycin-d(ATGCAT)2 appears to be favored over the three other possible species. This is because of the greater intermolecular binding for this species rather than lower helix distortion energies. The molecular modeling is also in accord with the experimentally determined nondistortive nature of the anthramycin-d-(ATGCAT)2 adduct.
• Boyd, F. L., Stewart, D., Remers, W. A., Barkley, M. D., & Hurley, L. H. (1990). Characterization of a unique tomaymycin-d(CICGAATTCICG)₂ adduct containing two drug molecules per duplex by NMR, fluorescence, and molecular modeling studies. Biochemistry, 29(9), 2387-2403.
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  PMID: 2337606;Abstract: Tomaymycin is a member of the pyrrolo[l,4]benzodiazepine [P(1,4)B] antitumor antibiotic group. This antibiotic is proposed to react with the exocyclic 2-amino group (N2) of guanine to form a covalent adduct that lies snugly within the minor groove of DNA. While DNA-footprinting experiments using methidiumpropyl-EDTA have revealed the favored bonding sequences for tomaymycin and related drugs on DNA, the stereochemistry at the covalent bonding site (C-11) and orientation in the minor groove were not established by these experiments. In previous studies using a combined fluorescence, high-field NMR, and molecular modeling approach, we have shown that for tomaymycin there are two diastereomeric species (11R and 11S) on both calf thymus DNA and d(ATGCAT)2. Although we were able to infer the identity (stereochemistry at C-11 and orientation in the minor groove) of the two species on d(ATGCAT)2 by high-field NMR and fluorescence studies, in combination with molecular mechanics calculations, definitive experimental evidence was lacking. We have designed and synthesized a self-complementary 12-mer [d(CICGAATTCICG)2] based on the Dickerson dodecamer [d(CGCGAATTCGCG)2] that bonds identically two tomaymycin molecules, each having a defined orientation and stereochemistry. Thus the bis(tomaymycin)-12-mer adduct maintains the self-complementarity of the original duplex molecule. Two-dimensional proton J-correlated spectroscopy (COSY) of the bis(tomaymycin)-d(CICGAATTCICG)2 adduct (I = inosine) unequivocally shows that C-11 of tomaymycin covalently bonds through N2 of guanine with an 11S stereochemistry in the sequence 5′-CGA-3′. Fluorescence studies confirm the "S" stereochemistry at C-11, and two-dimensional proton nuclear Overhauser (NOESY) experiments assign the orientation of the drug molecule in the minor groove of DNA, i.e., with the aromatic ring of tomaymycin to the 3′ side of covalently modified guanine. Molecular modeling experiments with AMBER are consistent with the identification of the species of tomaymycin (11S with 3′ orientation) bound to the 12-mer. This species and the other 11S species are favored over the two 11R species due to a combination of steric and electrostatic interactions. Analysis of two-dimensional COSY and NOESY experiments on the bis(tomaymycin)-d-(CICGAATTCICG)2 adducts reveals minimal effect of covalent bonding on local helix structure. From these experiments the modest but most pronounced distortion is at the deoxyribose attached to the modified guanine and both the phosphate and adjacent deoxyribose to the 5′ side. The distortion of this phosphate between the covalently modified guanine and the 5′ nucleoside is supported by its downfield-shifted phosphorus NMR resonance signal. The discrepancy between the pairs of most energetically favored species of tomaymycin-DNA adducts on d(ATGCAT)2 and the 12-mer is explained by examining individual drugnucleotide interactions. The results presented in this study together with previous investigations show that the orientation of the drug molecule in the minor groove, and stereochemistry at the covalent linkage site, is dependent upon both the flanking sequence and drug structure. This conclusion mandates caution be used in rationalizing the biochemical and biological effects of P(1,4)B bonding to DNA until precise structural information is established. © 1990 American Chemical Society.
• Hurley, L. H., Warpehoski, M. A., Lee, J. C., McGovren, B. P., Scahill, T. A., Kelly, R. C., Mitchell, M. A., Wicnienski, N. A., Gebhard, I., Johnson, P. D., & Bradford, V. S. (1990). Sequence specificity of DNA alkylation by the unnatural enantiomer of CC-1065 and its synthetic analogues. Journal of the American Chemical Society, 112(12), 4633-4649.
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  Abstract: (-)-CC-1065, the unnatural enantiomer of the potent and sequence-selective, DNA-reactive antibiotic, (+)-CC-1065, was prepared by synthesis and its covalent reaction with DNA was studied and compared to that of the natural product. Although (-)-CC-1065 also formed covalent adducts in which the cyclopropyl carbon was bonded to the N3 atom of adenine, and the thermal strand breakage that it produced paralleled that seen for (+)-CC-1065, it lay in the opposite direction along the minor groove and exhibited a markedly different sequence requirement for the covalently modified adenine. While (-)-CC-1065 and its full carbon framework analogue, (-)-AB'C', reacted readily at adenines near to, but generally distinct from, (+)CC-1065-reactive adenines and exhibited potent cytotoxicity, their simpler analogues did not alkylate DNA under the conditions employed and were biologically nonpotent At relatively high concentrations, the smallest such analogue, (-)-A, reacted detectably only at the same sites selected by (+)-CC-1065. An analysis of the reactivity patterns of (+)- and (-)-CC-1065 and their analogues with DNA restriction fragments supported the conclusion that the mode of sequence recognition for (-)-CC-1065 adduct formation is fundamentally different from that of (+)-CC-1065 and is primarily controlled by specific minor groove, AT-selective binding interactions, rather than by sequence requirements of the covalent step, as occurs for (+)-CC-1065 and the (+)-CPI analogues. Models are proposed comparing the interactions of the enantiomeric alkylating moieties variously oriented in the minor groove at potential reaction sites. The evolutionary significance of both the alkylating moiety and the minor groove binding segments of the natural product is discussed.
• Lin, C. H., & Hurley, L. H. (1990). Determination of the major tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA adduct by ¹H and ¹⁵N NMR studies. Biochemistry, 29(41), 9503-9507.
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  PMID: 2271598;Abstract: (+)-CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of the drug are thought to be due to the formation of a covalent adduct with DNA through N3 of adenine. Although the covalent linkage sites between (+)-CC-1065 and DNA have been determined, the tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA duplex adduct was not defined. A [6-15N]deoxyadenosine-labeled 12 base pair non-self-complementary oligomer, d(GGCGGAGTT*AGG)·d(CCTAACTCCGCC) (asterisk indicates 15N-labeled base), containing the (+)-CC-1065 most preferred binding sequence 5′AGTTA, was synthesized and modified with (+)-CC-1065. This [6-15N]deoxyadenosine-labeled 12-mer duplex adduct was then studied by 1H and 15N NMR. One-dimensional NOE difference and two-dimensional NOESY 1H NMR experiments on the nonisotopically labeled 12-mer duplex adduct demonstrate that the 6-amino protons of the covalently modified adenine exhibit two signals at 9.19 and 9.08 ppm. Proton NMR experiments on the [6-15N]deoxyadenosine-labeled 12-mer duplex adduct show that the two resonance signals for adenine H6 observed on the nonisotopically labeled duplex adduct were split, into doublets by the 15N nucleus with coupling constants of 91.3 Hz for non-hydrogen-bonded and 86.8 Hz for hydrogen-bonded amino protons. Parallel 15N NMR experiments on the [6-15N]deoxyadenosine-labeled (+)-CC-1065-12-mer duplex adduct show a triplet-like signal around -276.9 ppm and coupling constants of 91.5 and 85.6 Hz. The large downfield shift (24.05 ppm) of the 15N signal of the (+)-CC-1065-12-mer duplex adduct is in accord with the formation of an extra positive charge on the exocyclic nitrogen at the N6 position and a partial double bond character between N6 and C6 atoms of the covalently modified adenine upon drug modification. We conclude that the covalently modified adenine N6 of the (+)-CC-1065-12-mer duplex adduct is predominantly in the doubly protonated form, in which calculations predict that the C6-N6 bond is shortened and the positive charge is delocalized over the entire adenine molecule. © 1990 American Chemical Society.
• Hurley, L. H. (1989). DNA and associated targets for drug design. Journal of Medicinal Chemistry, 32(9), 2027-2033.
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  PMID: 2671370;
• Cheatham, S., Kook, A., Hurley, L. H., Barkley, M. D., & Remers, W. (1988). One- and two-dimensional ¹H NMR, fluorescence, and molecular modeling studies on the tomaymycin-d(ATGCAT)₂ Adduct. Evidence for two covalent adducts with opposite orientations and stereochemistries at the covalent linkage site. Journal of Medicinal Chemistry, 31(3), 583-590.
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  PMID: 3346874;Abstract: Tomaymycin is a member of the pyrrolo[1,4] benzodiazepine antitumor-antibiotic group that binds covalently to the exocyclic 2-amino group of guanine in DNA. Previous correlation of fluorescence and NMR data suggested that the 11R,11aS and the 11S,11aS diastereomers of tomaymycin could bind to DNA in two orientations relative to the covalently modified guanine (Barkley, M. D.; Cheatham, S.; Thurston, D. E.; Hurley, L. H. Biochemistry 1986, 25, 3021-3031). We now report on fluorescence, one- and two-dimensional proton NMR, and molecular modeling studies of the tomaymycin-d(ATGCAT)2 adduct, which corroborate these earlier observations. Fluorescence measurements show that there are two species of tomaymycin bound to d(ATGCAT)2, which are tentatively identified as the 11R,11aS and 11S,11aS diastereomers. Two distinct sets of signals for the tomaymycin molecule are present in the proton NMR spectrum of the tomaymycin-d(ATGCAT)2 duplex adduct. Two-dimensional correlation spectroscopy (2D-COSY) studies also show connectivities for four cytosine H5-H6 and eight thymine methyl-H6 protons and thus clearly establish the presence of two distinct species of tomaymycin-d(ATGCAT)2 adducts in solution. A single scalar 11-11a 1H NMR coupling in the 2D-COSY spectrum is indicative of an adduct species that has an S configuration at the C-11 position. Two-dimensional nuclear Overhauser effect (NOESY) spectra of the tomaymycin-d(ATGCAT)2 duplex adduct show that the adducts are relatively nondistortive. In a NOESY experiment, cross-peaks were identified between both the aromatic H9 proton and the ethylidine methyl protons of tomaymycin and two different adenine H2 protons of d(ATGCAT)2. Molecular mechanics calculations with AMBER show that the two species with the thermodynamically most favorable binding energies are the 11R,11aS and 11S,11aS isomers with their aromatic rings to the 5′ and 3′ sides of the covalently bound guanine, respectively. The NOEs observed between tomaymycin protons and adenine H2 protons are in accord with molecular modeling studies. Taken together, these results strongly suggest that the two forms of tomaymycin bound to d(ATGCAT)2 are the 11S,11aS and 11R,11aS species, oriented with their aromatic rings to the 3′ and 5′ sides, respectively, of the covalently modified guanines. © 1988 American Chemical Society.
• Hurley, L. H., & Boyd, F. (1988). DNA as a target for drug action. Trends in Pharmacological Sciences, 9(11), 402-407.
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  PMID: 3078076;Abstract: DNA is the presumed target for a number of clinically useful anticancer drugs. In this review, Laurence Hurley and Leslie Boyd discuss the appropriateness of the term 'receptor' for DNA and outline the forseeable problems in designing drugs that will produce a defined pharmacological response through interaction with DNA. They describe the structural features which present DNA as an attractive target for drug design, and the possible characteristics of drugs that react with DNA to produce a predetermined biochemical response. Finally, they outline modern approaches to elucidating the structural and biological consequences of drug modification. © 1998.
• Hurley, L. H., Lee, C., McGovren, J. P., Warpehoski, M. A., Mitchell, M. A., Kelly, R. C., & Aristoff, P. A. (1988). Molecular basis for sequence-specific DNA alkylation by CC-1065. Biochemistry, 27(10), 3886-3892.
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  PMID: 3408734;Abstract: CC-1065 is a potent antitumor antibiotic that binds covalently to N3 of adenine in the minor groove of DNA. The CC-1065 molecule is made up of three repeating pyrroloindole subunits, one of which (the left-hand one or A subunit) contains a reactive cyclopropyl function. The drug reacts with adenines in DNA in a highly sequence-specific manner, overlapping four base pairs to the 5′-side of the covalently modified base. Concomitant with CC-1065 covalent binding to DNA is an asymmetric effect on local DNA structure which extends more than one helix turn to the 5′-side of the covalent binding site. The DNA alkylation, sequence specificity, and biological potency of CC-1065 and a select group of trimeric synthetic analogues were evaluated. The results suggest that (a) noncovalent interactions between this series of compounds and DNA do not lead to the formation of complexes stable enough to be detected by footprinting methods, (b) sequence specificity and alkylation intensity can be modulated by the substituents on the nonreactive middle and right-hand segments, and (c) biological potency correlates well with ability to alkylate DNA. In addition, the extent and the sequence specificity of covalent adduct formation between linear DNA fragments and three analogues comprised of the CC-1065 alkylating subunit linked to zero (analogue A), one (analogue AB), or two (analogue ABC) nonreactive indole subunits were compared. The results suggest that specificity of covalent binding of this analogue series is controlled not by the noncovalent interactions of the B and C subunits with the minor groove but by sequence-dependent reactivity of adenines with the alkylating (A) subunit. However, the B and C subunits markedly increase the apparent rate constant of the reaction with "susceptible" adenines, suggesting that these moieties facilitate noncovalent interactions preceding covalent bond formation. Covalent binding of the analogue consisting only of the alkylating subunit of CC-1065 (analogue A) was associated with the same large asymmetric effect on DNA structure as the entire CC-1065 molecule. This altered local DNA structure could be a consequence of adduct formation. Alternatively, it may be indirect evidence of a particular DNA conformation which existed prior to covalent bond formation and which was "trapped" by the drug. It is proposed that certain adenine-containing sequences have an increased propensity to undergo such a local conformational change and that this is the molecular basis for sequence specificity of these DNA-reactive compounds. These results provide strong experimental evidence for the importance of sequence-dependent site reactivity, rather than noncovalent minor groove interactions, in determining the alkylation specificity of some DNA-reactive molecules. © 1988 American Chemical Society.
• Hurley, L. H., Lee, C., McGovren, J., Warpehoski, M. A., Mitchell, M. A., Kelly, R. C., & Aristoff, P. A. (1988). Reaction of CC-1065 and select synthetic analogs with DNA. Biochemical Pharmacology, 37(9), 1795-1796.
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  PMID: 3377837;
• Hurley, L. H., Reck, T., Thurston, D. E., Langley, D. R., Holden, K. G., Hertzberg, R. P., R., J., Gallagher Jr., G., Faucette, L. F., Mong, S., & Johnson, R. K. (1988). Pyrrolo[1,4]benzodiazepine antitumor antibiotics: Relationship of DNA alkylation and sequence specificity to the biological activity of natural and synthetic compounds. Chemical Research in Toxicology, 1(5), 258-268.
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  PMID: 2979741;Abstract: The DNA alkylation and sequence specificity of a group of natural and synthetic pyrrolo-[1,4]benzodiazepines [P(1,4)Bs] were evaluated by using an exonuclease III stop assay, and the results were compared with in vitro and in vivo biological potency and antitumor activity. The P(1,4)B antibiotics are potent antitumor agents produced by various Actinomycetes, which are believed to mediate their cytotoxic effects by covalent bonding through N-2 of guanine in the minor groove of DNA. In this article we describe the results of a sensitive DNA alkylation assay using exonuclease III which permits both estimation of the extent of DNA modification as well as location of the precise guanines to which the drugs are covalently bound. Using this assay, we have evaluated a series of natural and synthetic compounds of the P(1,4)B class for their ability to bind to DNA and also determined their DNA sequence preference. The compounds included in this study are P(1,4)Bs carrying different substituents in the aromatic ring, having varying degrees of saturation in the five-membered ring, or differing in the stereochemistry at C-11a. These same compounds were evaluated for in vitro cytotoxic activity against B16 melanoma cells, for potency in vivo in B6D2F1 mice (LD50), and for antitumor activity (ILSmax) against P388 leukemia cells. A good correlation was found between extent of DNA alkylation and in vitro and in vivo potency. Furthermore, on the basis of electronic and steric considerations, it was possible to rationalize why those compounds that showed negligible biological activity were unable to bond covalently to DNA. Last, we have determined that the degree of saturation in the five-membered ring of the P(1,4)Bs has a significant effect on the DNA bonding reactivity and biological activity of this class of compounds. © 1988 American Chemical Society.
• Tang, M., Lee, C., Doisy, R., Ross, L., Needham-VanDevanter, D. R., & Hurley, L. H. (1988). Recognition and repair of the CC-1065-(N3-adenine)-DNA adduct by the UVRABC nucleases. Biochemistry, 27(3), 893-901.
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  PMID: 2966637;Abstract: The recognition and repair of the helix-stabilizing and relatively nondistortive CC-1065-(N3-adenine)-DNA adduct by UVRABC nuclease has been investigated both in vivo with ΦX174 RFI DNA by a transfection assay and in vitro by a site-directed adduct in a 117 base pair fragment from M13mpl. CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis which binds within the minor groove of DNA through N3 of adenine. In contrast to the helix-destabilizing and distortive modifications of DNA caused by ultraviolet light or N-acetoxy-2-(acetylamino)fluorene, CC-1065 increases the melting point of DNA and decreases the S1 nuclease activity. Using a viral DNA-Escherichia coli transfection system, we have found that the uvrA, uvrB, and uvrC genes, which code for the major excision repair proteins for UV- and NAAAF-induced DNA damage, are also involved in the repair of CC-1065-DNA adducts. In contrast, the uvrD gene product, which has been found to be involved in the repair of UV damage, has no effect in repairing CC-1065-DNA adducts. Purified UVRA, UVRB, and UVRC proteins must work in concert to incise the drug-modified ΦX174 RFI DNA. Using a site-directed and multiple CC-1065 modified (MspI-BstNI) 117 base pair fragment from M13mpl, we have found that UVRABC nuclease incises at the eighth phosphodiester bond on the 5′ side of the CC-1065-DNA adduct on the drug-modified strand. The enzymes do not cut the noncovalently modified strand. At low drug binding ratios, of the four CC-1065 binding sites identified in the (MspI-BstNI) 117 base pair fragment, GATTA*, GGAAA*, GATAA*, and TTTTA* (* indicates the covalently modified adenine), only the adduct at the high-affinity binding site, GATTA*, is incised by the UVRABC nucleases. No difference in the effect of CC-1065 on local DNA structure, as determined by the DNase I cleavage pattern, was evident among these sites. At high drug binding ratios, a fifth drug binding site, AGCTA*, is identified. At this concentration UVRABC nucleases are unable to incise any of these five CC-1065-DNA adducts. The DNA sequence and/or helix-stabilizing effect of multiple adducts may determine the recognition and/or incision of the drug-DNA adduct by UVRABC nuclease. These results are discussed in relation to the structure of the CC-1065-DNA adduct and the effect of drug binding on local DNA structure. © 1988 American Chemical Society.
• Warpehoski, M. A., & Hurley, L. H. (1988). Sequence selectivity of DNA covalent modification. Chemical Research in Toxicology, 1(6), 315-333.
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  PMID: 2979748;
• Hurley, L. H. (1987). Molecular biology and medicinal chemistry.. Journal of Medicinal Chemistry, 30(10), 7A-8A.
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  PMID: 3656367;
• Hurley, L. H., & Boyd, F. L. (1987). Chapter 26 Approaches Toward the Design of Sequence-Specific Drugs for DNA. Annual Reports in Medicinal Chemistry, 22(C), 259-268.
• Hurley, L. H., Needham-VanDevanter, D., & Lee, C. (1987). Demonstration of the asymmetric effect of CC-1065 on local DNA structure using a site-directed adduct in a 117-base-pair fragment from M13mp1. Proceedings of the National Academy of Sciences of the United States of America, 84(18), 6412-6416.
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  PMID: 2819875;PMCID: PMC299086;Abstract: Using DNase I and Alu I endonuclease analysis of a site-directed CC-1065-[N3-adenine]DNA adduct in a 117-base-pair fragment from M13mp1 DNA, we have demonstrated that CC-1065 produces an asymmetric effect on DNA conformation that extends more than one helix turn to the 5' side of the covalently modified adenine. CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis, which is believed to mediate its cytotoxic effects through covalent binding to DNA. Previous studies have demonstrated that CC-1065 binds covalently to N3 of adenine and lies within the minor groove of DNA spanning a 4-base-pair sequence to the 5' side of the modified adenine. DNase I footprinting of this site-directed CC-1065-DNA adduct on the noncovalently modified strand shows that inhibition of cleavage occurs over a 12-base region, which is bordered on the 3' side by a site of 2-fold enhancement of cleavage. On the covalently modified strand a much less pronounced inhibition/enhancement pattern of cleavage occurs as far as 11 bases to the 5' side of the covalently modified adenine. While Hae III is able to cleave the DNA on both strands on the 3' side of the covalently modified adenine, Alu I is only able to cleave the covalently modified strand on the 5' side of the adduct. By taking into account the recently published structure of DNase I, we are able to interpret these results and develop a model for the effect of CC-1065 on local DNA structure. In this model, we propose selective drug-induced distortion of the covalently modified strand as a consequence of the alkylation of adenine by CC-1065.
• Barkley, M. D., Cheatham, S., Thurston, D. E., & Hurley, L. H. (1986). Pyrrolo[1,4]benzodiazepine antitumor antibiotics: Evidence for two forms of tomaymycin bound to DNA. Biochemistry, 25(10), 3021-3031.
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  PMID: 3718937;Abstract: Tomaymycin is an antibiotic belonging to the pyrrolo[1,4]benzodiazepine group of antitumor compounds. Previous studies have shown that tomaymycin and other members of this group, which include anthramycin, sibiromycin, and the neothramycins, bind covalently through N-2 of guanine and lie within the minor groove of DNA. Two fluorescent ground-state species of tomaymycin were observed in protic solvents and on DNA. 1H NMR studies showed that the two fluorescent species in methanol are the 11R,11aS and 115,11aS diastereomeric 11-methyl ethers of tomaymycin. On the basis of epimerization experiments and exchange of carbon-13 from 13CH3OH into the C-11 methoxy group of the tomaymycin methyl ether, a mechanism is proposed for their interconversion via 10,11-anhydrotomaymycin. Coupling information revealed that the solution conformations of the two diastereomers differ, with the C-5 carbonyl lying closer to the plane of the aromatic ring in the 11R, 11aS diastereomer. The fluorescence excitation and emission spectra of the two emitting species in methanol were separated by time-resolved fluorescence spectroscopy and were associated with the diastereomeric forms identified by 1H NMR. Time-resolved fluorescence studies of tomaymycin in protic solvents and on DNA indicated that the absorption spectrum of the longer lifetime component (11R,11aS form) is red-shifted relative to the absorption spectrum of the shorter lifetime component (11S,11aS form), consistent with more extensive conjugation. The two conformational forms of tomaymycin on DNA were tentatively identified as the 11S, 11aS and 11R,11aS diastereomeric adducts, which bind in opposite orientations in the minor groove. This proposal is supported by molecular modeling studies using a 6-mer duplex adduct of d(ATGCAT)2. © 1986 American Chemical Society.
• Hertzberg, R. P., Hecht, S. M., Reynolds, V. L., Molineux, I. J., & Hurley, L. H. (1986). DNA sequence specificity of the pyrrolo[1,4]benzodiazepine antitumor antibiotics. Methidiumpropyl-EDTA-iron(II) footprinting analysis of DNA binding sites for anthramycin and related drugs. Biochemistry, 25(6), 1249-1258.
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  PMID: 3008824;Abstract: Anthramycin, tomaymycin, and sibiromycin are members of the pyrrolo[1,4]benzodiazepine [P(1,4)B] antitumor antibiotic group. These drugs bind covalently through N2 of guanine and lie within the minor groove of DNA [Petrusek, R. L., Anderson, G. L., Garner, T. F., Fannin, Q. L., Kaplan, D. J., Zimmer, S. G., & Hurley, L. H. (1981) Biochemistry 20, 1111-1119]. The DNA sequence specificity of the P(1,4)B antibiotics has been determined by a footprinting method using methidiumpropyl-EDTA-iron(II) [MPE·Fe(H)], and the results show that each of the drugs has a two to three base pair sequence specificity that includes the covalently modified guanine residue. While 5′PuGPu is the most preferred binding sequence for the P(1,4)Bs, 5′PyGPy is the least preferred sequence. Footprinting analysis by MPE·Fe(II) reveals a minimum of a three to four base pair footprint size for each of the drugs on DNA with a larger than expected offset (two to three base pairs) on opposite strands to that observed in previous analyses of noncovalently bound small molecules. There is an extremely large enhancement of MPE·Fe(II) cleavage between drug binding sites in AT rich regions, probably indicating a drug-induced change in the conformational features of DNA which encourages interaction with MPE·Fe(II). In the presence of sibiromycin or tomaymycin the normally guanine-specific methylene blue reaction used in Maxam and Gilbert sequencing cleaves at other bases in defined positions relative to the drug binding sites. Finally, modeling studies are used to rationalize the differences and similarities in sequence specificities between the various drugs in the P(1,4)B group and their reactions with DNA. © 1986 American Chemical Society.
• Hurley, L. H., & Needham-VanDevanter, D. (1986). Sequence specificity and biological consequences of drugs that bind covalently in the minor groove of DNA.. Basic life sciences, 38, 203-210.
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  PMID: 3741332;
• Hurley, L. H., & Needham-VanDevanter, D. R. (1986). Covalent binding of antitumor antibiotics in the minor groove of DNA. Mechanism of action of CC-1065 and the pyrrolo (1,4) benzodiazepines. Accounts of Chemical Research®, 19(8), 230-237.
• Jacobson, M. K., Twehous, D., & Hurley, L. H. (1986). Depletion of nicotinamide adenine dinucleotide in normal and xeroderma pigmentosum fibroblast cells by the antitumor drug CC-1065. Biochemistry, 25(20), 5929-5932.
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  PMID: 3790494;Abstract: CC-1065 is an extremely potent antitumor antibiotic that forms a well-defined adduct with DNA in which the molecule lies within the minor groove and is covalently attached through N3 of adenine. Addition of CC-1065 to human fibroblast cells produced a prolonged depletion of the nicotinamide adenine dinucleotide (NAD) pool even at extremely low drug concentrations (0.01 μg/mL). The depletion of NAD by CC-1065 was blocked by 3-aminobenzamide, which is consistent with a NAD depletion mechanism involving poly-(ADP-ribose) synthesis in response to a repair-induced DNA strand breakage event. Significantly, similar extents of NAD depletion were also evident in xeroderma pigmentosum cells of complementation groups A and D following exposure to CC-1065. Since this NAD depletion is presumably associated with repair-induced incision, the repair of CC-1065-DNA adducts can probably take place by a pathway distinct from that involved in repair of more conventional bulky DNA adducts. The prolonged depletion of NAD, even at low doses of drug, suggests that CC-1065 causes DNA damage that results in a delay or block in DNA excision repair between the excision and ligation steps. © 1986 American Chemical Society.
• Needham-VanDevanter, D. R., & Hurley, L. H. (1986). Construction and characterization of a site-directed CC-1065-N3-adenine adduct within a 117 base pair DNA restriction fragment. Biochemistry, 25(26), 8430-8436.
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  PMID: 3030397;Abstract: The design, construction, and characterization of a site-directed CC-1065-N3-adenine adduct in a 117 base pair segment of M13mpI DNA are described. CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have demonstrated that the cyclopropyl ring of CC-1065 reacts quite specifically with N3 of adenine in double-stranded DNA to form a CC-1065-DNA adduct. Following alkylation, the drug molecule lies snugly within the minor groove of DNA, overlapping with five base pairs for which a marked sequence preference exists [Hurley, L. H., Reynolds, V. R., Swenson, D. H., Petzold, G. L., & Scahill, T. A. (1984) Science (Washington, D.C.) 226, 843-844]. On the basis of the unique characteristics of the reaction of CC-1065 with DNA and the structure of the resulting DNA adduct, we have designed a general strategy to construct a site-directed CC-1065-DNA adduct in a restriction fragment. The presence of unique AluI and HaeIII restriction enzymes sites on each side of a high-affinity CC-1065 binding sequence (5′-GATTA) permitted the preparation of a partial duplex DNA molecule containing the CC-1065 binding sequence in the duplex DNA region. Since CC-1065 only binds to duplex DNA, potential CC-1065 binding sequences in the long single-stranded regions were protected from drug binding during the construction process. After purification of the CC-1065 partial duplex DNA adduct by differential melting of the modified and unmodified partial duplex DNA, DNA polymerase I was used to generate the full duplex DNA molecule, which contained a single site-directed CC-1065-N3-adenine adduct at adenine 6229 of the 117 base pair MspI-BstNI DNA restriction fragment of the Escherichia coli lac insert of M13mpI DNA. A CC-1065 thermal strand scission assay was used to confirm the unique binding site on the covalently modified strand. Methidiumpropyl-EDTA-iron(II) [MPE-Fe(II)] digestions were used to locate the binding site and the orientation of CC-1065 in the minor groove of DNA. MPE-Fe(11) footprinting revealed a slight enhancement of digestion on both DNA strands, but just to one side of the CC-1065-DNA adduct. © 1986 American Chemical Society.
• Reynolds, V. L., McGovren, J. P., & Hurley, L. H. (1986). The chemistry, mechanism of action and biological properties of CC-1065, a potent antitumor antibiotic. Journal of Antibiotics, 39(3), 319-334.
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  PMID: 3516958;
• Hurley, L. H., & Needham-VanDevanter, D. (1985). Sequence specificity and biologic consequences of drugs that bind covalently in the minor groove of DNA. Cancer Bulletin, 37(4), 183-186.
• Reynolds, V. L., Molineux, I. J., Kaplan, D. J., Swenson, D. H., & Hurley, L. H. (1985). Reaction of the antitumor antibiotic CC-1065 with DNA. Location of the site of thermally induced strand breakage and analysis of DNA sequence specificity. Biochemistry, 24(22), 6228-6237.
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  PMID: 3002434;Abstract: CC-1065 is a unique antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of this drug are thought to be due to its ability to form a covalent adduct with DNA through N3 of adenine. Thermal treatment of CC-1065-DNA adducts leads to DNA strand breakage. We have shown that the CC-1065 structural modification of DNA that leads to DNA strand breakage is related to the primary alkylation site on DNA. The thermally induced DNA strand breakage occurs between the deoxyribose at the adenine covalent binding site and the phosphate on the 3′ side. No residual modification of DNA is detected on the opposite strand around the CC-1065 lesion. Using the early promoter element of SV40 DNA as a target, we have examined the DNA sequence specificity of CC-1065. A consensus sequence analysis of CC-1065 binding sites on DNA reveals two distinct classes of sequences for which CC-1065 is highly specific, i.e., 5′PuNTTA and 5′AAAAA. The orientation of the DNA sequence specificity relative to the covalent binding site provides a basis for predicting the polarity of drug binding in the minor groove. Stereo drawings of the CC-1065-DNA adduct are proposed that are predictive of features of the CC-1065-DNA adduct elucidated in this investigation. © 1985 American Chemical Society.
• Brahme, N. M., Gonzalez, J. E., Mizsak, S., Rolls, J. R., Hessler, E. J., & Hurley, L. H. (1984). Biosynthesis of the lincomycins. 2. Studies using stable isotopes on the biosynthesis of methylthiolincosaminide moiety of lincomycin A. Journal of the American Chemical Society, 106(25), 7878-7883.
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  Abstract: Lincomycin is an antibiotic produced by Streptomyces lincolnensis and consists of a unique aminooctose moiety, α-methylthiolincosaminide (MTL), attached via an amide linkage to a propylhygric acid unit. The biosynthesis of the MTL moiety of lincomycin has been investigated by using both specifically carbon-13 labeled substrates and uniformly carbon-13 labeled D-glucose. In the latter case 13C-13C spin coupling patterns in lincomycin and MTL were used to determine those carbon atoms from glucose that remained intact during their conversion to the antibiotic. By combination of the biosynthetic information obtained from the 13C-13C spin coupling patterns with that from those carbon atoms in MTL which were enriched from carbon-13 specifically labeled molecules, conclusions can be drawn about likely pathways and intermediates between glucose and MTL. The C8-carbon skeleton of MTL is assembled through condensation of a pentose unit (C5) and a C3 unit. The C5 unit can be assembled in two ways. Either it is derived from glucose via the hexose monophosphate shunt (HMPS) as an intact unit or it is assembled from condensation of a C3 unit (glyceraldehyde 3-phosphate) with a C2-unit donor such as sedoheptulose 7-phosphate (SH7P) via a transketolase reaction. The C3 unit, which combines with the C5 unit, is likely contributed from a suitable donor molecule such as SH7P via a transaldolase reaction. Dependent upon the origin of the C3-unit donor, this unit may consist either of an intact C3 unit or a C2 unit combined with a C1 unit. The octase produced from condensation of a C5 unit and a C3 unit can then be converted by unexceptional means to MTL. © 1984 American Chemical Society.
• Brahme, N. M., Gonzalez, J. E., Rolls, J. P., Hessler, E. J., Mizsak, S., & Hurley, L. H. (1984). Biosynthesis of the lincomycins. 1. Studies using stable isotopes on the biosynthesis of the propyl- and ethyl-L-hygric acid moieties of lincomycins A and B. Journal of the American Chemical Society, 106(25), 7873-7878.
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  Abstract: The biosynthesis of the propyl-L-hygric acid and ethyl-L-hygric acid moieties of lincomycins A and B has been examined by using deuterated and carbon-13 labeled precursors in combination with carbon-13 NMR and mass spectral analysis. The results, using specifically deuterated tyrosine, DOPA, and methionine, demonstrate that tyrosine is converted via DOPA to an intermediate that undergoes aromatic ring cleavage most probably via a 2,3-extradiol cleavage reaction. An experiment using D-(13C6)glucose in combination with analysis of the 13C-13C spin-coupling patterns in lincomycin A and propyl-L-hygric acid permits the determination of those carbon atoms that remain together during the biosynthesis of lincomycin A. Glucose is converted via glycolysis and the hexose monophosphate shunt to phosphoenolpyruvate and erythrose 4-phosphate, respectively, which are in turn converted via the shikimic acid pathway to tyrosine and hence into DOPA. The subsequent reactions after DOPA are consistent with the 2,3-extradiol cleavage pathway and an addition of a C-1 unit from methionine to give rise to the terminal methyl group of the propyl side chain. These results are now consistent with those obtained for the C2- and C3-proline moieties found in anthramycin, tomaymycin, and sibiromycin that are also biosynthetically derived from tyrosine. © 1984 American Chemical Society.
• Graves, D. E., Pattaroni, C., Krishnan, B. S., Ostrander, J. M., Hurley, L. H., & Krugh, T. R. (1984). The reaction of anthramycin with DNA. Proton and carbon nuclear magnetic resonance studies on the structure of the anthramycin-DNA adduct. Journal of Biological Chemistry, 259(13), 8202-8209.
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  PMID: 6736032;Abstract: Nuclear magnetic resonance techniques are used to confirm the points of attachment of anthramycin to DNA. Using 13C NMR spectroscopy, the C-11 resonance of anthramycin is shown to undergo a 16-ppm upfield shift upon formation of a covalent bond with DNA, indicative of an animal linkage at that position. The site of attachment on the DNA is determined using the self-complementary oligodeoxyribonucleotide d-(ApTpGpCpApT) as a DNA model. Proton NMR, both in H2O and D2O solutions, provides a direct characterization of the anthramycin-oligonucleotide adduct. Upon covalent attachment to the duplex, a loss in the helical symmetry is observed, resulting in a doubling of several of the oligonucleotide resonances. Examination of the data confirms that the point of attachment of the anthramycin to the d-(ApTpGpCpApT) is at the guanine-NH2-position, consistent with the model proposed by Hurley and Petrusek (Hurley, L.H., and Petrusek, R.L. (1979) Nature (Lond.) 282, 529-531) and Petrusek et al. (Petrusek, R.L., Anderson, G.L., Garner, T.F., Fannin, Q.L., Kaplan, D.J., Zimmer, S.G., and Hurley, L.H. (1981) Biochemistry 20, 1111-1119).
• Hurley, L. H., & Reynolds, V. L. (1984). Reaction of the antitumor antibiotic CC-1065 with DNA: Structure of a DNA adduct with DNA sequence specificity. Science, 226(4676), 843-844.
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  PMID: 6494915;Abstract: Sequence-dependent variations in DNA revealed by x-ray crystallographic studies have suggested that certain DNA-reactive drugs may react preferentially with defined sequences in DNA. Drugs that wind around the helix and reside within one of the grooves of DNA have perhaps the greatest chance of recognizing sequence-dependent features of DNA. The antitumor antibiotic CC-1065 covalently binds through N-3 of adenine and resides within the minor groove of DNA. This drug overlaps with five base pairs for which a high sequence specificity exists.
• Hurley, L. H., & Thurston, D. E. (1984). Pyrrolo(1,4)benzodiazepine antitumor antibiotics: Chemistry, interaction with DNA, and biological implications. Pharmaceutical Research, NO. 2, 52-59.
• Needham-vandevanter, D. R., Hurley, L. H., Reynolds, V. L., Theriault, N. Y., Krueger, W. C., & Wierenga, W. (1984). Characterization of an adduct between CC-1065 and a defined oligodeoxynucleotide duplex. Nucleic Acids Research, 12(15), 6159-6168.
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  PMID: 6473105;PMCID: PMC320064;Abstract: CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis. The drug binds covalently through N-3 of adenine and lies within the minor groove of DNA. Previous studies indicated that CC-1065 reacted with adenine in DNA to yield a thermally labile product that could be used to reveal its sequence specificity. These studies also provided insight into a DNA sequence (5′-CGGAGTTAGGGGCG-3′) which should bind one molecule of CC-1065 in an unambiguous manner. This sequence, which contains the CC-1065 adenine binding site within the sequence 5′-TTA-3′ was chemically synthesized together with the complementary strand. CC-1065 reacted with the oligoduplex to give an adduct that maintained the B-DNA form and had a final CD spectrum similar to those of the CC-1065 comp1exes formed with calf thymus DNA. The above l4mer was 5′ end-labelled with 32P, annealed with its complementary strand, reacted with CC-1065 and heated. Drug-mediated strand breakage was evaluated on a sequencing gel. A single break occurred in the labelled strands to give a fragment that migrated as an 8.5mer; subsequent piperidine treatment produced a fragment that migrated as a 7mer, which is the size expected from the known binding of CC-1065 at adenine in 5′-TTA-3′ sequences. © 1984 IRL Press Limited.
• Thurston, D. E., Kaumaya, P. T., & Hurley, L. H. (1984). Limitations and factors affecting the lactam reduction approach to the synthesis of anthramycin analogs. Tetrahedron Letters, 25(25), 2649-2652.
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  Abstract: The limitations and factors affecting the hydride reduction of pyrrolo [1,4]benzodiazepine-5,10-diones to anthramycin-type analogs have been explored. © 1984.
• Hurley, L. H., & Rokem, J. S. (1983). Biosynthesis of the antitumor antibiotic CC-1065 by Streptomyces zelensis. Journal of Antibiotics, 36(4), 383-390.
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  PMID: 6406412;Abstract: The biosynthesis is the antitumor antibiotic, CC-1065, has been investigated by radioactive isotope techniques, in combination with chemical degradation of CC-1065. Tyrosine, dopa, serine and methionine (S-CH3 group) have been shown to be precursors of CC-1065. Tyrosine is proposed to be a precursor of all three benzodipyrrole subunits, while dopa is only apparently incorporated into subunits B and C. Serine is postulated to contribute three 2C units, with loss of C-1, to all three subunits of CC-1065. The S-CH3 group of methionine probably contributes four C-1 units to CC-1065 of which one is incorporated with considerable loss of tritium, most probably into the cyclopropane ring of subunit A.
• Thurston, D. E., & Hurley, L. H. (1983). A rational basis for development of antitumor agents in the pyrrolo[1,4]benzodiazepine group. Drugs of the Future, 8(11), 957-971.
• Thurston, D. E., & Hurley, L. H. (1983). Tomaymycin. Drugs of the Future, 8(11), 945-946.
• Petrusek, R. L., Uhlenhopp, E. L., Duteau, N., & Hurley, L. H. (1982). Reaction of anthramycin with DNA. Biological consequences of DNA damage in normal and xeroderma pigmentosum cell lines. Journal of Biological Chemistry, 257(11), 6207-6216.
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  PMID: 7076670;Abstract: Anthramycin, an antitumor antibiotic produced by Streptomyces refuineus, produces a well defined covalent adduct with DNA and lies within the narrow groove of DNA, attached through a thermal-labile covalent aminal linkage to the exocyclic amino group of guanine, without detectable distortion of the helix. This paper describes results in which the biological consequences of DNA damage and repair by repair-proficient and a repair-deficient xeroderma pigmentosum (XP 12RO) cell line are presented. Anthramycin has been shown to produce excision-dependent single and double strand breaks in DNA, both of which appear to persist many hours after removal of the drug from the media. The lower ability of the xeroderma pigmentosum cell line to remove anthramycin lesions from DNA is correlated with a decreased cell survival. The biological consequences of DNA damage (genetic effects, DNA strand breakage, and cytotoxicity) are discussed with respect to the defined structure and stability of the anthramycin-deoxyguanosine adduct.
• Reynolds, V. L., & Hurley, L. H. (1982). Comparison of properties of the in vitro and cellular anthramycin-DNA adducts and characterization of the reaction of anthramycin with chromatin. Chemico-Biological Interactions, 42(2), 141-151.
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  PMID: 7151226;Abstract: The reaction of anthramycin with DNA has been examined to determine the chemical identity of the adduct which forms in a living cell and to observe the effects of the nucleosome structure of chromatin on drug binding. The chemical identity of the cellular adduct was probed by comparing various properties of the cellular adduct to properties of the known, in vitro adduct. The effect of the histones on anthramycin binding was investigated by time-course binding reactions. Results indicate that the properties of the cellular anthramycin-DNA adduct are similar to the in vitro adduct. The histone proteins associated with DNA in chromatin were found to decrease both the reaction kinetics and the final levels of anthramycin binding. Anthramycin reacts appreciably with nucleosome core DNA, but appears to exhibit a preference for linker DNA. © 1982.
• Swenson, D. H., Li, L. H., Hurley, L. H., Rokem, J. S., Petzold, G. L., Dayton, B. D., Wallace, T. L., Lin, A. H., & Krueger, W. C. (1982). Mechanism of interaction of CC-1065 (NSC 298223) with DNA. Cancer Research, 42(7), 2821-2828.
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  PMID: 7083173;Abstract: CC-1065 (NSC 298223), a potent new antitumor antibiotic produced by Streptomyces zelensis, interacts strongly with double-stranded DNA and appears to exert its cytotoxic effects through disruption of DNA synthesis. We undertook this study to elucidate the sites and mechanisms of CC-1065 interaction with DNA. The binding of CC-1065 to synthetic and native DNA was examined by differential circular dichroism or by Sephadex chromatography with photometric detection. The binding of CC-1065 with calf thymus DNA was rapid, being complete within 2 hr, and saturated at 1 drug per 7 to 11 base pairs. The interaction of CC-1065 with synthetic DNA polymers indicated a specificity for adenine- and thymine-rich sites. Agarose gel electrophoresis of CC-1065-treated supercoiled DNA showed that CC-1065 did not intercalate. Site exclusion studies using substitutions in the DNA grooves showed CC-1065 to bind primarily in the minor groove. CC-1065 did not cause DNA breaks; it inhibited susceptibility of DNA to nuclease S1 digestion. It raised the thermal melting temperature of DNA, and it inhibited the thiolium-induced unwinding of DNA. Thus, in contrast to many antitumor agents, CC-1065 stabilized the DNA helix. DNA helix overstabilization may be relevant to the mechanism of action of CC-1065.
• Hurley, L. H., & Petrusek, R. L. (1981). Rationalization of the biological consequences of DNA damage produced by anthramycin in repair proficient and deficient human cell lines. Journal of Supramolecular and Cellular Biochemistry, 15(Suppl.5), No. 511.
• Kaplan, D. J., & Hurley, L. H. (1981). Anthramycin binding to deoxyribonucleic acid-mitomycin C complexes. Evidence for drug-induced deoxyribonucleic acid conformational change and cooperativity in mitomycin C binding. Biochemistry, 20(26), 7572-7580.
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  PMID: 6798992;Abstract: Anthramycin and mitomycin C (MC) are two DNA reactive drugs, which bind covalently to GC pairs producing different effects on DNA: anthramycin stiffening and MC distorsion. This paper describes experiments in which we have used anthramycin as a probe to sense quantitatively the effects on DNA of MC binding. Saturation binding experiments show that both anthramycin and MC partially inhibit the binding of the other drug to DNA (maximum inhibition by MC and anthramycin, 22.4% and 19.7%, respectively) but by a mechanism other than direct site exclusion. This suggests that MC binds in the major groove of DNA, since anthramycin is known to bind in the minor groove. An abrupt reduction in the binding of anthramycin to DNA-MC complexes occurs between MC binding ratios of 0.030 and 0.035, which parallels and probably results from sudden intensification of a MC-induced DNA conformational change occurring between these binding ratios. Dialysis measurements indicate that anthramycin is very possibly binding at sites distant from MC sites and suggest a clustering of closely bound MC chromophores resulting from possible cooperative binding. S1 nuclease digest experiments demonstrate an initial enhancement of nuclease activity in DNA-MC complexes, the magnitude of which correlates well with the reduction of anthramycin binding, relative to the degree of MC binding. The enhanced nuclease activity in these complexes indicates regions of exposed DNA or helix base distortion which is related to or is the result of conformational change. © 1981 American Chemical Society.
• Malhotra, R. K., Ostrander, J. M., Hurley, L. H., McInnes, A. G., Smith, D. G., Walter, J. A., & Wright, J. L. (1981). Chemical conversion of anthramycin 11-methyl ether to didehydroanhydroanthramycin and its utilization in studies of the biosynthesis and mechanism of action of anthramycin. Journal of Natural Products, 44(1), 38-44.
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  PMID: 7217948;Abstract: Reaction of anthramycin 11-methyl ether (AME) with trifluoroacetic acid results in formation of (1,11a)-didehydroanhydroanthramycin (DAA). Anthramycin biosynthetically labelled from DL-[3′RS(3′-3H)]; DL-[3′S(3′-3H)] and DL-[3′R(3′-3H)]tyrosine each lose approximately 50% of their tritium during this conversion to DAA confirming the labelling pattern of 3′-tritiated species of tyrosine in AME. As expected negligible losses of tritium occurred from AME biosynthetically labelled from L-[2- or 6-3H] or L-[3- or 5-3H]tyrosine. DAA did not form a stable adduct with DNA in accord with the postulated mechanism of action of anthramycin.
• Ostrander, J. M., Hurley, L. H., Balakrishnan, M. S., & Krugh, T. (1981). Determination of the structure of the anthramycin-DNA adduct by ¹H and ¹³C-NMR spectroscopy. Journal of Supramolecular and Cellular Biochemistry, 15(Suppl.5), No. 446.
• Petrusek, R. L., Anderson, G. L., Garner, T. F., Fannin, Q. L., Kaplan, D. J., Zimmer, S. G., & Hurley, L. H. (1981). Pyrrolo[1,4]benzodiazepine antibiotics. Proposed structures and characteristics of the in vitro deoxyribonucleic acid adducts of anthramycin, tomaymycin, sibiromycin, and neothramycins A and B. Biochemistry, 20(5), 1111-1119.
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  PMID: 6261786;Abstract: The pyrrolo[1,4]benzodiazepine antibiotics anthramycin, tomaymycin, sibiromycin, and neothramycins A and B are potent antitumor agents that bind to DNA in a unique manner, resulting in some unusual biological consequences. This paper describes results on which the points of covalent linkage between the drugs (carbinolamine carbon atom) and DNA (N-2 of guanine) are deduced, as well as Corey-Pauling-Koltun (CPK) models for the various drug-DNA adducts. Predictions based upon these CPK models have been tested, and the results are reported in this paper. These tested experimental predictions include (1) instability of the drug-DNA adducts to denaturation of DNA, (2) saturation binding limits, (3) effect of drug binding on the structure of DNA, (4) lack of unwinding and in vitro strand breakage of closed-circular supercoiled simian virus 40 (SV-40) DNA, (5) sensitivity of the secondary structure of DNA to drug binding, (6) hydrodynamic properties of the drug-DNA adducts, (7) hydrogen bonding of the 9-phenolic proton in anthramycin to DNA, (8) structure-activity relationships, and (9) biological consequences of DNA damage, including cumulative damage and slow excision repair, double-strand breaks in DNA in repair-proficient cells, and the selective inhibition of H-strand DNA synthesis in mitochondria. The results are completely in accord with our postulated space-filling models. © 1981 American Chemical Society.
• Rokem, J. S., & Hurley, L. H. (1981). Sensitivity and permeability of the anthramycin producing organism Streptomyces refuineus to anthramycin and structurally related antibiotics. Journal of Antibiotics, 34(9), 1171-1174.
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  PMID: 6895748;Abstract: S. refuineus, the microorganism which produces the DNA reactive antibiotic anthramycin, has been shown to possess a quite specific mechanism to survive and grow in the presence of this antibiotic. Stationary phase cells are insensitive to anthramycin since the antibiotic is prevented from entering these cells. However, cells in early log phase are inhibited by concentrations of anthramycin that are later produced by these same cells. Significantly, sibiromycin, a closely related antibiotic, is taken up by cells of S. refuineus independent of the age of the culture. Anthramycin reacts in vitro equally as well with DNA isolated from S. refuineus and other procaryotic and eucaryotic cells. When S. refuineus has reached the production phase the anthramycin is probably biosynthesized outside the cell membrane which also becomes specifically impermeable to anthramycin.
• Hannan, M. A., Estes, R., & Hurley, L. H. (1980). Induction and potentiation of lethal and genetic effects of ultraviolet light by tobacco smoke condensates in yeast. Environmental Research, 21(1), 97-107.
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  PMID: 6993205;Abstract: Tobacco smoke condensates (TSC) were tested for DNA repair inhibition in both repair proficient and different classes of repair deficient strains of Saccharomyces cerevisiae. TSC was also tested for induction and potentiation of mutations and mitotic gene conversion in unirradiated and uv-irradiated yeast cells. TSC was found to sensitize all the strains of yeast to uv-inactivation indicating that it acts in a nonspecific manner and does not specifically inhibit a particular repair pathway. Genetic studies showed that TSC, without exogenous metabolic activation, failed to produce mutations while it induced mitotic gene conversion in the diploid strain. At specific concentrations, TSC potentiated both mutagenic and gene convertogenic effects uv-light while at higher concentrations of TSC a reduction of mutations was observed. The results are discussed as they relate to carcinogenesis and cocarcinogenesis/tumor promotion. © 1980.
• Hurley, L. H. (1980). DNA as a mobile target for drug action. Pharmacy International, 1(9), 178-181.
• Hurley, L. H. (1980). Elucidation and formulation of novel biosynthetic pathways leading to the pyrrolo[1,4]benzodiazepine antibiotics anthramycin, tomaymycin, and sibiromycin. Accounts of Chemical Research, 13(8), 263-269.
• Hurley, L. H., Rokem, J., & Petrusek, R. L. (1980). Proposed structures of the pyrrolo(1,4)benzodiazepine antibiotic-deoxyribonucleic acid adducts. Biochemical Pharmacology, 29(9), 1307-1310.
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  PMID: 7397015;
• Lubawy, W. C., Dallam, R. A., & Hurley, L. H. (1980). Protection against anthramycin-induced toxicity in mice by coenzyme Q₁₀. Journal of the National Cancer Institute, 64(1), 105-109.
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  PMID: 6928034;Abstract: Pretreatment of Swiss Webster mice with coenzyme Q10 (CoQ) markedly reduced the lethality of the antitumor antibiotic anthramycin as well as its ability to decrease ventricular weights. In tumor-bearing mice CoQ pretreatment did not produce any consistent alteration of radioactivity levels in blood, heart, tumor, lungs, kidneys, liver, muscles, brain, or spleen after [15-3H]anthramycin administration. Gross alterations in anthramycin distribution is probably not the mechanism by which CoQ alters the cardiotoxicity and lethality of anthramycin.
• Ostrander, J. M., Hurley, L. H., McInnes, A. G., Smith, D. G., Walter, J. A., & Wright, J. L. (1980). Proof for the biosynthetic conversion of L-[indole- ¹⁵N]tryptophan to [10- ¹⁵N]anthramycin using ( ¹³C, ¹⁵N) labelling in conjunction with ¹³C-NMR and mass spectral analysis. Journal of Antibiotics, 33(10), 1167-1171.
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  PMID: 7451368;
• Otsuka, H., Mascaretti, O. A., Hurley, L. H., & Floss, H. G. (1980). Stereochemical aspects of the biosynthesis of spectinomycin. Journal of the American Chemical Society, 102(22), 6817-6820.
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  Abstract: The biosynthesis of spectinomycin (1) has been studied with specifically and stereospecifically labeled glucose as precursors. The results further define the mode of conversion of glucose into the actinamine (2) moiety of 1 and show that the formation of the cyclitol portion by myo-inositol-1-phosphate synthase involves stereospecific loss of the pro-R hydrogen from C-6 of glucose 6-phosphate. The TDP-glucose oxidoreductase reaction is implicated in the formation of the 4,6-dideoxyhexose moiety of 1 by the demonstration of an intramolecular hydrogen transfer from C-4 to C-6 of the hexose, which occurs with the same stereochemistry, i.e., replacement of OH at C-6 by H-4 in an inversion mode, that has been demonstrated for the enzyme from E. coli and from another streptomycete. © 1980 American Chemical Society.
• Allen, C. S., Lubawy, W. C., & Hurley, L. H. (1979). Pyrrolol(1,4)benzodiazepine antitumor antibiotics. The anthramycin-DNA conjugate; its in vitro and in vivo stability and its potential application as an anthramycin prodrug. Journal of Natural Products, 42(6), 694-.
• Dallam, R. A., Lubawy, W. C., & Hurley, L. H. (1979). Pyrrolo(1,4)benzodiazepine antitumor antibiotics. Combined protective therapy with co-enzyme Q₁₀ or vitamin E and the antitumor antibiotic anthramycin. Journal of Natural Products, 42(6), 693-.
• Hurley, L. H. (1979). Pyrrolo[1,4]benzodiazepine antibiotics. Biosynthesis of the antitumor antibiotic sibiromycin by streptosporangium sibiricum. Biochemistry, 18(19), 4225-4229.
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  PMID: 582800;Abstract: The biosynthesis of the antitumor antibiotic sibiromycin by Streptosporangium sibiricum requires the construction of four units: the amino sugar from glucose; the anthranilate ring from DL-tryptophan probably via kynurenine; the aromatic methyl group from methionine; the propylidene proline from L-tyrosine with the loss of two aromatic carbons and addition of a C-1 from methionine. Retention of tritium from DL-[5-3H]tryptophan in sibiromycin suggests an NIH shift during hydroxylation of an intermediate. © 1979 American Chemical Society.
• Hurley, L. H. (1979). Pyrrolo[1,4]benzodiazepine antibiotics. Biosynthetic conversion of tyrosine to the C₂- and C₃-proline moieties of anthramycin, tomaymycin, and sibiromycin. Biochemistry, 18(19), 4230-4237.
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  PMID: 582801;Abstract: This paper describes biosynthetic labeling experiments on the conversion of tyrosine to the C2- and C3-proline units of anthramycin, tomaymycin, and sibiromycin. The biosynthetic fate of all of the aromatic and side-chain hydrogens has been determined in each antibiotic by using dual tagged (3H/14C) and 2H-labeled tyrosine molecules. In addition, experiments using [15N]tyrosine and the tritiated D and L isomers of tyrosine have shed some light on the biochemical reactions which take place at the α position of tyrosine. On the basis of results of all these experiments, a biosynthetic scheme has been proposed to rationalize the apparent inconsistencies which occur between the results for the three antibiotics. This scheme proposes that a common main pathway involving proximal extradiol cleavage of Dopa and condensation to form the pyrrolo ring leads ultimately to a C-7 branch point compound. Parallel pathways from this central branch point compound lead by well-known biochemical transformations to the C2- and C3-proline units of anthramycin, tomaymycin, and sibiromycin. The reactions in these parallel pathways are suggested to be "cosmetic or after events". © 1979 American Chemical Society.
• Hurley, L. H., & Gairola, C. (1979). Pyrrolo (1,4) benzodiazepine antitumor antibiotics: Biosynthetic studies on the conversion of tryptophan to the anthranilic acid moieties of sibiromycin and tomaymycin. Antimicrobial Agents and Chemotherapy, 15(1), 42-45.
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  PMID: 581831;PMCID: PMC352597;Abstract: Biosynthetic intermediates between tryptophan and the anthranilate mioeties of tomaymycin and sibiromycin have been suggested, based upon a combination of feeding experiments with either carbon-14-labeled substrates or competition experiments between radiolabeled tryptophan and unlabeled intermediates. In the case of sibiromycin and tomaymycin, substitution of the aromatic ring most likely takes place at the kynurenine stage. Feeding experiments with the antramycin culture were inconclusive, most likely because of the cell impermeability.
• Hurley, L. H., & Petrusek, R. (1979). Proposed structure of the anthramycin-DNA adduct. Nature, 282(5738), 529-531.
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  PMID: 503235;
• Hurley, L. H., Allen, C. S., Feola, J. M., & Lubawy, W. C. (1979). In vitro and in vivo stability of anthramycin-DNA conjugate and its potential application as an anthramycin prodrug. Cancer Research, 39(8), 3134-3140.
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  PMID: 455297;Abstract: Anthramycin-DNA adducts, produced in vitro by reaction of anthramycin with calf thymus DNA, have been shown to be stable only as long as the secondary structure of DNA is maintained. Denaturation either by heat or enzymatic degradation of the DNA adduct, with DNase I and snake venom phosphodiesterase, leads to the release of significant amounts of the bound drug as unchanged anthramycin. These observations led us to suspect that the DNA adduct might be a suitable prodrug system for anthramycin, which might be more efficacious and less toxic than the administration of the free drug. In order to test this hypothesis, the ability of the adduct versus free drug to inhibit DNA synthesis and induce unshceduled DNA synthesis in a human cell line was evaluated. The results demonstrated that the anthramycin-DNA adduct was less potent than the free drug in these systems in both respects. The anthramycin and anthramycin-DNA conjugate were compared in mice for lethality, tisue levels, alteration of hexobarbital sleeping times, and efficacy against a mouse ascites tumor model. These results showed that the DNA adduct was three times more lethal and produced similar increases in sleeping times at equitoxic doses. The increase in lethality of the anthramycin-DNA adduct could be explained by elevated and more prolonged blood and tissue levels following administration of the DNA conjugate as compared to free anthramycin. When tested for efficacy against a mouse ascites tumor line, the anthramycin-DNA adduct was found to be less efficacious than the free drug.
• Hurley, L. H., Chandler, C., Garner, T. F., Petrusek, R., & Zimmer, S. G. (1979). DNA binding, induction of unscheduled DNA synthesis, and excision of anthramycin from DNA in normal and repair-deficient human fibroblasts.. Journal of Biological Chemistry, 254(3), 605-608.
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  PMID: 762084;Abstract: The reaction of the antitumor antibiotic anthramycin with cellular DNA and the ability of normal human fibroblasts cells and xeroderma pigmentosum (XP) cells to respond to this injury has been evaluated. The binding of [15-3H]anthramycin to cellular DNA in human skin fibroblasts occurred in a linear manner up to 6 h. Treatment with unlabeled antibiotic resulted in unscheduled (repair) DNA synthesis in human skin fibroblasts maintained in hydroxyurea, whereas negligible unscheduled DNA synthesis was observed in cells of an excision-defective strain of XP. Confluent nondividing normal skin fibroblast cells were able to remove 86% of the bound anthramycin within 72 h, however XP cells were only able to remove 49% during the same incubation period. These results are discussed in terms of the types of DNA damage produced by anthramycin in vitro and the likely repair pathways involved in removing lesions produced on DNA by anthramycin.
• Lubawy, W. C., Whaley, J., & Hurley, L. H. (1979). Coenzyme Q₁₀ or α-tocopherol reduce the acute toxicity of anthramycin in mice. Research Communications in Chemical Pathology and Pharmacology, 24(2), 401-404.
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  PMID: 461995;
• Malhotra, R. K., Ostrander, J. M., & Hurley, L. H. (1979). Pyrrolo(1,4)benzodiazepine antibiotics. Chemical conversion of anthramycin into didehydroanhydroanthramycin and its utilization in biosynthetic and mode of action studies. Journal of Natural Products, 42(6), 679-680.
• Ostrander, J. M., & Hurley, L. H. (1979). Pyrrolo(1,4)benzodiazepine antibiotics. Biosynthetic conversion of (D) and (L) tyrosine, (¹⁵N, 3- and 5-²H₂ 1-¹⁴C)tyrosine and L-(5-³H, indole-¹⁵N)tryptophan into anthramycin and sibiromycin. Journal of Natural Products, 42(6), 693-.
• Petrusek, R., Garner, T., & Hurley, L. H. (1979). Pyrrolo(1,4)benzodiazepine antibiotics. Molecular factors affecting the excision of the antitumor agent anthramycin from DNA in human cell lines. Journal of Natural Products, 42(6), 679-.
• Hannan, M. A., & Hurley, L. H. (1978). Pathways to DNA repair operating in yeast treated with the pyrrolo(1,4)-benzodiazepine antitumor antibiotics. Journal of Antibiotics, 31(9), 911-913.
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  PMID: 361666;
• Hannan, M. A., Hurley, L. H., & Gairola, C. (1978). Mutagenic and recombinogenic effects of the antitumor antibiotic anthramycin. Cancer Research, 38(9), 2795-2799.
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  PMID: 354779;Abstract: Anthramycin, one of the pyrrolo(1,4)benzodiazepine antibiotics with potent antitumor activity, was tested for its effects on a number of genetic parameters. The results show that this antibiotic is nonmutagenic in the Ames strains of Salmonella typhimurium while mutagenic in only one and antimutagenic in the rest of the genes tested in the eukaryotic organism Saccharomyces cerevisiae. The antibiotic is, however, a potent recombinogen in as much as it reduced mitotic crossing over, mitotic gene conversion, and possibly other chromosomal alterations in a diploid strain of S. cerevisiae. These studies emphasize the need for a battery of test systems including eukaryotic organisms to detect the genetic activity of certain antitumor drugs. The importance of considering data distinguishing between highly mutagenic and poorly mutagenic cancer chemotherapeutic agents is also discussed.
• Hurley, L. H. (1977). Pyrrolo(1,4)benzodiazepine antitumor antibiotics. Comparative aspects of anthramycin, tomaymycin and sibiromycin. Journal of Antibiotics, 30(5), 349-370.
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  PMID: 328469;Abstract: The pyrrolo(1,4)benzodiazepine antibiotics comprise the antibiotics, anthramycin, tomaymycin, sibiromycin, dextrochrysin and the neothramycins A and B. Structure activity relationships indicate that in addition to a pyrrolo(1,4)benzodiazepine nucleus, these compounds also require a 10,11 carbinolamine and an unsaturated side chain at C-2 to retain their potent biological activities. The antibiotics within this group show antiviral and most significantly antitumor activity. Anthramycin and sibiromycin have been shown to have a wide range of antitumor activity in animal models, and in the case of anthramycin, activity against both solid and liquid tumors in man. Tryptophan, methionine and tyrosine supply the biosynthetic building blocks for anthramycin, tomaymycin and the aglycone moiety of sibiromycin. Parallel pathways from tryptophan, via kynurenine lead to the anthranilate moieties of these three antibiotics. Conversion of tyrosin, via dopa, to the C2 and C3-proline moieties of anthramycin, tomaymycin and sibiromycin involves meta cleavage of the aromatic ring of a tyrosine metabolite and loss of two aromatic carbon atoms. Addition of the pyrrolo(1,4)benzodiazepine antibiotics to bacterial or mammalian cells leads to a potent inhibition of RNA and DNA synthesis, while protein synthesis is virtually unaffected at these same antibiotic concentrations. Overwhelming evidence suggests that the pyrrolo(1,4)benzodiazepine antibiotics form a covalent linkage with DNA. No major modification of the pyrrolo(1,4)benzodiazepine antibiotics occurs upon reaction with DNA, and intact antibiotic can be released from their respective DNA adducts under certain acidic conditions. Structure activity relationships lead to the conclusion that the DNA reactive site on the pyrrolo(1,4)benzodiazepine antibiotics is the 10,11-carbinolamine. Most likely the covalent linkage with DNA involves a nucleophilic attack originating from a basic group on DNA at C-11 of the antibiotics resulting in loss of the conjugate acid.
• Hurley, L. H., Gairola, C., & Zmijewski, M. (1977). Pyrrolo(1,4)benzodiazepine antitumor antibiotics In vitro interaction of anthramycin, sibiromycin and tomaymycin with DNA using specifically radiolabelled molecules. BBA Section Nucleic Acids And Protein Synthesis, 475(3), 521-535.
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  PMID: 15599;Abstract: Anthramycin, tomaymycin and sibiromycin are pyrrolo(1,4)benzodiazepine antitumor antibiotics. These compounds react with DNA and other guanine-containing polydeoxynucleotides to form covalently bound antibiotic · polydeoxynucleotide complexes. Experiments utilizing radiolabelled antibiotics have led to the following conclusions: 1. 1. Sibiromycin reacts much faster than either anthramycin or tomaymycin with DNA. 2. 2. At saturation binding the final antibiotic to base ratios for sibiromycin, anthramycin and tomaymycin are 1 : 8.8, 1 : 12.9, and 1 : 18.2 respectively. 3. 3. No reaction with RNA or protein occurs with the pyrrolo(1,4)benzodiazepine antibiotics. 4. 4. Sibiromycin effectively competes for the same DNA binding sites as anthramycin and tomaymycin; however, there is only partial overlap for the same binding sites between anthramycin and tomaymycin. 5. 5. Whereas all three pyrrolo(1,4)benzodiazepine antibiotic · DNA complexes are relatively stable to alkaline conditions, their stability under acidic conditions increases in the order tomaymycin, anthramycin and sibiromycin. 6. 6. No loss of non-exchangeable hydrogens in either the pyrrol ring or the side chains of these antibiotics occurs upon formation of their complexes with DNA. 7. 7. Unchanged antibiotic has been demonstrated to be released upon acid treatment of the anthramycin · DNA and tomaymycin · DNA complexes. 8. 8. A Schiffbase linkage between the antibiotics and DNA has been eliminated. The comparative reactivity of the three antibiotics towards DNA and the stability of their DNA complexes is discussed in relation to their structures. A working hypothesis for the formation of the antibiotic · DNA covalent complexes is proposed based upon the available information. © 1977.
• Chang, C., Floss, H. G., Hurley, L. H., & Zmijewski, M. (1976). Application of long-range spin-spin couplings in biosynthetic studies. Journal of Organic Chemistry, 41(17), 2932-2934.
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  PMID: 956946;Abstract: The long-range 13C-1H couplings of anthramycin and pyrrolnitrin are utilized to locate the 13C-enriched carbon atoms of the biosynthetically labeled antibiotics which were isolated from feeding experiments with L-[Me-13C]methionine and DL-[alanine-3-13C]tryptophan.
• Gairola, C., & Hurley, L. (1976). The mechanism for the methionine mediated reduction of anthramycin yields in Streptomyces refuineus fermentations. European Journal of Applied Microbiology, 2(2), 95-101.
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  Abstract: The addition of L-methionine to cultures of Streptomyces refuineus producing anthramycin, results in an apparent decrease in the antibiotic titer. It has been shown that this effect is not due to the inhibition of tyrosinase, an enzyme implicated in anthramycin biosynthesis. The inhibitory effect also appears unlikely to be due to a regulatory role played by methionine in a non-precursor fashion. Experiments with radiolabeled anthramycin have revealed that the reduced yields of the antibiotic are due to the interaction of anthramycin with generated reactive metabolites produced in the methionine-supplemented cultures. © 1976 Springer-Verlag.
• Hurley, L. H. (1976). Pyrrolo[1,4]benzodiazepine antibiotics. Biosynthesis of the antitumor antibiotic 11-demethyltomaymycin and its biologically inactive metabolite oxotomaymycin by streptomyces achromogenes. Biochemistry, 15(17), 3760-3769.
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  PMID: 1085163;Abstract: 11-Demethyltomaymycin, an antitumor antibiotic produced by Streptomyces achromogenes, and its biologically inactive metabolite oxotomaymycin are biosynthesized from L-tyrosine, DL-tryptophan, and L-methionine. The anthranilate part of 11-demethyltomaymycin is derived from tryptophan probably via the kynurenine pathway. The predominant loss of tritium from DL-[5-3H]tryptophan, during its conversion to 11-demethyltomaymycin and oxotomaymycin is interpreted to mean by NIH shift rules, that the main pathway to the 5-methoxy-4-hydroxy anthranilate moiety is through hydroxylation at C-8 prior to hydroxylation at C-7. The methoxy carbon is derived from the S-methyl group of methionine by transfer of an intact methyl group. The ethylideneproline moiety of 11-demethyltomaymycin is biosynthesized from tyrosine, without a 1-carbon unit from methionine. The results of biosynthetic feeding experiments with L-[1-14C, 3- or 5-3H] tyrosine are consistent with a "meta" or extradiol cleavage of 6, 7-dihydroxycyclodopa as has also been demonstrated previously for anthramycin and lincomycin A. An experiment in which L-[1-14C, Ala-2,3-3H]tyrosine was fed showed that both of the β hydrogens of this amino acid are retained in 11-demethyltomaymycin. It has been demonstrated in cultures and washed cell preparations that 11-demethyltomaymycin is enzymatically converted to oxotomaymycin by an intracellular constitutive enzyme. Conversion of oxotomaymycin to 11-demethyltomaymycin by these same preparations could not be demonstrated. The enzymatic activity associated with the conversion of 11 -demethyltomaymycin to oxotomaymycin is not limited to the 11-demethyltomaymycin production phase, since trophophase cells and even cells from 11-demethyltomaymycin nonproducing cultures of S. achromogenes were equally active in converting 11-demethyltomaymycin to oxotomaymycin.
• Hurley, L., Das, N., Gairola, C., & Zmijewski, M. (1976). Biosynthetic incorporation of DL-tryptophan-(5-³H) into anthramycin, sibiromycin and tomaymycin : N.I.H. shift produced by actinomycetes. Tetrahedron Letters, 17(18), 1419-1422.
• Hurley, L. H., Gairola, C., & Zmijewski Jr., M. J. (1975). Biosynthesis of the antiviral antibiotic 11-demethyltomaymycin by Streptomyces achromogenes. Journal of the Chemical Society, Chemical Communications, 120-121.
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  Abstract: The building blocks for 11-demethyltomay-mycin have been established as trytophan, tyrosine and a one carbon unit via methionine.
• Hurley, L. H., Zmijewski, M., & Chang, C. (1975). Biosynthesis of anthramycin. Determination of the labeling pattern by the use of radioactive and stable isotope techniques. Journal of the American Chemical Society, 97(15), 4372-4378.
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  PMID: 1141599;Abstract: The building blocks for anthramycin, an antitumor antibiotic produced by a strain of Streptomyces refuineus, have been shown to be L-tryptophan, probably via 3-hydroxyanthranilic acid, L-tyrosine which loses two of its aromatic carbons, and L-methionine which contributes two methyl groups. While one of the two methyl groups is transferred intact, the other loses all of its hydrogens and becomes the carbonyl of an amide group. A mechanism involving extradiol cleavage of Dopa is proposed on the basis of double labeling and stable isotope experiments. A general scheme for the biosynthetic origin of the C3-proline moieties of anthramycin, lincomycin A, and sibiromycin and the C2-proline moieties of tomaymycin and lincomycin B is proposed.
• Hurley, L. H., & Bialek, D. (1974). Regulation of antibiotic production: catabolite inhibition and the dualistic effect of glucose on indolmycin production. Journal of Antibiotics, 27(1), 49-56.
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  PMID: 4210488;
• Hurley, L. H., & Zmijewski, M. (1974). Biosynthesis of the antitumor antibiotic anthramycin by Streptomyces refuineus. Journal of the Chemical Society, Chemical Communications, 337-338.
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  Abstract: The biogenetic building blocks for anthramycin have been established as tryptophan, tyrosine, and two one-carbon units via methionine.
• Hornemann, U., Hurley, L. H., Speedie, M. K., & Floss, H. G. (1971). The biosynthesis of indolmycin. Journal of the American Chemical Society, 93(12), 3028-3035.
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  PMID: 5095271;Abstract: Indolmycin, an antibiotic produced by a strain of Streptomyces griseus, is formed from (S)-tryptophan, which loses from its side chain the amino nitrogen atom, the hydrogen atom from C-2, and one of the hydrogen atoms from C-3, two intact methyl groups of (S)-methionine, and the guanido carbon atom of (S)-arginine. (R)-β-Methylindolepyruvate and (2S,3R)-indolmycenic acid are intermediates in the biosynthesis. The absolute configuration of indolmycin has been determined by chemical correlation with (-)-(R)-indoleisopropionic acid. Studies with cell-free extracts of S. griseus revealed the presence of a transaminase which converts (S)-tryptophan into indolepyruvate and a methyltransferase which C-methylates indolepyruvate.
• Hornemann, U., Hurley, L. H., Speedie, M. K., & Floss, H. G. (1970). Isolation and absolute configuration of indolmycenic acid, an intermediate in the biosynthesis of indolmycin by streptomyces griseus.. Tetrahedron Letters, 11(26), 2255-2258.
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  PMID: 5447649;
• Hornemann, U., Speedie, M. K., Hurley, L. H., & Floss, H. G. (1970). Demonstration of a C-methylating enzyme in cell free extracts of indolmycin-producing Streptomycesgriseus. Biochemical and Biophysical Research Communications, 39(4), 594-599.
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  PMID: 5490210;Abstract: A cell free extract of an indolmycin-producing strain of Streptomycesgriseus is shown to catalyze the transfer of a methyl group from S-adenosylmethionine-(methyl-14C) to indolepyruvate to form radioactive β-methylindolepyruvate, an intermediate in indolmycin biosynthesis. The reaction product β-methylindolepyruvate has been unequivocally identified by chemical correlations and it has been shown to have the S configuration. © 1970.
• Hornemann, U., Hurley, L. H., Speedie, M. K., Guenther, H. F., & Floss, H. G. (1969). Biosynthesis of the antibiotic indolmycin by Streptomyces griseus. C-Methylation at the β-carbon atom of the tryptophan side-chain. Journal of the Chemical Society D: Chemical Communications, 245-246.
• Hornemann, U., Speedie, M. K., Kelley, K. M., Hurley, L. H., & Floss, H. G. (1969). Biosynthesis of indoleisopropionic acid by Claviceps. Biological C-methylation involving an intact methyl group. Archives of Biochemistry and Biophysics, 131(2), 430-440.
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  PMID: 5787216;Abstract: (R)-Indoleisopropionic acid (I) [(2R)-(3-indolyl)-propionic acid], a metabolite of a Claviceps strain, is formed from l-tryptophan and the intact methyl group of l-methionine. Indoleacetic acid is not incorporated into indoleisopropionic acid. (2R,S), (3S,R)-3-methyltryptophan (β-methyltryptophan, isomer B) was efficiently incorporated, but no evidence for its formation by the organism could be obtained. A hypothetical scheme for the biosynthesis of indoleisopropionic acid is presented. © 1969.

Presentations

• Hurley, L. (2018, August). How Promoter G- and C-Quadruplexes Work Together to Control Gene Expression in Cells and Can Be Targeted by Small Molecules. Lisbon, Portugal.
• Hurley, L. (2018, Fall). The Interplay of Supercoiling and C- and G-Quadruplexes in Promoters and the Control of Gene Expression. BIONIC 2018: Biology of Non-Canonical Nucleic Acids International Symposium, Padua, Italy, September 26-28, 2018.
• Hurley, L. (2018, Spring). How the G- and C-Quadruplexes in the MYC Promoter Control Gene Expression and Can Be Targeted by Small Molecules to Turn ON and OFF Gene Expression. NCI Frontiers in Targeting MYC: Expression, Regulation, and Degradation Symposium, April 9–18, 2018. Bethesda, MD.