Vijay Gokhale
- Associate Research Professor
- Member of the Graduate Faculty
Contact
- (520) 626-4224
- TW Keating Bioresearch Bldg., Rm. 437
- Tucson, AZ 85721
- gokhale@arizona.edu
Bio
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Interests
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Courses
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Scholarly Contributions
Chapters
- Gokhale, V., & Maggiora, G. (2016). Non-Specificity of Drug-Target Interactions – Consequences for Drug Discovery. In ACS Symposium Series 1222: Frontiers in Molecular Design and Chemical Information Science(pp 91-142). American Chemical Society. doi:10.1021/bk-2016-1222.ch007
Journals/Publications
- Gokhale, V. (2022). Quindoline-derivatives display potent G-quadruplex-mediated antiviral activity against herpes simplex virus 1.. Antiviral Research, 208, 105432.
- Cai, S., Moutal, A., Yu, J., Chew, L. A., Isensee, J., Chawla, R., Gomez, K., Luo, S., Zhou, Y., Chefdeville, A., Madura, C., Perez-Miller, S., Bellampalli, S. S., Dorame, A., Scott, D. D., François-Moutal, L., Shan, Z., Woodward, T., Gokhale, V., , Hohmann, A. G., et al. (2021). Selective targeting of NaV1.7 via inhibition of the CRMP2-Ubc9 interaction reduces pain in rodents. Science translational medicine, 13(619), eabh1314.More info[Figure: see text].
- François-Moutal, L., Miranda, V. G., Mollasalehi, N., Gokhale, V., & Khanna, M. (2021). Targeting of the Long Noncoding RNA MALAT1. ACS medicinal chemistry letters, 12(6), 915-921.More infoRNA targeting has gained traction over the past decade. It has become clear that dysregulation of RNA can be linked to many diseases, leading to a need for new scaffolds recognizing RNA specifically. Long noncoding RNAs are emerging as key controllers of gene expression and potential therapeutic targets. However, traditional targeting methods have overwhelmingly been focused on proteins. In this study, we used a protein computational tool and found several possible targetable pockets in a structurally characterized long noncoding RNA, MALAT1. Screening against those identified pockets revealed several hit compounds. We tested the binding of those compounds to MALAT1 RNA and tRNA as a negative control, using SPR. While several compounds were nonspecific binders, others were able to recognize MALAT1 specifically. One of them, MTC07, has an apparent affinity of 400.2 ± 14.4 μM. Although it has weak affinity, MTC07 is the first compound targeting MALAT1 originating from docking.
- Gokhale, V. (2021). Selective targeting of NaV1.7 via inhibition of the CRMP2-Ubc9 interaction reduces pain in rodents.. Science Translational Medicine. doi:10.1126/scitranslmed.abh1314More infoClinical and preclinical data identified the voltage-gated sodium channel 1.7 (NaV1.7) as one of the most promising targets for treating acute and chronic pain. However, the lack of selectivity of current NaV1.7 channel blockers hindered the development of effective treatments. Here, Cai et al. showed that preventing SUMOylation of the NaV1.7-interacting protein CRMP2 using a small molecule (termed 194) reduced NaV1.7 surface expression and current density. 194 showed high selectivity for NaV1.7 and reduced acute and chronic pain in rodent models, after oral administration, without triggering adverse events. The results suggest that targeting posttranslational mechanisms might be an effective approach to selectively block NaV1.7 and obtain antinociceptive effects.
- Miranti, C. K., Moore, S., Kim, Y., Chappeta, V. R., Wu, K., De, B., Gokhale, V., Hurley, L. H., & Reyes-Reyes, E. M. (2020). Erratum: Nucleolin represses transcription of the androgen receptor gene through a G-quadruplex. Oncotarget, 11(26), 2586.More info[This corrects the article DOI: 10.18632/oncotarget.27589.].
- Miranti, C. K., Moore, S., Kim, Y., Chappeta, V. R., Wu, K., De, B., Gokhale, V., Hurley, L. H., & Reyes-Reyes, E. M. (2020). Nucleolin represses transcription of the androgen receptor gene through a G-quadruplex. Oncotarget, 11(19), 1758-1776.More infoThe androgen receptor (AR) is a major driver of prostate cancer development and progression. Men who develop advanced prostate cancer often have long-term cancer control when treated with androgen-deprivation therapies (ADT). Still, their disease inevitably becomes resistant to ADT and progresses to castration-resistant prostate cancer (CRPC). ADT involves potent competitive AR antagonists and androgen synthesis inhibitors. Resistance to these types of treatments emerges, primarily through the maintenance of AR signaling by ligand-independent activation mechanisms. There is a need to find better ways to block AR to overcome CRPC. In the findings reported here, we demonstrate that the nuclear scaffold protein, nucleolin (NCL), suppresses the expression of AR. NCL binds to a G-rich region in the AR promoter that forms a G-quadruplex (G4) structure. Binding of NCL to this G4-element is required for NCL to suppress AR expression, specifically in AR-expressing tumor cells. Compounds that stabilize G4 structures require NCL to associate with the G4-element of the promoter in order to decrease AR expression. A newly discovered G4 compound that suppresses AR expression demonstrates selective killing of AR-expressing tumor cells, including CRPC lines. Our findings raise the significant possibility that G4-stabilizing drugs can be used to increase NCL transcriptional repressor activity to block AR expression in prostate cancer. Our studies contribute to a clearer understanding of the mechanisms that control AR expression, which could be exploited to overcome CRPC.
- Mollasalehi, N., Francois-Moutal, L., Scott, D. D., Tello, J. A., Williams, H., Mahoney, B., Carlson, J. M., Dong, Y., Li, X., Miranda, V. G., Gokhale, V., Wang, W., Barmada, S. J., & Khanna, M. (2020). An Allosteric Modulator of RNA Binding Targeting the N-Terminal Domain of TDP-43 Yields Neuroprotective Properties. ACS chemical biology, 15(11), 2854-2859.More infoIn this study, we targeted the N-terminal domain (NTD) of transactive response (TAR) DNA binding protein (TDP-43), which is implicated in several neurodegenerative diseases. docking of 50K compounds to the NTD domain of TDP-43 identified a small molecule (nTRD22) that is bound to the N-terminal domain. Interestingly, nTRD22 caused allosteric modulation of the RNA binding domain (RRM) of TDP-43, resulting in decreased binding to RNA . Moreover, incubation of primary motor neurons with nTRD22 induced a reduction of TDP-43 protein levels, similar to TDP-43 RNA binding-deficient mutants and supporting a disruption of TDP-43 binding to RNA. Finally, nTRD22 mitigated motor impairment in a model of amyotrophic lateral sclerosis. Our findings provide an exciting way of allosteric modulation of the RNA-binding region of TDP-43 through the N-terminal domain.
- Wang, W., Gokhale, V., Dong, Y., Mollasalehi, N., Francois-Moutal, L., Scott, D. D., Tello, J. A., Williams, H., Mahoney, B., Carlson, J. M., Li, X., Miranda, V. G., Barmada, S. J., & Khanna, M. (2020). An Allosteric Modulator of RNA Binding Targeting the N-Terminal Domain of TDP-43 Yields Neuroprotective Properties. ACS Chemical Biology, 15(11), 2854-2859. doi:10.1021/acschembio.0c00494
- François-Moutal, L., Felemban, R., Scott, D. D., Sayegh, M. R., Miranda, V. G., Perez-Miller, S., Khanna, R., Gokhale, V., Zarnescu, D. C., & Khanna, M. (2019). Small Molecule Targeting TDP-43's RNA Recognition Motifs Reduces Locomotor Defects in a Model of Amyotrophic Lateral Sclerosis (ALS). ACS chemical biology, 14(9), 2006-2013.More infoRNA dysregulation likely contributes to disease pathogenesis of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. A pathological form of the transactive response (TAR) DNA binding protein (TDP-43) binds to RNA in stress granules and forms membraneless, amyloid-like TDP-43 aggregates in the cytoplasm of ALS motor neurons. In this study, we hypothesized that by targeting the RNA recognition motif (RRM) domains of TDP-43 that confer a pathogenic interaction between TDP-43 and RNA, motor neuron toxicity could be reduced. docking of 50000 compounds to the RRM domains of TDP-43 identified a small molecule (rTRD01) that (i) bound to TDP-43's RRM1 and RRM2 domains, (ii) partially disrupted TDP-43's interaction with the hexanucleotide RNA repeat of the disease-linked gene, but not with (UG) canonical binding sequence of TDP-43, and (iii) improved larval turning, an assay measuring neuromuscular coordination and strength, in an ALS fly model based on the overexpression of mutant TDP-43. Our findings provide an instructive example of a chemical biology approach pivoted to discover small molecules targeting RNA-protein interactions in neurodegenerative diseases.
- Gokhale, V., Taylor, B., Hurley, L. H., Gokhale, V., Ferdosi, S. R., Dhruv, H., Bollam, S., & Berens, M. E. (2019). CBMT-23. NON-CANONICAL FUNCTIONS OF TERT IN GLIOBLASTOMA. Neuro-oncology, 21(Supplement_6), vi38-vi38. doi:10.1093/neuonc/noz175.145More infoAbstract Telomerase is an enzyme with a catalytic subunit, telomerase reverse transcriptase (TERT), that is in charge of telomere elongation in the nucleus. Promotor region of TERT is commonly mutated across cancers, especially in glioblastoma (GBM) with over 80% frequency. In the absence of any effective molecular targeting therapy for GBM, elucidating oncogenic signaling of TERT could open new avenues in GBM treatment. Canonically, mutations of TERT, which result in TERT upregulation, maintain telomere length in the nucleus and promote indefinite proliferation of cancer cells. However, a non-canonical function of TERT in the mitochondria has recently been suggested. We screened GBM cell models against a novel small molecule inhibitor (RG1534, Reglagene Inc.) that interferes with the functionality of a mutated hTERT promoter. RG1534 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes. More interestingly, RG1534 treatment leads to rapid apoptosis induction in glioma cell lines that does not correlate with the time course of the telomere shortening effect. We further validated this rapid apoptosis behavior in glioma cell lines using siRNA and CRISPR/Cas-9 mediated hTERT knockdown. We also measured the protein expression of TERT in subcellular fractions of glioma cell lines and demonstrated the presence of higher TERT expression in mitochondrial extract compared to the nucleus. Finally, using MitoSOX dye we assessed ROS generation in glioma cells in response to an oxidant with or without TERT expresssion. In summary, our results demonstrate that non-canonical functions of TERT may play critical role in glioma pathobiology and require more detail investigation.
- Gokhale, V., Taylor, B., Peng, S., Hurley, L. H., Gokhale, V., Ferdosi, S. R., Dhruv, H., Bollam, S., & Berens, M. E. (2019). Abstract 5226: Non-canonical functions of TERT in glioblastoma pathobiology. Cancer Research, 79, 5226-5226. doi:10.1158/1538-7445.sabcs18-5226More infoMutations in the promotor region of TERT are the most common non-coding mutations across cancers, especially in glioblastoma (GBM), which has a TERT mutation frequency rate over 80%. In the absence of any effective molecular targeting therapy for GBM, elucidating oncogenic signaling of TERT could open new avenues in GBM treatment. Canonically, mutations of TERT, which result in TERT upregulation, maintain telomere length in the nucleus and promote indefinite proliferation of cancer cells. However, a non-canonical function of TERT in the mitochondria has recently been suggested. We screened GBM cell models against a novel small molecule inhibitor (RG1534, Reglagene Inc.) that interferes with the functionality of a mutated hTERT promoter. RG1534 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes. More interestingly, RG1534 treatment leads to rapid apoptosis induction in glioma cell lines that does not correlate with the time course of the telomere shortening effect. We further validated this rapid apoptosis behavior in glioma cell lines using siRNA and CRISPR/Cas-9 mediated hTERT knockdown. We also measured the protein expression of TERT in subcellular fractions of glioma cell lines and demonstrated the presence of higher TERT expression in mitochondrial extract compared to the nuclear extract. Finally, using MitoSOX dye we demonstrated significant increase of ROS generation in glioma cells treated with RG1534 as compared to control. In summary, our results demonstrate that non-canonical functions of TERT may play critical role in glioma pathobiology and require more detail investigation. Citation Format: Shayesteh R. Ferdosi, Saumya Bollam, Sen Peng, Brett Taylor, Vijay Gokhale, Laurence Hurley, Michael Berens, Harshil Dhruv. Non-canonical functions of TERT in glioblastoma pathobiology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5226.
- Khanna, R., Yu, J., Yang, X., Moutal, A., Chefdeville, A., Gokhale, V., Shuja, Z., Chew, L. A., Bellampalli, S. S., Luo, S., François-Moutal, L., Serafini, M. J., Ha, T., Perez-Miller, S., Park, K. D., Patwardhan, A. M., Streicher, J. M., Colecraft, H. M., & Khanna, M. (2019). Targeting the CaVα-CaVβ interaction yields an antagonist of the N-type CaV2.2 channel with broad antinociceptive efficacy. Pain, 160(7), 1644-1661.More infoInhibition of voltage-gated calcium (CaV) channels is a potential therapy for many neurological diseases including chronic pain. Neuronal CaV1/CaV2 channels are composed of α, β, γ and α2δ subunits. The β subunits of CaV channels are cytoplasmic proteins that increase the surface expression of the pore-forming α subunit of CaV. We targeted the high-affinity protein-protein interface of CaVβ's pocket within the CaVα subunit. Structure-based virtual screening of 50,000 small molecule library docked to the β subunit led to the identification of 2-(3,5-dimethylisoxazol-4-yl)-N-((4-((3-phenylpropyl)amino)quinazolin-2-yl)methyl)acetamide (IPPQ). This small molecule bound to CaVβ and inhibited its coupling with N-type voltage-gated calcium (CaV2.2) channels, leading to a reduction in CaV2.2 currents in rat dorsal root ganglion sensory neurons, decreased presynaptic localization of CaV2.2 in vivo, decreased frequency of spontaneous excitatory postsynaptic potentials and miniature excitatory postsynaptic potentials, and inhibited release of the nociceptive neurotransmitter calcitonin gene-related peptide from spinal cord. IPPQ did not target opioid receptors nor did it engage inhibitory G protein-coupled receptor signaling. IPPQ was antinociceptive in naive animals and reversed allodynia and hyperalgesia in models of acute (postsurgical) and neuropathic (spinal nerve ligation, chemotherapy- and gp120-induced peripheral neuropathy, and genome-edited neuropathy) pain. IPPQ did not cause akinesia or motor impairment, a common adverse effect of CaV2.2 targeting drugs, when injected into the brain. IPPQ, a quinazoline analog, represents a novel class of CaV2.2-targeting compounds that may serve as probes to interrogate CaVα-CaVβ function and ultimately be developed as a nonopioid therapeutic for chronic pain.
- Montoya, J. J., Turnidge, M. A., Wai, D. H., Patel, A. R., Lee, D. W., Gokhale, V., Hurley, L. H., Arceci, R. J., Wetmore, C., & Azorsa, D. O. (2019). In vitro activity of a G-quadruplex-stabilizing small molecule that synergizes with Navitoclax to induce cytotoxicity in acute myeloid leukemia cells. BMC cancer, 19(1), 1251.More infoAcute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that arise from genomic alterations in the expression of key growth regulatory genes causing cells to assume an undifferentiated state and continue to proliferate. Recent efforts have focused on developing therapies that target specific protein products of aberrantly expressed genes. However, many of the identified proteins are difficult to target and thought to be "undrugable" because of structural challenges, protein overexpression, or mutations that confer resistance to therapy. A novel technology that circumvents some of these issues is the use of small molecules that stabilize secondary DNA structures present in the promoters of many potential oncogenes and modulate their transcription.
- Song, J. H., Kang, H. J., Luevano, L. A., Gokhale, V., Wu, K., Pandey, R., Sherry Chow, H. H., Hurley, L. H., & Kraft, A. S. (2019). Small-Molecule-Targeting Hairpin Loop of hTERT Promoter G-Quadruplex Induces Cancer Cell Death. Cell chemical biology, 26(8), 1110-1121.e4.More infoIncreased telomerase activity is associated with malignancy and poor prognosis in human cancer, but the development of targeted agents has not yet provided clinical benefit. Here we report that, instead of targeting the telomerase enzyme directly, small molecules that bind to the G-hairpin of the hTERT G-quadruplex-forming sequence kill selectively malignant cells without altering the function of normal cells. RG260 targets the hTERT G-quadruplex stem-loop folding but not tetrad DNAs, leading to downregulation of hTERT expression. To improve physicochemical and pharmacokinetic properties, we derived a small-molecule analog, RG1603, from the parent compound. RG1603 induces mitochondrial defects including PGC1α and NRF2 inhibition and increases oxidative stress, followed by DNA damage and apoptosis. RG1603 injected as a single agent has tolerable toxicity while achieving strong anticancer efficacy in a tumor xenograft mouse model. These results demonstrate a unique approach to inhibiting the hTERT that functions by impairing mitochondrial activity, inducing cell death.
- Wai, D. H., Turnidge, M. A., Patel, A., Montoya, J. J., Lee, D. W., Hurley, L. J., Gokhale, V., & Azorsa, D. O. (2019). Abstract 2967: Activity of G-quadruplex stabilizing small molecule that downregulates MYC and synergizes with Navitoclax in acute myeloid leukemia cells. Cancer Research, 79, 2967-2967. doi:10.1158/1538-7445.sabcs18-2967More infoAcute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that can occur when genomic changes alter expression of key genes, causing cells to resume an undifferentiated state and proliferate. Ongoing efforts have focused on developing therapies that specifically target the protein products of aberrantly expressed genes. However, many of the identified proteins are difficult to target because of structural challenges, protein overexpression, or mutations that confer resistance to therapy. A type of therapy that circumvents these issues is the use of small molecules that stabilize DNA secondary structures called G-quadruplexes, which are present in the promoters of many potential oncogenes, and have regulatory roles in their transcription. This study analyzes the activity of G-quadruplex stabilizing small molecule GQC-05 that has been shown to down-regulate MYC, which is commonly misregulated in AML. Treatment of MYC-expressing AML cell lines KG-1a, CMK and TF-1 with GQC-05 resulted in decreased expression of MYC mRNA and protein, with the effect more pronounced in KG-1a cells. GQC-05 treatment of the AML cells decreased cell viability, while increasing apoptosis and DNA damage. Combinational drug screening was performed to identify compounds that potentiated the anti-proliferative effects of GQC-05. Results from the screen identified the BCL-2 inhibitor Navitoclax as a compound that potentiated GQC-05 activity. Co-treatment with GQC-05 and Navitoclax showed synergistic effect on cell viability of AML cells as determined by Chou-Talalay analysis. Furthermore, co-treatment with Navitoclax did not affect the downregulation of MYC by GQC-05, but did increase apoptosis and DNA damage leading to rapid cytotoxicity. These results indicate that the G-quadruplex stabilizing small molecule GQC-05 may function more as a DNA damaging agent in AML cells and that combining GQC-05 with a Bcl-2/Bcl-XLinhibitor such as Navitoclax can result in increased cytotoxic activity. Citation Format: Justin J. Montoya, Megan A. Turnidge, Daniel H. Wai, David W. Lee, Apruvi Patel, Vijay Gokhale, Laurence J. Hurley, David O. Azorsa. Activity of G-quadruplex stabilizing small molecule that downregulates MYC and synergizes with Navitoclax in acute myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2967.
- François-Moutal, L., Dustrude, E. T., Wang, Y., Brustovetsky, T., Dorame, A., Ju, W., Moutal, A., Perez-Miller, S., Brustovetsky, N., Gokhale, V., Khanna, M., & Khanna, R. (2018). Inhibition of the Ubc9 E2 SUMO-conjugating enzyme-CRMP2 interaction decreases NaV1.7 currents and reverses experimental neuropathic pain. Pain, 159(10), 2115-2127.More infoWe previously reported that destruction of the small ubiquitin-like modifier (SUMO) modification site in the axonal collapsin response mediator protein 2 (CRMP2) was sufficient to selectively decrease trafficking of the voltage-gated sodium channel NaV1.7 and reverse neuropathic pain. Here, we further interrogate the biophysical nature of the interaction between CRMP2 and the SUMOylation machinery, and test the hypothesis that a rationally designed CRMP2 SUMOylation motif (CSM) peptide can interrupt E2 SUMO-conjugating enzyme Ubc9-dependent modification of CRMP2 leading to a similar suppression of NaV1.7 currents. Microscale thermophoresis and amplified luminescent proximity homogeneous alpha assay revealed a low micromolar binding affinity between CRMP2 and Ubc9. A heptamer peptide harboring CRMP2's SUMO motif, also bound with similar affinity to Ubc9, disrupted the CRMP2-Ubc9 interaction in a concentration-dependent manner. Importantly, incubation of a tat-conjugated cell-penetrating peptide (t-CSM) decreased sodium currents, predominantly NaV1.7, in a model neuronal cell line. Dialysis of t-CSM peptide reduced CRMP2 SUMOylation and blocked surface trafficking of NaV1.7 in rat sensory neurons. Fluorescence dye-based imaging in rat sensory neurons demonstrated inhibition of sodium influx in the presence of t-CSM peptide; by contrast, calcium influx was unaffected. Finally, t-CSM effectively reversed persistent mechanical and thermal hypersensitivity induced by a spinal nerve injury, a model of neuropathic pain. Structural modeling has now identified a pocket-harboring CRMP2's SUMOylation motif that, when targeted through computational screening of ligands/molecules, is expected to identify small molecules that will biochemically and functionally target CRMP2's SUMOylation to reduce NaV1.7 currents and reverse neuropathic pain.
- François-Moutal, L., Jahanbakhsh, S., Nelson, A. D., Ray, D., Scott, D. D., Hennefarth, M. R., Moutal, A., Perez-Miller, S., Ambrose, A. J., Al-Shamari, A., Coursodon, P., Meechoovet, B., Reiman, R., Lyons, E., Beilstein, M., Chapman, E., Morris, Q. D., Van Keuren-Jensen, K., Hughes, T. R., , Khanna, R., et al. (2018). A Chemical Biology Approach to Model Pontocerebellar Hypoplasia Type 1B (PCH1B). ACS chemical biology, 13(10), 3000-3010.More infoMutations of EXOSC3 have been linked to the rare neurological disorder known as Pontocerebellar Hypoplasia type 1B (PCH1B). EXOSC3 is one of three putative RNA-binding structural cap proteins that guide RNA into the RNA exosome, the cellular machinery that degrades RNA. Using RNAcompete, we identified a G-rich RNA motif binding to EXOSC3. Surface plasmon resonance (SPR) and microscale thermophoresis (MST) indicated an affinity in the low micromolar range of EXOSC3 for long and short G-rich RNA sequences. Although several PCH1B-causing mutations in EXOSC3 did not engage a specific RNA motif as shown by RNAcompete, they exhibited lower binding affinity to G-rich RNA as demonstrated by MST. To test the hypothesis that modification of the RNA-protein interface in EXOSC3 mutants may be phenocopied by small molecules, we performed an in-silico screen of 50 000 small molecules and used enzyme-linked immunosorbant assays (ELISAs) and MST to assess the ability of the molecules to inhibit RNA-binding by EXOSC3. We identified a small molecule, EXOSC3-RNA disrupting (ERD) compound 3 (ERD03), which ( i) bound specifically to EXOSC3 in saturation transfer difference nuclear magnetic resonance (STD-NMR), ( ii) disrupted the EXOSC3-RNA interaction in a concentration-dependent manner, and ( iii) produced a PCH1B-like phenotype with a 50% reduction in the cerebellum and an abnormally curved spine in zebrafish embryos. This compound also induced modification of zebrafish RNA expression levels similar to that observed with a morpholino against EXOSC3. To our knowledge, this is the first example of a small molecule obtained by rational design that models the abnormal developmental effects of a neurodegenerative disease in a whole organism.
- François-Moutal, L., Scott, D. D., Perez-Miller, S., Gokhale, V., Khanna, M., & Khanna, R. (2018). Chemical shift perturbation mapping of the Ubc9-CRMP2 interface identifies a pocket in CRMP2 amenable for allosteric modulation of Nav1.7 channels. Channels (Austin, Tex.), 12(1), 219-227.More infoDrug discovery campaigns directly targeting the voltage-gated sodium channel NaV1.7, a highly prized target in chronic pain, have not yet been clinically successful. In a differentiated approach, we demonstrated allosteric control of trafficking and activity of NaV1.7 by prevention of SUMOylation of collapsin response mediator protein 2 (CRMP2). Spinal administration of a SUMOylation incompetent CRMP2 (CRMP2 K374A) significantly attenuated pain behavior in the spared nerve injury (SNI) model of neuropathic pain, underscoring the importance of SUMOylation of CRMP2 as a pathologic event in chronic pain. Using a rational design strategy, we identified a heptamer peptide harboring CRMP2's SUMO motif that disrupted the CRMP2-Ubc9 interaction, inhibited CRMP2 SUMOylation, inhibited NaV1.7 membrane trafficking, and specifically inhibited NaV1.7 sodium influx in sensory neurons. Importantly, this peptide reversed nerve injury-induced thermal and mechanical hypersensitivity in the SNI model, supporting the practicality of discovering pain drugs by indirectly targeting NaV1.7 via prevention of CRMP2 SUMOylation. Here, our goal was to map the unique interface between CRMP2 and Ubc9, the E2 SUMO conjugating enzyme. Using computational and biophysical approaches, we demonstrate the enzyme/substrate nature of Ubc9/CRMP2 binding and identify hot spots on CRMP2 that may form the basis of future drug discovery campaigns disrupting the CRMP2-Ubc9 interaction to recapitulate allosteric regulation of NaV1.7 for pain relief.
- Gokhale, V., Peng, S., Kang, H., Hurley, L. H., Gokhale, V., Dhruv, H., Bollam, S. R., & Berens, M. E. (2018). Abstract 4798: Targeting hTERT for treatment of glioblastoma (GBM). Cancer Research, 78, 4798-4798. doi:10.1158/1538-7445.am2018-4798More infoApproximately 86% of GBM tumors exhibit a mutation at -124 or -146 bp upstream from the ATG start site in the transcription activating promoter region of Human telomerase reverse transcriptase (hTERT). Mutations in the hTERT promoter are known to impair repression, leading to overexpression of hTERT and tumor maintenance. Yet, complete understanding of mechanistic implications is still necessary. While overexpressed hTERT is associated with oncogenesis and resistance to apoptosis, we also observe phenotypes unrelated to the reverse transcription function. We characterized long-term glioma cell lines and glioma PDX models by hTERT promoter mutation status, hTERT mRNA expression, and hTERT protein expression in subcellular fractions. The -124 and -146 mutations are located in the major 5-12 G-quadruplex and result in misfolding of the silencer element, thus causing over-expression of hTERT. Using a diverse small molecule library, we identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, TG-4260, which binds to the 26 mer base-pair heteroduplex loop, which is the nucleation site for cooperative folding of the major 5-12 G-quadruplex. The chaperone effect of TG-4260 corrects DNA hTERT G-quadruplex misfolding resulting from the promoter mutations and restores the silencer function of the G-quadruplex. TG-4260 directly decreases the transcription activity of the −124, −124/125, −138/139, and −146 mutants to a similar extent and suppresses the downstream gene BCL2, which activates caspase-3 and produces cell-cycle arrest, leading to cell death. This is the first example of the use of a pharmacoperone molecule to correct the misfolding of a DNA G-quadruplex element resulting from mutations in an early folding intermediate. We screened GBM cell models against this novel small molecule inhibitor that interferes with mutated hTERT promoter and demonstrated that TG-4260 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes. Finally, telomere phenotypes from treated cells indicate non-canonical functions of telomerase may also contribute to glioma pathogenesis. Citation Format: Saumya Reddy Bollam, Hyun-Jin Kang, Sen Peng, Vijay Gokhale, Laurence Hurley, Michael Berens, Harshil Dhruv. Targeting hTERT for treatment of glioblastoma (GBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4798.
- Lee, D. W., Gokhale, V., Wai, D. H., Turnidge, M. A., Patel, A., Montoya, J. J., Lee, D. W., Hurley, L. H., Gokhale, V., Azorsa, D. O., & Arceci, R. J. (2018). Abstract B04: Direct downregulation of MYC in AML cells using promoter G-quadruplex interacting small molecules. Cancer Research, 78. doi:10.1158/1538-7445.pedca17-b04More infoAcute myeloid leukemia (AML) is a disease that can affect both children and adults and is characterized by complex and heterogeneous genomic changes, including MYC overexpression driven in part by the bromodomain (BRD) and extraterminal (BET) protein family of epigenetic readers. Current limitations in treatment approaches, including protein overexpression and mutations that confer resistance to therapy, lead to a wide range of response variability to conventional therapy, excessive treatment-related toxicity, and an overall poor outcome. A new therapeutic strategy involves stabilizing the MYC promoter G-quadruplex to downregulate expression. We hypothesize that direct downregulation of MYC in AML cells using MYC-promoter G-quadruplex interacting drugs (GQID) is a feasible novel approach in targeted therapy of AML. Treatment of AML cell lines KG-1a and UT-7epo with the MYC-promoter GQID, GQC-05 resulted in decreased MYC expression and induced a more significant decrease in c-Myc protein than treatment with the BET inhibitor (+)-JQ1. GQC-05 also decreased cell viability and increased apoptosis. RNA-seq of GQC-05-treated KG-1a cells identified 947 significantly differentially expressed genes compared to DMSO. The expression of several c-MYC target genes (including E2F1, GNL3, HSPA8, MAT2A, RCC1, SHMT1, and TFRC) was significantly decreased concomitant with MYC downregulation. The 947 genes were compared with genes with putative quadruplex sequences (PQS) from the literature. Of the 17 genes in common, MYC, NOTCH1, PIM1, and RHOU are significantly downregulated following GQC-05 treatment. These findings were validated by Q-RT-PCR in KG-1a and several other AML cell lines. Drug sensitivity and resistance (DSR) screening on a panel of AML cell lines using a library of 55 anticancer and targeted agents identified a range of sensitivities to GQC-05 (AUC range: 1.65-2.99; DMSO AUC ~ 4). These were compared to microarray gene-expression profiles and, as expected, GQC-05 sensitivity was not correlated with MYC expression. Nevertheless, ANOVA identified 1,049 significantly differentially expressed probe sets between the most sensitive vs. the least sensitive cell lines. Functional annotation revealed over-representation of cadherin, Wnt, and p53 signaling pathways as well over genes involved in hemostasis and hematopoietic cell lineage. In conclusion, directly targeting the MYC promoter G-quadruplex in AML cells leads to knockdown of MYC expression and induces apoptosis. These results further support the development of GQID for targeting key genetic drivers in AML, and lay the groundwork for advances in treatment of other cancers driven by G-quadruplex regulated oncogenes. Citation Format: Megan A. Turnidge, Daniel H. Wai, Apurvi Patel, Justin J. Montoya, David W. Lee, Laurence H. Hurley, Vijay Gokhale, Robert J. Arceci, David O. Azorsa. Direct downregulation of MYC in AML cells using promoter G-quadruplex interacting small molecules [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B04.
- Moutal, A., Li, W., Wang, Y., Ju, W., Luo, S., Cai, S., François-Moutal, L., Perez-Miller, S., Hu, J., Dustrude, E. T., Vanderah, T. W., Gokhale, V., Khanna, M., & Khanna, R. (2018). Homology-guided mutational analysis reveals the functional requirements for antinociceptive specificity of collapsin response mediator protein 2-derived peptides. British journal of pharmacology, 175(12), 2244-2260.More infoN-type voltage-gated calcium (Ca 2.2) channels are critical determinants of increased neuronal excitability and neurotransmission accompanying persistent neuropathic pain. Although Ca 2.2 channel antagonists are recommended as first-line treatment for neuropathic pain, calcium-current blocking gabapentinoids inadequately alleviate chronic pain symptoms and often exhibit numerous side effects. Collapsin response mediator protein 2 (CRMP2) targets Ca 2.2 channels to the sensory neuron membrane and allosterically modulates their function. A 15-amino-acid peptide (CBD3), derived from CRMP2, disrupts the functional protein-protein interaction between CRMP2 and Ca 2.2 channels to inhibit calcium influx, transmitter release and acute, inflammatory and neuropathic pain. Here, we have mapped the minimal domain of CBD3 necessary for its antinociceptive potential.
- Song, J. H., Gokhale, V., Hurley, L. H., Kraft, A. S., Song, J. H., Luevano, L. A., Kraft, A. S., Kang, H., Hurley, L. H., & Gokhale, V. (2018). Abstract 4841: Prostate cancer cell death triggered by a small molecule interacting with the hTERT G-quadruplex. Cancer Research, 78, 4841-4841. doi:10.1158/1538-7445.am2018-4841More infoHuman telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase, is widely overexpressed in human cancers including prostate cancer. Along with its regulation of the telomere, hTERT was also shown to regulate various signal transduction mechanisms involving DNA damage response, cell cycle checkpoint, and apoptosis. The majority of human solid tumors display telomerase activity, which was found to be well correlated with hTERT expression, and hTERT inhibition led to reduced telomerase activity and telomere length. However, approaches that directly inhibit the action of telomerase have not produced a therapeutically useful response because of long lag times for subsequent cell division to produce telomere shortening. Alternatively, recent studies demonstrated that this delay problem can be solved through downregulation of hTERT transcription. It was shown that telomeric DNA is capable of folding into four-stranded guanine quadruplex (G4) structures, which then lock the hTERT promoter activity that leads to robust telomere shortening. Our new approach has focused on the discovery of small molecules that could stabilize G-quadruplexes by interacting with the telomeric G-rich overhang stem loop, producing a lead candidate compound that enhances the kinetic folding rate of the G4 silencer element. Our in vitro studies demonstrate that this singular mechanism of action permitted reduced telomerase production and telomere shortening. Significantly, hTERT downregulation with the identified molecule caused prostate cancer cell death, which was characterized by activation of the caspase cascade and increased Annexin V- positive cells. In contrast, this compound did not harm normal prostate epithelial cells. Similarly, mouse prostate cancer cells that do not contain a G-quadruplex were not killed, demonstrating that the unique mechanism of action is mediated through the hTERT promoter G-quadruplex element. Our in vivo study demonstrated that administration of this significantly delayed tumor growth on xenografted mice. The rapid onset of cell death by G4 stands in contrast to the delayed action of classic telomerase inhibition. Therefore, our current study provides important data to support developing inhibitors of hTERT as a cancer drug target. Citation Format: Jin H. Song, Libia A. Luevano, Hyunjin Kang, Vijay Gokhale, Laurence H. Hurley, Andrew S. Kraft. Prostate cancer cell death triggered by a small molecule interacting with the hTERT G-quadruplex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4841.
- 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.More infoSequences 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.
- Branca, C., Shaw, D. M., Belfiore, R., Gokhale, V., Shaw, A. Y., Foley, C., Smith, B., Hulme, C., Dunckley, T., Meechoovet, B., Caccamo, A., & Oddo, S. (2017). Dyrk1 inhibition improves Alzheimer's disease-like pathology. Aging cell, 16(5), 1146-1154.More infoThere is an urgent need for the development of new therapeutic strategies for Alzheimer's disease (AD). The dual-specificity tyrosine phosphorylation-regulated kinase-1A (Dyrk1a) is a protein kinase that phosphorylates the amyloid precursor protein (APP) and tau and thus represents a link between two key proteins involved in AD pathogenesis. Furthermore, Dyrk1a is upregulated in postmortem human brains, and high levels of Dyrk1a are associated with mental retardation. Here, we sought to determine the effects of Dyrk1 inhibition on AD-like pathology developed by 3xTg-AD mice, a widely used animal model of AD. We dosed 10-month-old 3xTg-AD and nontransgenic (NonTg) mice with a Dyrk1 inhibitor (Dyrk1-inh) or vehicle for eight weeks. During the last three weeks of treatment, we tested the mice in a battery of behavioral tests. The brains were then analyzed for the pathological markers of AD. We found that chronic Dyrk1 inhibition reversed cognitive deficits in 3xTg-AD mice. These effects were associated with a reduction in amyloid-β (Aβ) and tau pathology. Mechanistically, Dyrk1 inhibition reduced APP and insoluble tau phosphorylation. The reduction in APP phosphorylation increased its turnover and decreased Aβ levels. These results suggest that targeting Dyrk1 could represent a new viable therapeutic approach for AD.
- 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.More infoThe 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.
- Gokhale, V., Peng, S., Kang, H., Hurley, L. H., Gokhale, V., Dhruv, H. D., Bollam, S. R., & Berens, M. E. (2017). Abstract 1169: mtTERT promoter as a target for treatment of glioblastoma. Cancer Research, 77, 1169-1169. doi:10.1158/1538-7445.am2017-1169
- Gokhale, V., Vanderah, T. W., Moutal, A., Li, W., Wang, Y., Ju, W., Luo, S., Cai, S., François-Moutal, L., Perez-Miller, S., Hu, J., Dustrude, E. T., Khanna, M., & Khanna, R. (2017). Homology-guided mutational analysis reveals the functional requirements for antinociceptive specificity of collapsin response mediator protein 2-derived peptides: Mapping of CRMP2-derived antinociceptive peptides. British Journal of Pharmacology, 175(12), 2244-2260. doi:10.1111/bph.13737
- Hurley, L. H., Gokhale, V., Kendrick, S., Muranyi, A., & 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. doi:10.1021/acs.jmedchem.7b00298
- 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.More infoSecondary 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.
- Lee, D. W., Lee, D. W., Gokhale, V., Gokhale, V., Wai, D. H., Wai, D. H., Turnidge, M. A., Turnidge, M. A., Patel, A. R., Patel, A. R., Montoya, J. J., Montoya, J. J., Lee, D. W., Lee, D. W., Hurley, L. H., Hurley, L. H., Gokhale, V., Gokhale, V., Azorsa, D. O., , Azorsa, D. O., et al. (2017). Abstract 5181: Targeted knockdown of MYC in AML cells using G-quadruplex interacting small molecules. Cancer Research, 77, 5181-5181. doi:10.1158/1538-7445.am2017-5181More infoAcute Myeloid Leukemia (AML) is a disease that occurs when genomic changes alter expression of key genes, causing cells to resume an undifferentiated state, proliferate, and maintain tumor growth throughout the body. Recently, there has been an increase in efforts toward developing therapies that specifically target the protein products of these aberrantly expressed genes. However, many of the proteins are difficult to target because of structural challenges, protein overexpression that requires high drug doses, or mutations that confer resistance to therapy. One potential type of targeted therapy that circumvents these issues is the use of small molecules that stabilize DNA secondary structures called G-quadruplexes. G-quadruplexes are present in the promoters of many potential oncogenes, and have regulatory roles in their transcription. This study analyzes the therapeutic potential of compounds that target the regulatory G-quadruplex in the MYC oncogene, which is commonly misregulated in AML. Treatment of the MYC expressing AML cell lines KG-1 and UT-7epo with the compound GQC-05 resulted in decreased expression of MYC mRNA, as determined by qPCR. Moreover, GQC-05 treatment induced a more significant decrease in c-Myc protein expression than similar treatment with JQ-1(+), as shown by western blot analysis. Treatment of AML cells with GQC-05 also decreased cell viability and increased apoptosis. Concurrent treatment of AML cells with GQC-05 and JQ-1(+) showed an antagonistic effect, indicating potential competition in the silencing of MYC. These results demonstrate that targeting the MYC promoter G-quadruplex in AML cells leads to knockdown of MYC expression and induces apoptosis. These results further support the development of a novel mechanism for targeting key genetic drivers in AML, and lay the groundwork for advances in treatment of other cancers driven by G-quadruplex regulated oncogenes. Citation Format: Megan A. Turnidge, Apurvi Patel, Justin J. Montoya, David W. Lee, Daniel H. Wai, Vijay Gokhale, Laurence Hurley, Robert J. Arceci, David O. Azorsa. Targeted knockdown of MYC in AML cells using G-quadruplex interacting small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5181. doi:10.1158/1538-7445.AM2017-5181
- Maggiora, G., & Gokhale, V. (2017). A simple mathematical approach to the analysis of polypharmacology and polyspecificity data. F1000Research, 6.More infoThere many possible types of drug-target interactions, because there are a surprising number of ways in which drugs and their targets can associate with one another. These relationships are expressed as polypharmacology and polyspecificity. Polypharmacology is the capability of a given drug to exhibit activity with respect to multiple drug targets, which are not necessarily in the same activity class. Adverse drug reactions ('side effects') are its principal manifestation, but polypharmacology is also playing a role in the repositioning of existing drugs for new therapeutic indications. Polyspecificity, on the other hand, is the capability of a given target to exhibit activity with respect to multiple, structurally dissimilar drugs. That these concepts are closely related to one another is, surprisingly, not well known. It will be shown in this work that they are, in fact, mathematically related to one another and are in essence 'two sides of the same coin'. Hence, information on polypharmacology provides equivalent information on polyspecificity, and . Networks are playing an increasingly important role in biological research. Drug-target networks, in particular, are made up of drug nodes that are linked to specific target nodes if a given drug is active with respect to that target. Such networks provide a graphic depiction of polypharmacology and polyspecificity. However, by their very nature they can obscure information that may be useful in their interpretation and analysis. This work will show how such latent information can be used to determine bounds for the degrees of polypharmacology and polyspecificity, and how to estimate other useful features associated with the lack of completeness of most drug-target datasets.
- Amin, A. D., Li, L., Rajan, S. S., Gokhale, V., Groysman, M. J., Pongtornpipat, P., Tapia, E. O., Wang, M., & Schatz, J. H. (2016). TKI sensitivity patterns of novel kinase-domain mutations suggest therapeutic opportunities for patients with resistant ALK+ tumors. Oncotarget, 7(17), 23715-29.More infoThe anaplastic lymphoma kinase (ALK) protein drives tumorigenesis in subsets of several tumors through chromosomal rearrangements that express and activate its C-terminal kinase domain. In addition, germline predisposition alleles and acquired mutations are found in the full-length protein in the pediatric tumor neuroblastoma. ALK-specific tyrosine kinase inhibitors (TKIs) have become important new drugs for ALK-driven lung cancer, but acquired resistance via multiple mechanisms including kinase-domain mutations eventually develops, limiting median progression-free survival to less than a year. Here we assess the impact of several kinase-domain mutations that arose during TKI resistance selections of ALK+ anaplastic large-cell lymphoma (ALCL) cell lines. These include novel variants with respect to ALK-fusion cancers, R1192P and T1151M, and with respect to ALCL, F1174L and I1171S. We assess the effects of these mutations on the activity of six clinical inhibitors in independent systems engineered to depend on either the ALCL fusion kinase NPM-ALK or the lung-cancer fusion kinase EML4-ALK. Our results inform treatment strategies with a likelihood of bypassing mutations when detected in resistant patient samples and highlight differences between the effects of particular mutations on the two ALK fusions.
- Gokhale, V., Dhruv, H., Peng, S., Kang, H., Hurley, L. H., Gokhale, V., Dhruv, H. D., & Berens, M. E. (2016). Exth-59. mtTert Promoter As ATarget For Treatment Of Glioblastoma. Neuro-oncology, 18.
- Gokhale, V., Peng, S., Kang, H., Hurley, L. H., Gokhale, V., Dhruv, H., Bollam, S., & Berens, M. E. (2016). OS01.3 mtTERT promoter as a target for treatment of Glioblastoma.. Neuro-oncology, 19(suppl_3), iii1-iii1. doi:10.1093/neuonc/nox036.002More infoAbstractApproximately 86% of GBM tumors exhibit mutation at -124 or -146 bases upstream of the ATG start site in the transcription activating promoter region of Human telomerase reverse transcriptase (hTERT). Mutation in the promoter region of hTERT impairs repression, leading to overexpression of hTERT; inappropriate hTERT is associated with oncogenesis, tumor maintenance, and resistance to apoptosis. We surveyed long-term glioma cell lines and glioma PDX models for mt-hTERT; mRNA and protein expression of hTERT were assessed by qPCR and western blot. The -124 and -146 mutations are located in the major 5–12 G-quadruplex and result in misfolding of the silencer element and consequent over-expression of hTERT. Using a diverse small molecule library, we identified a small drug-like pharmacological chaperone (pharmacoperone) molecule, TG-4260, which binds to the 26 mer base-pair heteroduplex loop, which is the nucleation site for cooperative folding of the major 5–12 G-quadruplex. The chaperone effect of TG-4260 corrects DNA hTERT G-quadruplex misfolding resulting from the somatic mutations and restores the silencer function of the G-quadruplex thereby reducing hTERT activity. TG-4260 directly decreases the transcription activity of the WT and the −124, −124/125, −138/139, and −146 mutants to a similar extent and suppresses the downstream gene BCL2, which activates caspase-3 and produces cell-cycle arrest, leading to cell death. Finally, TG-4260 significantly inhibits telomerase and shortens telomere length after five days of treatment and induces a senescence-like phenotype. This is the first example of the use of a pharmacoperone molecule to correct the misfolding of a DNA G-quadruplex element resulting from mutations in an early folding intermediate. Finally, we screened GBM cell models against a novel small molecule inhibitor that interferes with mutated hTERT promoter and demonstrated that TG-4260 selectively suppresses glioma cell viability without affecting non-transformed normal human astrocytes.
- 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, 138(41), 13673-13692.More infoActivation 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. L., Gokhale, V., Rimsza, L. M., Rimsza, L. M., Kendrick, S. L., Hurley, L. H., & Gokhale, V. (2016). Concurrent Targeting of BCL2 and MYC Transcription Leads to Chemo-Sensitization of Dual-Expressing Diffuse Large B-Cell Lymphoma In Vivo. Blood, 128(22), 4090-4090. doi:10.1182/blood.v128.22.4090.4090More infoTo date, there are no effective strategies to treat patients with a particularly lethal form of diffuse large B-cell lymphoma (DLBCL) that over-express both B-cell lymphoma gene-2 ( BCL2 ) and cellular-myelocytomatosis ( MYC ) oncogenes. Here, we provide a novel approach for simultaneously targeting BCL2 and MYC directly through secondary DNA structures. These structures act as molecular switches, turning gene expression on or off, and serve as scaffolds for protein and small molecule interaction. Previously identified compounds IMC-76 and GQC-05, recognize the BCL2 or MYC promoter region DNA structures, respectively, and independently lower mRNA and protein expression of each oncogene.For utility in sensitizing aggressive lymphoid tumors that concurrently dual express BCL2 and MYC, we examined the efficacy ofIMC-76 and GQC-05 in the presence and absence of a standard chemotherapy, cyclophosphamide (CPA)to inhibit tumor growth in a severe combinedimmunodeficient U2932 DLBCLxenograft mouse model. Unlike IMC-76, effects of GQC-05 in a xenograftmouse model are unknown; therefore, our initial in vivo studies involved GQC-05 alone and with IMC-76 in combination with CPA to determine the concentration for MYC down-regulation and a maximum tolerated dose (MTD). At 2.5 and 5 mg/kg GQC-05 with 50 mg/kg CPA, we observed no effect on mean mouse weight and while the 5 mg/kg GQC-05-CPA combination group exhibited some mouse death, LD50 was not reached. Tumors from mice treated with GQC-05 and CPA were compared to diluent or CPA alone treated mice for MYC mRNA level and found to have significantly less MYC expression. As expected, there was no effect on BCL2 mRNA levels. Next, we carried over the 5 mg/kg GQC-05 to a subsequent in vivo experiment where we also tested a 10 mg/kg GQC-05 dose and included IMC-76 at 10 mg/kg, a previously established efficacious dose, to determine if we would reach an MTD, but still retain the repression of MYC . While the mice were able to tolerate the 5 mg/kg GQC-05 dose when IMC-76 and/or CPA was added to the treatment regimen, the LD50 was reached and surpassed in mice treated with the 10 mg/kg of GQC-05 combinations. However, both concentrations of GQC-05 when co-treated with IMC-76 and CPA resulted in a notable decrease of tumor MYC expression relative to untreated and CPA only treated mice as well as a detectable inhibition of BCL2 . We then conducted a follow-up study to evaluate effects on tumor burden using concentrations of GQC-05 below the LD50, 5 mg/kg and 7.5 mg/kg, and also reduced the dose of CPA to 30 mg/kg. The co-treatment of mice with IMC-76 (10 mg/kg) and GQC-05 (7.5 mg/kg) in combination with CPA delayed tumor growth and decreased tumor size by 50%, 46%, and 37% at 9, 12, and 15 days post-first-day drug administration compared to the diluent and 50%, 67%, and 44% smaller tumors relative to CPA only treated mice ( Figure 1 ). Overall, there was a prominent decrease in tumor burden in these mice as determined by area under the curveand the tumors displayed a lower expression of BCL2 and MYC at both the mRNA and protein levels. Interestingly, tumors of mice that received IMC-76 and GQC-05 also showed knock-down of BCL2 and MYC , but no effect on tumor burden unless CPA was present indicating these transcriptional inhibitors act as chemo-sensitizers. None of the various drug regimens resulted in a significant loss of mouse weight and while survival for GQC-05 7.5 mg/kg treated mice was reduced, LD50 was not reached. These are the first studies to demonstrate two differentpromoterDNA secondary structures can be targeted at the same time for direct transcriptional inhibition that leads tochemo-sensitization and slowed tumor growth in mice.Our findings demonstrate the potential utility of a dual-targeted, precision medicine-based strategy to improve the response of DLBCL patients to current chemotherapy and overcome resistant disease. ![Figure][1] Figure Tumor burden of DLBCL xenograft mice Disclosures Gokhale: Tetragene: Equity Ownership. Hurley: Tetragene: Consultancy, Equity Ownership. Rimsza: NCI/NIH: Patents & Royalties: L.M. Rimsza is a co-inventor on a provisional patent, owned by the NCI of the NIH, using Nanostring technology for determining cell of origin in DLBCL.. [1]: pending:yes
- 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.
- Lee, D. W., Gokhale, V., Wai, D. H., Turnidge, M., Patel, A., Montoya, J. J., Lee, D. W., Hurley, L. H., Gokhale, V., Azorsa, D. O., Arceci, R. J., & Aleem, E. (2016). Abstract 3766: Targeting promoter regions of c-Myc and Bcl-2 in AML cells using G-quadruplex interacting drugs. Cancer Research, 76, 3766-3766. doi:10.1158/1538-7445.am2016-3766More infoCancer is a disease that can be characterized by overexpression of key oncogenic drivers that support tumor development and maintenance. In many instances, these oncogenic drivers are ‘undruggable’ because of structural challenges, the inability to effectively inhibit high concentrations of overexpressed proteins, and the development of drug resistance mutations. An alternative therapeutic approach is to directly inhibit gene transcription by targeting unique secondary DNA structures, called G-quadruplexes, that are associated with subsets of promoters. We investigated the activity of a class of compounds termed G-quadruplex Interacting Drugs (GQIDs) that are able to shut down the expression of specific oncogenic drivers, such as c-Myc and Bcl-2, used by tumor cells for growth and survival. We tested the activity of two GQIDs, GQC-05 and GSA-1103, on cell growth of a panel of eight pediatric and eight adult acute myeloid leukemia (AML) cell lines. Drug dose response analysis showed IC50 values ranging from approximately 10 nM to 1 μM for GQC-05 and from 40 nM to 2 μM for GSA-1103. The AML cell lines had different sensitivities to each GQID indicating a different mechanism of action for each compound. Three AML cell lines that were highly resistant to cytarabine were among the more sensitive to GQC-05. Four cell lines sensitive to GQC-05 had high expression of either c-Myc and/or Bcl-2, both of which are potential targets of these two GQIDs. Furthermore, treatment of the highly sensitive line MV-4-11 with GQC-05 showed a 3-4 fold decrease in expression of c-Myc and Bcl-2 mRNA. Furthermore, GQC-05 treatment resulted in decreased levels of both c-Myc and Bcl-2 proteins. These studies will help define a novel approach of repressing key drivers of leukemia by targeting selective promoter structures. Citation Format: Megan Turnidge, Justin J. Montoya, Apurvi Patel, David W. Lee, Eiman Aleem, Daniel H. Wai, Laurence H. Hurley, Vijay Gokhale, Robert J. Arceci, David O. Azorsa. Targeting promoter regions of c-Myc and Bcl-2 in AML cells using G-quadruplex interacting drugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3766.
- Wai, D. H., Turnidge, M. A., Sabir, M. S., Patel, A., Montoya, J. J., Lee, D. W., Hurley, L. J., Gokhale, V., Azorsa, D. O., Arceci, R. J., & Aleem, E. (2016). Abstract B24: Targeting promoter regions of oncogenic drivers in pediatric AML cells using G-quadruplex Interacting Drugs (GQIDs). Cancer Research, 76. doi:10.1158/1538-7445.pedca15-b24More infoCancer is primarily a disease characterized by aberrant gene expression that is manifested by the overexpression of key genes that support tumor development and maintenance. In many instances, oncogenic drivers are frequently ‘undruggable’ because of structural challenges, the inability to effectively inhibit high concentrations of overexpressed proteins, and the development of drug resistance mutations. An alternative therapeutic approach is to directly inhibit gene transcription by targeting unique secondary DNA structures, called G-quadruplexes, that are associated with subsets of promoters. Our laboratory investigated the activity of a class of compounds termed G-quadruplex Interacting Drugs (GQIDs) that are able to shut down the expression of specific genes used by tumor cells for growth and survival. We tested the activity of two GQIDs GQC-05 and GSA-1103 on cell growth of a panel of eight pediatric and eight adult AML cell lines. Drug dose response analysis showed IC50 values ranging from approximately 10 nM to 1 µM for GQC-05 and from 40 nM to 2 µM for GSA-1103. The AML cell lines had different sensitivities to each GQID indicating a different mechanism of action for each compound. Three AML cell lines that were highly resistant to cytarabine were among the more sensitive to GQC-05. Four cell lines sensitive to GQC-05 had high expression of either c-myc and/or bcl-2, both of which are potential targets of these two GQIDs. Furthermore, treatment of cells with the GQC-05 decreased expression of c-myc and bcl-2. These studies will help define a novel approach of repressing key drivers of leukemia by targeting selective promoter structures. Validation of such an approach for AML will have important implications for testing this therapeutic approach to other childhood cancers in order to improve the length and quality of survival. Citation Format: Apurvi Patel, Justin J. Montoya, Megan Turnidge, Marya Sabir, Daniel H. Wai, David W. Lee, Vijay Gokhale, Eiman Aleem, Laurence J. Hurley, Robert J. Arceci, David O. Azorsa. Targeting promoter regions of oncogenic drivers in pediatric AML cells using G-quadruplex Interacting Drugs (GQIDs). [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B24.
- Davis, A. L., Qiao, S., Lesson, J. L., Rojo de la Vega, M., Park, S. L., Seanez, C. M., Gokhale, V., Cabello, C. M., & Wondrak, G. T. (2015). The quinone methide aurin is a heat shock response inducer that causes proteotoxic stress and Noxa-dependent apoptosis in malignant melanoma cells. The Journal of biological chemistry, 290(3), 1623-38.More infoPharmacological induction of proteotoxic stress is rapidly emerging as a promising strategy for cancer cell-directed chemotherapeutic intervention. Here, we describe the identification of a novel drug-like heat shock response inducer for the therapeutic induction of proteotoxic stress targeting malignant human melanoma cells. Screening a focused library of compounds containing redox-directed electrophilic pharmacophores employing the Stress & Toxicity PathwayFinder(TM) PCR Array technology as a discovery tool, a drug-like triphenylmethane-derivative (aurin; 4-[bis(p-hydroxyphenyl)methylene]-2,5-cyclohexadien-1-one) was identified as an experimental cell stress modulator that causes (i) heat shock factor transcriptional activation, (ii) up-regulation of heat shock response gene expression (HSPA6, HSPA1A, DNAJB4, HMOX1), (iii) early unfolded protein response signaling (phospho-PERK, phospho-eIF2α, CHOP (CCAAT/enhancer-binding protein homologous protein)), (iv) proteasome impairment with increased protein-ubiquitination, and (v) oxidative stress with glutathione depletion. Fluorescence polarization-based experiments revealed that aurin displays activity as a geldanamycin-competitive Hsp90α-antagonist, a finding further substantiated by molecular docking and ATPase inhibition analysis. Aurin exposure caused caspase-dependent cell death in a panel of human malignant melanoma cells (A375, G361, LOX-IMVI) but not in non-malignant human skin cells (Hs27 fibroblasts, HaCaT keratinocytes, primary melanocytes) undergoing the aurin-induced heat shock response without impairment of viability. Aurin-induced melanoma cell apoptosis depends on Noxa up-regulation as confirmed by siRNA rescue experiments demonstrating that siPMAIP1-based target down-regulation suppresses aurin-induced cell death. Taken together, our data suggest feasibility of apoptotic elimination of malignant melanoma cells using the quinone methide-derived heat shock response inducer aurin.
- Shen, K., Ramirez, B., Mapes, B., Shen, G. R., Gokhale, V., Brown, M. E., Santarsiero, B., Ishii, Y., Dudek, S. M., Wang, T., & Garcia, J. G. (2015). Structure-Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants. PloS one, 10(6), e0130515.More infoThe MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.
- Gokhale, V., Stratton, S. P., Stratton, S. P., Shaw, A. Y., Hulme, C., & Gokhale, V. (2014). Abstract 2524: Promising early-stage novel androgen receptor antagonists in head-head comparisons with Enzalutamide and Bicalutamide. Cancer Research, 74, 2524-2524. doi:10.1158/1538-7445.am2014-2524More infoProstate cancer is the most common male cancer and the second leading cause of cancer-related deaths in developed countries. The androgen receptor (AR) is a key molecular target in the etiology and progression of the disease where the early stages of prostate cancer tumor growth are androgen dependent and respond well to androgen ablation and androgen receptor antagonists. However, castration-resistant prostate cancer (CRPC) typically develops in 1-2 years after androgen deprivation therapy and AR antagonist administration. Despite promising results from the recently approved second-generation AR antagonist Enzalutamide (MDV3100), CRPC remains a largely unmet medical need with less than a 15% 5-year survival rate. This poster introduces the discovery of a novel series of significantly structurally distinct small-molecule androgen antagonists from current therapeutics with promising biological profiles in (1) an FP binding assay, (2) a GeneBlazer TM functional assay and (3) a study of anti-proliferative activity against LNCaP cell growth. One of several promising compounds from a current collection of ∼100 analogs is exemplified by AR002. In summary, a novel class of AR antagonists has been identified, encouragingly demonstrating potent activity against AR and inhibition of cell growth in the prostate specific line LNCaP. Citation Format: Arthur Y. Shaw, Vijay Gokhale, Christopher Hulme, Steven P. Stratton. Promising early-stage novel androgen receptor antagonists in head-head comparisons with Enzalutamide and Bicalutamide. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2524. doi:10.1158/1538-7445.AM2014-2524
- 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.More infoIt 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.
- Gokhale, V., Smith, B., Shaw, A. Y., Medda, F., Hulme, C., Gokhale, V., & Dunckley, T. (2013). Beyond secretases: Kinase inhibitors for the treatment of Alzheimer's disease. Annual Reports in Medicinal Chemistry, 48(48), 57-71. doi:10.1016/b978-0-12-417150-3.00005-3More infoAbstract Alzheimer’s disease (AD) is the most prevalent form of dementia in old age. Recent data indicate that 24.3 million people worldwide suffer from AD. Hyperphosphorylation of tau, a protein normally involved in microtubule stabilization, has been identified as an important pathological contributor to AD development. In AD brains, hyperphosphorylation of tau leads to its aggregation, misfolding, and formation of neurofibrillary tangles, one common hallmark of AD. Specific protein kinases, such as GSK-3β, CDK5, and DYRK1A, are involved in tau hyperphosphorylation and have been identified as potential targets for the development of novel therapeutic agents for the treatment of AD cognitive deficits. We herein review the current state of the art in the development of small molecule inhibitors of GSK-3β, CDK5, DYRK1A, and other protein kinases involved in tau phosphorylation. Only recently developed compounds with cellular and/or in vivo activity will be discussed.
- 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, 1-21.More infoG-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.
- Medda, F., Sells, E., Chang, H. H., Dietrich, J., Chappeta, S., Smith, B., Gokhale, V., Meuillet, E. J., & Hulme, C. (2013). Synthesis and biological activity of aminophthalazines and aminopyridazines as novel inhibitors of PGE2 production in cells. Bioorganic & medicinal chemistry letters, 23(2), 528-31.More infoThis Letter reports the synthesis and biological evaluation of a collection of aminophthalazines as a novel class of compounds capable of reducing production of PGE(2) in HCA-7 human adenocarcinoma cells. A total of 28 analogs were synthesized, assayed for PGE(2) reduction, and selected active compounds were evaluated for inhibitory activity against COX-2 in a cell free assay. Compound 2xxiv (R(1)=H, R(2)=p-CH(3)O) exhibited the most potent activity in cells (EC(50)=0.02 μM) and minimal inhibition of COX-2 activity (3% at 5 μM). Furthermore, the anti-tumor activity of analog 2vii was analyzed in xenograft mouse models exhibiting good anti-cancer activity.
- Boddupally, P. V., Hahn, S., Beman, C., De, B., 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), 6076-86.More infoThis 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.
- Chang, H. H., Song, Z., Wisner, L., Tripp, T., Gokhale, V., & Meuillet, E. J. (2012). Identification of a novel class of anti-inflammatory compounds with anti-tumor activity in colorectal and lung cancers. Investigational new drugs, 30(5), 1865-77.More infoChronic inflammation is associated with 25% of all cancers. In the inflammation-cancer axis, prostaglandin E(2) (PGE(2)) is one of the major players. PGE(2) synthases (PGES) are the enzymes downstream of the cyclooxygenases (COXs) in the PGE(2) biosynthesis pathway. Microsomal prostaglandin E(2) synthase 1 (mPGES-1) is inducible by pro-inflammatory stimuli and constitutively expressed in a variety of cancers. The potential role for this enzyme in tumorigenesis has been reported and mPGES-1 represents a novel therapeutic target for cancers. In order to identify novel small molecule inhibitors of mPGES-1, we screened the ChemBridge library and identified 13 compounds as potential hits. These compounds were tested for their ability to bind directly to the enzyme using surface plasmon resonance spectroscopy and to decrease cytokine-stimulated PGE(2) production in various cancer cell lines. We demonstrate that the compound PGE0001 (ChemBridge ID number 5654455) binds to human mPGES-1 recombinant protein with good affinity (K(D) = 21.3 ± 7.8 μM). PGE0001 reduces IL-1β-induced PGE(2) release in human HCA-7 colon and A549 lung cancer cell lines with EC(50) in the sub-micromolar range. Although PGE0001 may have alternative targets based on the results from in vitro assays, it shows promising effects in vivo. PGE0001 exhibits significant anti-tumor activity in SW837 rectum and A549 lung cancer xenografts in SCID mice. Single injection i.p. of PGE0001 at 100 mg/kg decreases serum PGE(2) levels in mice within 5 h. In summary, our data suggest that the identified compound PGE0001 exerts anti-tumor activity via the inhibition of the PGE(2) synthesis pathway.
- Gokhale, V., Meuillet, E. J., Sells, E., Meuillet, E. J., Medda, F., Hulme, C., Gokhale, V., & Chang, H. (2012). Abstract 2834: Discovery of a novel class of prostaglandin e2 synthesis inhibitors with anti-tumor activity in colorectal cancer. Cancer Research, 72, 2834-2834. doi:10.1158/1538-7445.am2012-2834More infoProstaglandin E 2 (PGE 2 ), a key mediator of inflammation, is the most common prostanoid with various bioactivities and has been associated with many pathologies. Thus, inhibition of PGE 2 synthesis and its action has been suggested in the treatment of inflammatory-associated diseases, including cancer. Microsomal Prostaglandin E 2 Synthase-1 (mPGES-1) is an enzyme downstream of the cyclooxygenases (COXs) in the PGE 2 biosynthesis pathway. mPGES-1, which is one of the three PTGES isoforms, is constitutively expressed in many cancers, including colon cancer. The potential role for this enzyme in tumorigenesis has been reported. Since inhibition of COXs lead to side effects due to the global reduction of other key prostaglandins, targeting downstream m-PGES-1 might be a better therapeutic strategy for cancers. In order to identify novel small molecule inhibitors of mPGES-1, we have screened several libraries in silico and used published scaffolds exhibiting anti-inflammatory properties to initiate our study. As a result, 10 compounds were identified by molecular docking simulations using the reported crystal structure of mPGES-1. Subsequently a total of 32 analog compounds were synthesized or purchased and tested for their ability to inhibit COX-2 in vitro activity as well as for their ability to inhibit PGE 2 production in an HCA-7 colorectal cancer cell line. We show that 16 of the 32 compounds reduced PGE 2 synthesis by over 80% at 1 µM, with only one of these compounds inhibiting more than 30% of COX-2 activity at 5 µM in vitro. Furthermore, one compound, PGE0056 (EC 50 = 0.37 ± 0.06 µM), exhibited anti-tumor activity after 10 days at 100mg/kg i.p. in HCA-7 colon cancer xenograft mouse model. Following treatment with PGE0056, PGE 2 production was reduced in the blood of scid mice. In summary, our data suggest that these new identified compounds exert anti-tumor activity at least in part through inhibition of the COX2/mPGES-1 enzymes within the PGE 2 synthesis pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2834. doi:1538-7445.AM2012-2834
- Hurley, L. H., Gokhale, V., Gaerig, V., Yu, Z., Cui, Y., Kang, H., Zhao, Y., & 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. doi:10.1021/ja210399h
- Jagadish, B., Guntle, G. P., Zhao, D., Gokhale, V., Ozumerzifon, T. J., Ahad, A. M., Mash, E. A., & Raghunand, N. (2012). Redox-active magnetic resonance imaging contrast agents: studies with thiol-bearing 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid derivatives. Journal of medicinal chemistry, 55(23), 10378-86.More infoThe synthesis and structure-activity relationships of a homologous series of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid gadolinium(III) complexes bearing thiol-terminated alkyl side chains from three to nine carbons in length are reported. The observed binding with human serum albumin (HSA) of the compounds having C-3 through C-7 side chain lengths was inhibited by homocysteine in a manner consistent with single-site binding. The observed binding with HSA of the compounds having C-8 and C-9 side chain lengths was only partly inhibited by homocysteine, consistent with multisite binding. The binding affinity of the C-7 compound could be related to the HSA oxidation state. 2D 1H-1H NMR TOCSY provided evidence of covalent binding of the europium analog of the C-6 compound to HSA-Cys34. The longitudinal water-proton MRI relaxivities of the gadolinium complexes at 7 T increased upon binding to HSA. On the basis of these results, the C-6 and C-7 compounds were identified as promising redox-sensitive MRI contrast agents.
- Mash, E. A., Gokhale, V., Jagadish, B., Guntle, G. P., Zhao, D., Ozumerzifon, T. J., Ahad, A. M., & Raghunand, N. (2012). Redox-active Magnetic Resonance Imaging Contrast Agents: Studies with Thiol-bearing 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetracetic Acid Derivatives. Journal of Medicinal Chemistry, 55(23), 10378-10386. doi:10.1021/jm300736f
- Smith, B., Chang, H. H., Medda, F., Gokhale, V., Dietrich, J., Davis, A., Meuillet, E. J., & Hulme, C. (2012). Synthesis and biological activity of 2-aminothiazoles as novel inhibitors of PGE2 production in cells. Bioorganic & medicinal chemistry letters, 22(10), 3567-70.More infoThis Letter presents the synthesis and biological evaluation of a collection of 2-aminothiazoles as a novel class of compounds with the capability to reduce the production of PGE(2) in HCA-7 human adenocarcinoma cells. A total of 36 analogs were synthesized and assayed for PGE(2) reduction, and those with potent cellular activity were counter screened for inhibitory activity against COX-2 in a cell free assay. In general, analogs bearing a 4-phenoxyphenyl substituent in the R(2) position were highly active in cells while maintaining negligible COX-2 inhibition. Specifically, compound 5l (R(1)=Me, R(2)=4-OPh-Ph, R(3)=CH(OH)Me) exhibited the most potent cellular PGE(2) reducing activity of the entire series (EC(50)=90 nM) with an IC(50) value for COX-2 inhibition of >5 μM in vitro. Furthermore, the anti-tumor activity of analog 1a was analyzed in xenograft mouse models exhibiting promising anti-cancer activity.
- Smith, B., Medda, F., Gokhale, V., Dunckley, T., & Hulme, C. (2012). Recent advances in the design, synthesis, and biological evaluation of selective DYRK1A inhibitors: a new avenue for a disease modifying treatment of Alzheimer's?. ACS chemical neuroscience, 3(11), 857-72.More infoWith 24.3 million people affected in 2005 and an estimated rise to 42.3 million in 2020, dementia is currently a leading unmet medical need and costly burden on public health. Seventy percent of these cases have been attributed to Alzheimer's disease (AD), a neurodegenerative pathology whose most evident symptom is a progressive decline in cognitive functions. Dual specificity tyrosine phosphorylation regulated kinase-1A (DYRK1A) is important in neuronal development and plays a variety of functional roles within the adult central nervous system. The DYRK1A gene is located within the Down syndrome critical region (DSCR) on human chromosome 21 and current research suggests that overexpression of DYRK1A may be a significant factor leading to cognitive deficits in people with Alzheimer's disease (AD) and Down syndrome (DS). Currently, treatment options for cognitive deficiencies associated with Down syndrome, as well as Alzheimer's disease, are extremely limited and represent a major unmet therapeutic need. Small molecule inhibition of DYRK1A activity in the brain may provide an avenue for pharmaceutical intervention of mental impairment associated with AD and other neurodegenerative diseases. We herein review the current state of the art in the development of DYRK1A inhibitors.
- Song, Z., Smith, R. M., Moses, S. A., Meuillet, E. J., Gokhale, V., & Beyer, T. E. (2012). Abstract 2835: Discovery of novel inhibitors for ECT2 as a novel therapeutic strategy for lung cancer. Cancer Research, 72, 2835-2835. doi:10.1158/1538-7445.am2012-2835More infoThe Epithelial Cell Transforming sequence 2 (ECT2) proto-oncogene is a Guanine Exchange Factor (GEF) for RhoA, Rac1 and Cdc42 and is essential to the regulation of cytokinesis. ECT2 contains the Dbl homology and pleckstrin homology (PH) domains, which are the hallmarks of GEFs. ECT2 is over-expressed in primary non-small cell lung cancer (NSCLC) tumors, and injection of ECT2 transfectants into nude mice efficiently induces tumor formation. High level of ECT2 expression is associated with poor prognosis for patients with NSCLC. Knock down of ECT2 expression by small interfering RNAs effectively suppresses lung cancer cell growth, suggesting a specific role of ECT2 in lung cancer development. Taken together, ECT2 may represent an attractive molecular target for inhibiting lung tumor growth. Our studies are based on the hypothesis that ECT2 plays an important role in lung cancer progression and is a novel target for the development of drugs to treat lung cancer. We have built a model for ECT2 PH domain using protein homology modeling. Docking of new compounds selected from commercial drug-like libraries has led to the identification of novel inhibitors of ECT2 PH domain. We have identified several compounds that bind ECT2 PH domain in the low micromolar range (KD Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2835. doi:1538-7445.AM2012-2835
- 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.More infoThe 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.
- 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.More infoMost 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.
- 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.More infoB-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.
- Gokhale, V., Meuillet, E. J., Wisner, L., Tripp, T., Song, Z., Meuillet, E. J., Hulme, C., Gokhale, V., & Chang, H. (2010). Abstract 3565: Novel inhibitors of mPGES-1 exhibit anti-tumor activity in colorectal and lung cancers. Cancer Research, 70, 3565-3565. doi:10.1158/1538-7445.am10-3565More infoChronic inflammation is associated with 25% of all cancers. In the inflammation-cancer axis, prostaglandin E2 (PGE2) is one of the major players involved. Prostaglandin E synthase (PGES) is an enzyme downstream of the cyclooxygenases (COXs) in the PGE2 biosynthesis pathway. Among the three PGES isoforms, microsomal prostaglandin E synthase-1 (mPGES-1) is inducible by proinflammatory stimuli and constitutively expressed in colon, lung, and gastric cancers. The potential role for this enzyme in tumorigenesis has also been reported. Since inhibition of COXs may lead to some side effects due to the global reduction of other key prostaglandins, targeting downstream mPGES-1 might be a better therapeutic or chemopreventive strategy for cancers. In order to identify novel small molecule inhibitors of mPGES-1, we have screened in silico a small library of compounds. As a result, 14 lead compounds were identified by molecular docking simulations of the chemicals based on the crystal structure of mPGES-1. These compounds were tested for their ability to inhibit cell growth, induce apoptosis and decrease cytokine-stimulated PGE2 production in various colorectal and lung cancer cell lines. Using surface plasmon resonance (SPR) spectroscopy, we show the binding of the compounds PGE001 and PGE011 to human mPGES-1 recombinant protein with good affinity (KD at low micromolar range). These two compounds also showed the ability to reduce the IL-1beta-induced PGE2 production in HT29 and SW837 colon cancer cells and A549 lung cancer cell line with EC50 at low micromolar range. Furthermore, the compounds exhibit significant anti-tumor activity in the SW837 xenograft mouse model with T/C (relative size of treated and control tumors)=40.3% for compound PGE001 and T/C=68.7% for compound PGE011 (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3565.
- Nakanishi, M., Gokhale, V., Meuillet, E. J., & Rosenberg, D. W. (2010). mPGES-1 as a target for cancer suppression: A comprehensive invited review "Phospholipase A2 and lipid mediators". Biochimie, 92(6), 660-4.More infoProstaglandin E(2) (PGE(2)) is a bioactive lipid that can elicit a wide range of biological effects associated with inflammation and cancer. The physiological roles of PGE(2) are diverse, mediated in part through activation of key downstream signaling cascades via transmembrane EP receptors located on the cell surface. Elevated levels of COX-2 and concomitant overproduction of PGE(2) are often found in human cancers. These observations have led to the use of non-steroidal anti-inflammatory drugs (NSAIDs) as chemopreventive agents, particularly for colorectal cancer (CRC). Their long-term use, however, may be associated with gastrointestinal toxicity and increased risk of adverse cardiovascular events, prompting the development of other enzymatic targets in this pathway. This review will focus on recent efforts to target the terminal synthase, mPGES-1, for cancer chemoprevention. The role of mPGES-1 in the pathogenesis of various cancers is discussed. In addition, an overview of recent efforts to develop small molecule inhibitors that target the protein with high selectivity is also be reviewed.
- Raghunand, N., Guntle, G. P., Gokhale, V., Nichol, G. S., Mash, E. A., & Jagadish, B. (2010). Design, synthesis, and evaluation of 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid derived, redox-sensitive contrast agents for magnetic resonance imaging. Journal of medicinal chemistry, 53(18), 6747-57.More infoThe design and synthesis of three 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) derivatives bearing linkers with terminal thiol groups and a preliminary evaluation of their potential for use in assembling redox-sensitive magnetic resonance imaging contrast agents are reported. The linkers were selected on the basis of computational docking with a crystal structure of human serum albumin (HSA). Gd(III)-DO3A and Eu(III)-DO3A complexes were synthesized, and the structure of one complex was established by X-ray crystallographic analysis. The binding to HSA of a Gd(III)-DO3A complex bearing a thiol-terminated 3,6-dioxanonyl chain was competitively inhibited by homocysteine and by the corresponding Eu chelate. Binding to HSA was abolished when the terminal thiol group of this complex was absent. The longitudinal water-proton relaxivities (r(1)) of the three Gd(III)-DO3A complexes and of two Gd(III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexes were measured in saline at 7 T. The DO3A complexes exhibited smaller r(1) values, in both bound and free states, than the DOTA complexes.
- Dexheimer, T. S., Carey, S. S., Zuohe, S., Gokhale, V. M., Hu, X., Murata, L. B., Maes, E. M., Weichsel, A., Sun, D., Meuillet, E. J., Montfort, W. R., & Hurley, L. H. (2009). NM23-H2 may play an indirect role in transcriptional activation of c-myc gene expression but does not cleave the nuclease hypersensitive element III(1). Molecular cancer therapeutics, 8(5), 1363-77.More infoThe formation of G-quadruplex structures within the nuclease hypersensitive element (NHE) III(1) region of the c-myc promoter and the ability of these structures to repress c-myc transcription have been well established. However, just how these extremely stable DNA secondary structures are transformed to activate c-myc transcription is still unknown. NM23-H2/nucleoside diphosphate kinase B has been recognized as an activator of c-myc transcription via interactions with the NHE III(1) region of the c-myc gene promoter. Through the use of RNA interference, we confirmed the transcriptional regulatory role of NM23-H2. In addition, we find that further purification of NM23-H2 results in loss of the previously identified DNA strand cleavage activity, but retention of its DNA binding activity. NM23-H2 binds to both single-stranded guanine- and cytosine-rich strands of the c-myc NHE III(1) and, to a lesser extent, to a random single-stranded DNA template. However, it does not bind to or cleave the NHE III(1) in duplex form. Significantly, potassium ions and compounds that stabilize the G-quadruplex and i-motif structures have an inhibitory effect on NM23-H2 DNA-binding activity. Mutation of Arg(88) to Ala(88) (R88A) reduced both DNA and nucleotide binding but had minimal effect on the NM23-H2 crystal structure. On the basis of these data and molecular modeling studies, we have proposed a stepwise trapping-out of the NHE III(1) region in a single-stranded form, thus allowing single-stranded transcription factors to bind and activate c-myc transcription. Furthermore, this model provides a rationale for how the stabilization of the G-quadruplex or i-motif structures formed within the c-myc gene promoter region can inhibit NM23-H2 from activating c-myc gene expression.
- Vardanyan, R., Vijay, G., Nichol, G. S., Liu, L., Kumarasinghe, I., Davis, P., Vanderah, T., Porreca, F., Lai, J., & Hruby, V. J. (2009). Synthesis and investigations of double-pharmacophore ligands for treatment of chronic and neuropathic pain. Bioorganic & medicinal chemistry, 17(14), 5044-53.More infoAcids 9a-f as possible bivalent ligands designed as a structural combination of opioid mu-agonist (Fentanyl) and NSAID (Indomethacin) activities and produced compounds which were tested as analgesics. The obtained series of compounds exhibits low affinity and activity both at opioid receptors and as cyclooxygenase (COX) inhibitors. One explanation of the weak opioid activity could be stereochemical peculiarities of these bivalent compounds which differ significantly from the fentanyl skeleton. The absence of significant COX inhibitory properties could be explained by the required substitution of an acyl fragment in the indomethacin structure for 4-piperidyl.
- 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), 3617-23.More infoResistance 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.
- Erickson, R. P., McQueen, C. A., Chau, B., Gokhale, V., Uchiyama, M., Toyoda, A., Ejima, F., Maho, N., Sakaki, Y., & Gondo, Y. (2008). An N-ethyl-N-nitrosourea-induced mutation in N-acetyltransferase 1 in mice. Biochemical and biophysical research communications, 370(2), 285-8.More infoGenetic variation in human N-acetyltransferases (NAT) has been implicated in susceptibility to aromatic amine and hydrazine carcinogens and therapeutic drugs. There are mouse models for variability of human NAT1; however mice with genetic differences in Nat1 (corresponding to human NAT2), have not been available. N-Ethyl-N-nitrosourea (ENU) mutagenesis was used to create genetic variation in Nat1. Among a number of mutations identified, a base-pair change substituting threonine for isoleucine at position 95 was recovered and studied. Molecular models suggested that this substitution would alter substrate binding. Analysis of hepatic Nat1 activity with the selective substrate isoniazid showed that there was a significant reduction in enzymatic activity in the homozygous mutants compared to the parental strain.
- Gokhale, V., Sun, D., Hurley, L. H., Guo, K., & Gokhale, V. (2008). DNA structural polymorphism in the proximal promoter of the vascular endothelial growth factor gene. Cancer Research, 68, 3189-3189.More info3189 Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis. A polyguanine/polycytosine (polyG/polyC) tract in the proximal promoter of the VEGF gene is essential for its promoter activity. Because this polyG/polyC tract is very dynamic in nature, it is able to unwind into single-stranded DNA and adopt non-B-DNA conformations, which potentially play a role in VEGF transcriptional regulation. In this study, we characterized the secondary structures that are formed in the guanine-rich (G-rich) and cytosine-rich (C-rich) strands of this tract using various biochemical and biophysical techniques. It was revealed that, in the presence of 100 mM KCl, the G-rich strand is able to adopt two parallel G-quadruplex loop isomers containing three G-tetrads with three double-chain-reversal loops of either 1:4:1 or 1:2:3. Our CD melting experiment and polymerase stop assay suggested that the G-quadruplex containing 1:4:1 double-chain-reversal loops is the more thermodynamically stable conformation that the G-rich strand readily adopts. This provides strong evidence that the sequences and sizes of the loops are critical for the formation and stability of G-quadruplexes. Our molecular model showed that the guanines at the 5’-end and the 3’-end of the G-quadruplex provide a stable capping structure. The complementary C-rich strand is able to form an intramolecular i-motif structure at slightly acidic pH, which involves six C-C+ base pairs and three loops of 2:3:2. One guanine and cytosine in one of the loops stabilize the i-motif by stacking interactions. Taken together, our results demonstrate that the G-quadruplex and i-motif structures are able to form in the G-rich and C-rich strands respectively of the polyG/polyC tract in the VEGF proximal promoter under conditions that favor the transition of B-DNA to non-B-DNA conformations. These unique secondary structures may be novel targets for anti-angiogenesis therapy in cancer.
- 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), 4598-608.More infoA 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 Br(2) 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.
- Mahadevan, D., Powis, G., Mash, E. A., George, B., Gokhale, V. M., Zhang, S., Shakalya, K., Du-Cuny, L., Berggren, M., Ali, M. A., Jana, U., Ihle, N., Moses, S., Franklin, C., Narayan, S., Shirahatti, N., & Meuillet, E. J. (2008). Discovery of a novel class of AKT pleckstrin homology domain inhibitors. Molecular cancer therapeutics, 7(9), 2621-32.More infoAKT, a phospholipid-binding serine/threonine kinase, is a key component of the phosphoinositide 3-kinase cell survival signaling pathway that is aberrantly activated in many human cancers. Many attempts have been made to inhibit AKT; however, selectivity remains to be achieved. We have developed a novel strategy to inhibit AKT by targeting the pleckstrin homology (PH) domain. Using in silico library screening and interactive molecular docking, we have identified a novel class of non-lipid-based compounds that bind selectively to the PH domain of AKT, with "in silico" calculated K(D) values ranging from 0.8 to 3.0 micromol/L. In order to determine the selectivity of these compounds for AKT, we used surface plasmon resonance to measure the binding characteristics of the compounds to the PH domains of AKT1, insulin receptor substrate-1, and 3-phosphoinositide-dependent protein kinase 1. There was excellent correlation between predicted in silico and measured in vitro K(D)s for binding to the PH domain of AKT, which were in the range 0.4 to 3.6 micromol/L. Some of the compounds exhibited PH domain-binding selectivity for AKT compared with insulin receptor substrate-1 and 3-phosphoinositide-dependent protein kinase 1. The compounds also inhibited AKT in cells, induced apoptosis, and inhibited cancer cell proliferation. In vivo, the lead compound failed to achieve the blood concentrations required to inhibit AKT in cells, most likely due to rapid metabolism and elimination, and did not show antitumor activity. These results show that these compounds are the first small molecules selectively targeting the PH domain of AKT.
- Sun, D., Liu, W. J., 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), 880-9.More infoPrevious 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, TMPyP4 and 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.
- Fisher, A. A., Labenski, M. T., Malladi, S., Gokhale, V., Bowen, M. E., Milleron, R. S., Bratton, S. B., Monks, T. J., & Lau, S. S. (2007). Quinone electrophiles selectively adduct "electrophile binding motifs" within cytochrome c. Biochemistry, 46(39), 11090-100.More infoElectrophiles generated endogenously, or via the metabolic bioactivation of drugs and other environmental chemicals, are capable of binding to a variety of nucleophilic sites within proteins. Factors that determine site selective susceptibility to electrophile-mediated post-translational modifications, and the consequences of such alterations, remain largely unknown. To identify and characterize chemical-mediated protein adducts, electrophiles with known toxicity were utilized. Hydroquinone, and its mercapturic acid pathway metabolites, cause renal proximal tubular cell necrosis and nephrocarcinogenicity in rats. The adverse effects of HQ and its thioether metabolites are in part a consequence of their oxidation to the corresponding electrophilic 1,4-benzoquinones (BQ). We now report that BQ and 2-(N-acetylcystein-S-yl)benzoquinone (NAC-BQ) preferentially bind to solvent-exposed lysine-rich regions within cytochrome c. Furthermore, we have identified specific glutamic acid residues within cytochrome c as novel sites of NAC-BQ adduction. The microenvironment at the site of adduction governs both the initial specificity and the structure of the final adduct. The solvent accessibility and local pKa of the adducted and neighboring amino acids contribute to the selectivity of adduction. Postadduction chemistry subsequently alters the nature of the final adduct. Using molecular modeling, the impact of BQ and NAC-BQ adduction on cytochrome c was visualized, revealing the spatial rearrangement of critical residues necessary for protein-protein interactions. Consequently, BQ-adducted cytochrome c fails to initiate caspase-3 activation in native lysates and also inhibits Apaf-1 oligomerization into an apoptosome complex in a purely reconstituted system. In summary, a combination of mass spectroscopic, molecular modeling, and biochemical approaches confirms that electrophile-protein adducts produce structural alterations that influence biological function.
- Gokhale, V., Fisher, A. A., Labenski, M. T., Malladi, S., Bowen, M. E., Milleron, R. S., Bratton, S. B., Monks, T. J., & Lau, S. S. (2007). Quinone Electrophiles Selectively Adduct “Electrophile Binding Motifs” within Cytochrome c. Biochemistry, 46(39), 11090-11100. doi:10.1021/bi700613w
- Gokhale, V., Monks, T., Lau, S. S., Labenski, M. T., Gokhale, V., Fisher, A. A., & Bratton, S. B. (2007). Cytochrome c Arylation by 1,4-Benzoquinone and Its Quinol-Thioether Metabolites Results in A Loss of Protein Function. The FASEB Journal, 21(5). doi:10.1096/fasebj.21.5.a440More info1,4-Benzoquinone (BQ) and its quinol-thioether (QT) metabolites, including 2-(N-acetylcystein-S-yl)benzoquinone (NAC-BQ), cause renal proximal tubular cell necrosis. The adverse effects of BQ and i...
- 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), 10220-8.More infoA 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.
- 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), 7698-713.More infoThe 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.
- Song, Z., Saghafi, N., Gokhale, V., Brabant, M., & Meuillet, E. J. (2007). Regulation of the activity of the tumor suppressor PTEN by thioredoxin in Drosophila melanogaster. Experimental cell research, 313(6), 1161-71.More infoHuman Thioredoxin-1 (hTrx-1) is a small redox protein with a molecular weight of 12 kDa that contains two cysteine residues found in its catalytic site. HTrx-1 plays an important role in cell growth, apoptosis, and cancer patient prognosis. Recently, we have demonstrated that hTrx-1 binds to the C2 domain of the human tumor suppressor, PTEN, in a redox dependent manner. This binding leads to the inhibition of PTEN lipid phosphatase activity in mammalian tissue culture systems. In this study, we show that over-expression of hTrx-1 in Drosophila melanogaster promotes cell growth and proliferation during eye development as measured by eye size and ommatidia size. Furthermore, hTrx-1 rescues the small eye phenotype induced by the over-expression of PTEN. We demonstrate that this rescue of the PTEN-induced eye size phenotype requires cysteine-218 in the C2 domain of PTEN. We also show that hTrx-1 over-expression results in increased Akt phosphorylation in fly head extracts supporting our observations that the hTrx-1-induced eye size increase results from the inhibition of PTEN activity. Our study confirms the redox regulation of PTEN through disulfide bond formation with the hTrx-1 in Drosophila and suggests conserved mechanisms for thioredoxins and their interactions with the phosphatidylinositol-3-kinase signaling pathway in humans and fruit flies.
- Zhu, M., Gokhale, V. M., Szabo, L., Munoz, R. M., Baek, H., Bashyam, S., Hurley, L. H., Von Hoff, D. D., & Han, H. (2007). Identification of a novel inhibitor of urokinase-type plasminogen activator. Molecular cancer therapeutics, 6(4), 1348-56.More infoUrokinase-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, UK122, showed the highest inhibition of uPA activity. The IC(50) of UK122 in a cell-free indirect uPA assay is 0.2 micromol/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 (IC(50) >100 micromol/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.
- Gokhale, V., Monks, T. J., Lau, S. S., Labenski, M. T., Gokhale, V., Fisher, A. A., & Bratton, S. B. (2006). Arylation of cytochrome c by benzoquinone and benzoquinone-thioether causes a loss of protein function. The FASEB Journal, 20(4).
- 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), 2944-59.More infoCationic 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.
- Stephens, B. J., Han, H., Gokhale, V., & Von Hoff, D. D. (2005). PRL phosphatases as potential molecular targets in cancer. Molecular cancer therapeutics, 4(11), 1653-61.More infoThe phosphatase of regenerating liver (PRL) family of phosphatases, consisting of PRL-1, PRL-2, and PRL-3, represents an intriguing group of proteins being validated as biomarkers and therapeutic targets in cancer. Individual PRLs are overexpressed in a variety of cancer cell lines and tissues when compared with their normal counterparts. More importantly, several recent studies have shown that PRL-3 is expressed at higher levels and at a greater frequency in colorectal cancer metastases compared with primary colorectal tumors and normal colon tissue. Ectopic expression of PRLs in nontumorigenic cells can influence proliferation and the migratory and invasive properties of cells, while knockdown of endogenous PRL-3 or PRL-1 in cancerous cells using small interfering RNA can abrogate cell motility and ability to metastasize in a mouse model. However, the exact biological function and cellular substrates of the PRLs remain unclear. This review will discuss what is known about the PRLs, what makes the PRLs possible attractive targets for therapeutic intervention, and the possible future directions in PRL biology and inhibitor identification.
- 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), 8702-9.More infoThe 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.