Eli Chapman

Interests

Research

Chemical BiologyDrug DiscoveryProteostasisCellular Quality Control

Teaching

Medicinal ChemistryChemical Biology

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Scholarly Contributions

Journals/Publications

• Ambrose, A. J., Zerio, C. J., Sivinski, J., Schmidlin, C. J., Shi, T., Ross, A. B., Widrick, K. J., Johnson, S. M., Zhang, D. D., & Chapman, E. (2019). A high throughput substrate binding assay reveals hexachlorophene as an inhibitor of the ER-resident HSP70 chaperone GRP78. Bioorganic & medicinal chemistry letters, 29(14), 1689-1693.
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  Glucose-regulated protein 78 (GRP78) is the ER resident 70 kDa heat shock protein 70 (HSP70) and has been hypothesized to be a therapeutic target for various forms of cancer due to its role in mitigating proteotoxic stress in the ER, its elevated expression in some cancers, and the correlation between high levels for GRP78 and a poor prognosis. Herein we report the development and use of a high throughput fluorescence polarization-based peptide binding assay as an initial step toward the discovery and development of GRP78 inhibitors. This assay was used in a pilot screen to discover the anti-infective agent, hexachlorophene, as an inhibitor of GRP78. Through biochemical characterization we show that hexachlorophene is a competitive inhibitor of the GRP78-peptide interaction. Biological investigations showed that this molecule induces the unfolded protein response, induces autophagy, and leads to apoptosis in a colon carcinoma cell model, which is known to be sensitive to GRP78 inhibition.
• Dodson, M., de la Vega, M. R., Cholanians, A. B., Schmidlin, C. J., Chapman, E., & Zhang, D. D. (2019). Modulating NRF2 in Disease: Timing Is Everything. Annual review of pharmacology and toxicology, 59, 555-575.
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  The transcription factor nuclear factor erythroid 2 (NF-E2)-related factor 2 (NRF2) is a central regulator of redox, metabolic, and protein homeostasis that intersects with many other signaling cascades. Although the understanding of the complex nature of NRF2 signaling continues to grow, there is only one therapeutic targeting NRF2 for clinical use, dimethyl fumarate, used for the treatment of multiple sclerosis. The discovery of new therapies is confounded by the fact that NRF2 levels vary significantly depending on physiological and pathological context. Thus, properly timed and targeted manipulation of the NRF2 pathway is critical in creating effective therapeutic regimens. In this review, we summarize the regulation and downstream targets of NRF2. Furthermore, we discuss the role of NRF2 in cancer, neurodegeneration, and diabetes as well as cardiovascular, kidney, and liver disease, with a special emphasis on NRF2-based therapeutics, including those that have made it into clinical trials.
• Gaffney, D. O., Jennings, E. Q., Anderson, C. C., Marentette, J. O., Shi, T., Schou Oxvig, A. M., Streeter, M. D., Johannsen, M., Spiegel, D. A., Chapman, E., Roede, J. R., & Galligan, J. J. (2019). Non-enzymatic Lysine Lactoylation of Glycolytic Enzymes. Cell chemical biology.
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  Post-translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome. Many of these PTMs are derived from cellular metabolites and serve as feedback and feedforward mechanisms of regulation. We have identified a PTM that is derived from the glycolytic by-product, methylglyoxal. This reactive metabolite is rapidly conjugated to glutathione via glyoxalase 1, generating lactoylglutathione (LGSH). LGSH is hydrolyzed by glyoxalase 2 (GLO2), cycling glutathione and generating D-lactate. We have identified the non-enzymatic acyl transfer of the lactate moiety from LGSH to protein Lys residues, generating a "LactoylLys" modification on proteins. GLO2 knockout cells have elevated LGSH and a consequent marked increase in LactoylLys. Using an alkyne-tagged methylglyoxal analog, we show that these modifications are enriched on glycolytic enzymes and regulate glycolysis. Collectively, these data suggest a previously unexplored feedback mechanism that may serve to regulate glycolytic flux under hyperglycemic or Warburg-like conditions.
• Liu, P., Tian, W., Tao, S., Tillotson, J., Wijeratne, E. M., Gunatilaka, A. A., Zhang, D. D., & Chapman, E. (2019). Non-covalent NRF2 Activation Confers Greater Cellular Protection than Covalent Activation. Cell chemical biology, 26(10), 1427-1435.e5.
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  The transcription factor NRF2 confers cellular protection by maintaining cellular redox homeostasis and proteostasis. Basal NRF2 levels are normally low due to KEAP1-mediated ubiquitylation and subsequent proteasomal degradation. KEAP1, a substrate adaptor protein of a KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex, contains a critical cysteine (C151) that is modified by electrophiles or oxidants, resulting in inactivation of the E3 ligase and inhibition of NRF2 degradation. Currently, nearly all NRF2 inducers are electrophilic molecules that possess unwanted off-target effects due to their reactive nature. Here, we report a group of NRF2 inducers, ent-kaurane diterpenoid geopyxins, with and without C151 reactive electrophilic moieties. Among 16 geopyxins, geopyxin F, a non-electrophilic NRF2 activator, showed enhanced cellular protection relative to an electrophilic NRF2 activator, geopyxin C. To our knowledge, this is the first detailed structure-activity relationship study of covalent versus non-covalent NRF2 activators, showing the promise of non-covalent NRF2 activators as potential therapeutic compounds.
• Liu, P., de la Vega, M. R., Dodson, M., Yue, F., Shi, B., Fang, D., Chapman, E., Liu, L., & Zhang, D. D. (2019). Spermidine Confers Liver Protection by Enhancing NRF2 Signaling Through a MAP1S-Mediated Noncanonical Mechanism. Hepatology (Baltimore, Md.), 70(1), 372-388.
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  Spermidine (SPD), a naturally occurring polyamine, has been recognized as a caloric restriction mimetic that confers health benefits, presumably by inducing autophagy. Recent studies have reported that oral administration of SPD protects against liver fibrosis and hepatocarcinogenesis through activation of microtubule associated protein 1S (MAP1S)-mediated autophagy. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor that mediates cellular protection by maintaining the cell's redox, metabolic, and proteostatic balance. In this study, we demonstrate that SPD is a noncanonical NRF2 inducer, and that MAP1S is a component of this noncanonical pathway of NRF2 activation. Mechanistically, MAP1S induces NRF2 signaling through two parallel mechanisms, both resulting in NRF2 stabilization: (1) MAP1S competes with Kelch-like ECH-associated protein 1 (KEAP1) for NRF2 binding through an ETGE motif, and (2) MAP1S accelerates p62-dependent degradation of KEAP1 by the autophagy pathway. We further demonstrate that SPD confers liver protection by enhancing NRF2 signaling. The importance of both NRF2 and p62-dependent autophagy in SPD-mediated liver protection was confirmed using a carbon tetrachloride-induced liver fibrosis model in wild-type, Nrf2 , p62 and Nrf2 ;p62 mice, as the protective effect of SPD was significantly reduced in NRF2 or p62 single knockout mice, and completely abolished in the double knockout mice. Conclusion: Our results demonstrate the pivotal role of NRF2 in mediating the health benefit of SPD, particularly in the context of liver pathologies.
• Shi, T., Kaneko, L., Sandino, M., Busse, R., Zhang, M., Mason, D., Machulis, J., Ambrose, A. J., Zhang, D. D., & Chapman, E. (2019). One-Step Synthesis of Thieno[2,3-]pyrimidin-4(3)-ones via a Catalytic Four-Component Reaction of Ketones, Ethyl Cyanoacetate, S and Formamide. ACS sustainable chemistry & engineering, 7(1), 1524-1528.
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  Thieno[2,3-]pyrimidin-4(3)-ones are important pharmacophores that previously required a three step synthesis with two chromatography steps. We herein report a green approach to the synthesis of this pharmacologically important class of compounds via a catalytic four-component reaction using a ketone, ethyl cyanoacetate, S and formamide. The reported reaction is characterized by step economy, reduced catalyst loading and easy purification.
• Shi, T., Teng, S., Gopi Krishna Reddy, A., Guo, X., Zhang, Y., Moore, K. T., Buckley, T., Mason, D. J., Wang, W., Chapman, E., & Hu, W. (2019). Catalytic asymmetric synthesis of 2,5-dihydrofurans using synergistic bifunctional Ag catalysis. Organic & biomolecular chemistry, 17(38), 8737-8744.
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  We report a bifunctional Ag catalyst promoted intramolecular capture of oxonium ylides with alkynes for the enantioselective synthesis of 2,5-dihydrofurans. This represents unprecedented synergistic catalysis of a bifunctional Ag catalyst. Mechanistic studies revealed that [(R)-3,5-DM-BINAP](AgSbF) (9) is likely to be the active catalytic species and that the reaction involves second order kinetics with respect to 9, suggesting that two molecules of 9 are involved in the intramolecular trapping of a Ag-associated oxonium ylide with a Ag-activated alkyne. Based on our mechanistic hypothesis, we further optimized the reaction, rendering a facile approach to 2,5-dihydrofurans in good to excellent yields in a highly chemo- and enantioselective fashion.
• Shi, T., Wijeratne, E. M., Solano, C., Ambrose, A. J., Ross, A. B., Norwood, C., Orido, C. K., Grigoryan, T., Tillotson, J., Kang, M., Luo, G., Keegan, B. M., Hu, W., Blagg, B. S., Zhang, D. D., Gunatilaka, A. A., & Chapman, E. (2019). An Isoform-Selective PTP1B Inhibitor Derived from Nitrogen-Atom Augmentation of Radicicol. Biochemistry, 58(30), 3225-3231.
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  A library of natural products and their derivatives was screened for inhibition of protein tyrosine phosphatase (PTP) 1B, which is a validated drug target for the treatment of obesity and type II diabetes. Of those active in the preliminary assay, the most promising was compound containing a novel pyrrolopyrazoloisoquinolone scaffold derived by treating radicicol () with hydrazine. This nitrogen-atom augmented radicicol derivative was found to be PTP1B selective relative to other highly homologous nonreceptor PTPs. Biochemical evaluation, molecular docking, and mutagenesis revealed to be an allosteric inhibitor of PTP1B with a submicromolar . Cellular analyses using C2C12 myoblasts indicated that restored insulin signaling and increased glucose uptake.
• Shi, T., Zerio, C. J., Sivinski, J., Ambrose, A. J., Moore, K. T., Buckley, T., Kaneko, L., Zhang, M., Zhang, D. D., & Chapman, E. (2019). A one-step, atom economical synthesis of thieno[2,3-]pyrimidin-4-amine derivatives via a four-component reaction. European journal of organic chemistry, 20(2), 3269-3272.
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  A NaHPO-catalyzed four-component reaction between a ketone, malononitrile, S and formamide has been realized for the first time. This reaction provides a concise approach to thieno[2,3-]pyrimidin-4-amines, previously requiring 5 steps. The utility of this reaction was validated by preparing a multi-targeted kinase inhibitor and an inhibitor of the NRF2 pathway with excellent atom- and step-economy.
• Stevens, M., Abdeen, S., Salim, N., Ray, A. M., Washburn, A., Chitre, S., Sivinski, J., Park, Y., Hoang, Q. Q., Chapman, E., & Johnson, S. M. (2019). HSP60/10 chaperonin systems are inhibited by a variety of approved drugs, natural products, and known bioactive molecules. Bioorganic & medicinal chemistry letters, 29(9), 1106-1112.
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  All living organisms contain a unique class of molecular chaperones called 60 kDa heat shock proteins (HSP60 - also known as GroEL in bacteria). While some organisms contain more than one HSP60 or GroEL isoform, at least one isoform has always proven to be essential. Because of this, we have been investigating targeting HSP60 and GroEL chaperonin systems as an antibiotic strategy. Our initial studies focused on applying this antibiotic strategy for treating African sleeping sickness (caused by Trypanosoma brucei parasites) and drug-resistant bacterial infections (in particular Methicillin-resistant Staphylococcus aureus - MRSA). Intriguingly, during our studies we found that three known antibiotics - suramin, closantel, and rafoxanide - were potent inhibitors of bacterial GroEL and human HSP60 chaperonin systems. These findings prompted us to explore what other approved drugs, natural products, and known bioactive molecules might also inhibit HSP60 and GroEL chaperonin systems. Initial high-throughput screening of 3680 approved drugs, natural products, and known bioactives identified 161 hit inhibitors of the Escherichia coli GroEL chaperonin system (4.3% hit rate). From a purchased subset of 60 hits, 29 compounds (48%) re-confirmed as selective GroEL inhibitors in our assays, all of which were nearly equipotent against human HSP60. These findings illuminate the notion that targeting chaperonin systems might be a more common occurrence than we previously appreciated. Future studies are needed to determine if the in vivo modes of action of these approved drugs, natural products, and known bioactive molecules are related to GroEL and HSP60 inhibition.
• Tao, S., Wang, S., Moghaddam, S. J., Ooi, A., Chapman, E., Wong, P. K., & Zhang, D. D. (2019). Correction: Oncogenic KRAS Confers Chemoresistance by Upregulating NRF2. Cancer research, 79(5), 1015.
• Washburn, A., Abdeen, S., Ovechkina, Y., Ray, A. M., Stevens, M., Chitre, S., Sivinski, J., Park, Y., Johnson, J., Hoang, Q. Q., Chapman, E., Parish, T., & Johnson, S. M. (2019). Dual-targeting GroEL/ES chaperonin and protein tyrosine phosphatase B (PtpB) inhibitors: A polypharmacology strategy for treating Mycobacterium tuberculosis infections. Bioorganic & medicinal chemistry letters, 29(13), 1665-1672.
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  Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis - actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease - which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria.
• Abdeen, S., Kunkle, T., Salim, N., Ray, A. M., Mammadova, N., Summers, C., Stevens, M., Ambrose, A. J., Park, Y., Schultz, P. G., Horwich, A. L., Hoang, Q. Q., Chapman, E., & Johnson, S. M. (2018). Sulfonamido-2-arylbenzoxazole GroEL/ES Inhibitors as Potent Antibacterials against Methicillin-Resistant Staphylococcus aureus (MRSA). Journal of medicinal chemistry, 61(16), 7345-7357.
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  Extending from a study we recently published examining the antitrypanosomal effects of a series of GroEL/ES inhibitors based on a pseudosymmetrical bis-sulfonamido-2-phenylbenzoxazole scaffold, here, we report the antibiotic effects of asymmetric analogs of this scaffold against a panel of bacteria known as the ESKAPE pathogens ( Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). While GroEL/ES inhibitors were largely ineffective against K. pneumoniae, A. baumannii, P. aeruginosa, and E. cloacae (Gram-negative bacteria), many analogs were potent inhibitors of E. faecium and S. aureus proliferation (Gram-positive bacteria, EC values of the most potent analogs were in the 1-2 μM range). Furthermore, even though some compounds inhibit human HSP60/10 biochemical functions in vitro (IC values in the 1-10 μM range), many of these exhibited moderate to low cytotoxicity to human liver and kidney cells (CC values > 20 μM).
• Chapman, E. (2018). Hydroxybiphenylamide GroEL/ES Inhibitors Are Potent Antibacterials against Planktonic and Biofilm Forms of Staphylococcus aureus. Journal of Medicinal Chemistry.
• Chapman, E., Shi, T., Sandino, M., Busse, R., Zhang, M., Mason, D., Machulis, J., Ambrose, A. J., & Zhang, D. D. (2018). One-step synthesis of thieno[2,3-d]pyrimidin-4(3H)-ones via a catalytic four-component reaction of ketones, ethyl cyanoacetate, S8 and formamide. ACS Sustainable Chemistry & Engineering.
• Cunningham, T. A., Chapman, E., & Schatz, J. H. (2018). eIF4A inhibition: ready for primetime?. Oncotarget, 9(85), 35515-35516.
• Dodson, M., Liu, P., Jiang, T., Ambrose, A. J., Luo, G., Rojo de la Vega, M., Cholanians, A. B., Wong, P. K., Chapman, E., & Zhang, D. D. (2018). Increased O-GlcNAcylation of SNAP29 drives arsenic-induced autophagic dysfunction. Molecular and cellular biology.
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  Environmental exposure to arsenic is linked to adverse health effects including cancer and diabetes. Pleiotropic cellular effects are observed with arsenic exposure. Previously, we demonstrated that arsenic dysregulated the autophagy pathway at low, environmentally relevant concentrations. Here, we show that arsenic blocks autophagy by preventing autophagosome-lysosome fusion. Specifically, arsenic disrupts formation of the STX17-SNAP29-VAMP8 SNARE complex, where SNAP29 mediates vesicle fusion through bridging STX17-containing autophagosomes to VAMP8-bearing lysosomes. Mechanistically, arsenic inhibits SNARE complex formation, at least in part, by enhancing O-GlcNAcylation of SNAP29. Transfection of O-GlcNAcylation defective, but not wild type, SNAP29 into CRISPR-mediated knockout cells abolishes arsenic-mediated autophagy inhibition. These findings reveal a mechanism by which low-levels of arsenic perturb proteostasis through inhibition of SNARE complex formation, providing a possible therapeutic target for disease intervention in the more than 200 million people exposed to unsafe levels of arsenic.
• Dodson, M., de la Vega, M. R., Harder, B., Castro-Portuguez, R., Rodrigues, S. D., Wong, P. K., Chapman, E., & Zhang, D. D. (2018). Low-level arsenic causes proteotoxic stress and not oxidative stress. Toxicology and applied pharmacology, 341, 106-113.
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  Prolonged exposure to arsenic has been shown to increase the risk of developing a number of diseases, including cancer and type II diabetes. Arsenic is present throughout the environment in its inorganic forms, and the level of exposure varies greatly by geographical location. The current recommended maximum level of arsenic exposure by the EPA is 10μg/L, but levels>50-1000μg/L have been detected in some parts of Asia, the Middle East, and the Southwestern United States. One of the most important steps in developing treatment options for arsenic-linked pathologies is to understand the cellular pathways affected by low levels of arsenic. Here, we show that acute exposure to non-lethal, low-level arsenite, an environmentally relevant arsenical, inhibits the autophagy pathway. Furthermore, arsenite-induced autophagy inhibition initiates a transient, but moderate ER stress response. Significantly, low-level arsenite exposure does not exhibit an increase in oxidative stress. These findings indicate that compromised autophagy, and not enhanced oxidative stress occurs early during arsenite exposure, and that restoring the autophagy pathway and proper proteostasis could be a viable option for treating arsenic-linked diseases. As such, our study challenges the existing paradigm that oxidative stress is the main underlying cause of pathologies associated with environmental arsenic exposure.
• 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.
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  Mutations 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.
• Kunkle, T., Abdeen, S., Salim, N., Ray, A. M., Stevens, M., Ambrose, A. J., Victorino, J., Park, Y., Hoang, Q. Q., Chapman, E., & Johnson, S. M. (2018). Hydroxybiphenylamide GroEL/ES Inhibitors Are Potent Antibacterials against Planktonic and Biofilm Forms of Staphylococcus aureus. Journal of medicinal chemistry.
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  We recently reported the identification of a GroEL/ES inhibitor (1, N-(4-(benzo[ d]thiazol-2-ylthio)-3-chlorophenyl)-3,5-dibromo-2-hydroxybenzamide) that exhibited in vitro antibacterial effects against Staphylococcus aureus comparable to vancomycin, an antibiotic of last resort. To follow up, we have synthesized 43 compound 1 analogs to determine the most effective functional groups of the scaffold for inhibiting GroEL/ES and killing bacteria. Our results identified that the benzothiazole and hydroxyl groups are important for inhibiting GroEL/ES-mediated folding functions, with the hydroxyl essential for antibacterial effects. Several analogs exhibited >50-fold selectivity indices between antibacterial efficacy and cytotoxicity to human liver and kidney cells in cell culture. We found that MRSA was not able to easily generate acute resistance to lead inhibitors in a gain-of-resistance assay and that lead inhibitors were able to permeate through established S. aureus biofilms and maintain their bactericidal effects.
• Peters, T. L., Tillotson, J., Yeomans, A. M., Wilmore, S., Lemm, E., Jiménez-Romero, C., Amador, L. A., Li, L., Amin, A. D., Pongtornpipat, P., Zerio, C. J., Ambrose, A. J., Paine-Murrieta, G., Greninger, P., Vega, F., Benes, C. H., Packham, G., Rodríguez, A. D., Chapman, E., & Schatz, J. H. (2018). Target-Based Screening against eIF4A1 Reveals the Marine Natural Product Elatol as a Novel Inhibitor of Translation Initiation with Antitumor Activity. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(17), 4256-4270.
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  The DEAD-box RNA helicase eIF4A1 carries out the key enzymatic step of cap-dependent translation initiation and is a well-established target for cancer therapy, but no drug against it has entered evaluation in patients. We identified and characterized a natural compound with broad antitumor activities that emerged from the first target-based screen to identify novel eIF4A1 inhibitors. We tested potency and specificity of the marine compound elatol versus eIF4A1 ATPase activity. We also assessed eIF4A1 helicase inhibition, binding between the compound and the target including binding site mutagenesis, and extensive mechanistic studies in cells. Finally, we determined maximum tolerated dosing and assessed activity against xenografted tumors. We found elatol is a specific inhibitor of ATP hydrolysis by eIF4A1 with broad activity against multiple tumor types. The compound inhibits eIF4A1 helicase activity and binds the target with unexpected 2:1 stoichiometry at key sites in its helicase core. Sensitive tumor cells suffer acute loss of translationally regulated proteins, leading to growth arrest and apoptosis. In contrast to other eIF4A1 inhibitors, elatol induces markers of an integrated stress response, likely an off-target effect, but these effects do not mediate its cytotoxic activities. Elatol is less potent than the well-studied eIF4A1 inhibitor silvestrol but is tolerated at approximately 100× relative dosing, leading to significant activity against lymphoma xenografts. Elatol's identification as an eIF4A1 inhibitor with antitumor activities provides proof of principle for target-based screening against this highly promising target for cancer therapy. .
• Rojo de la Vega, M., Chapman, E., & Zhang, D. D. (2018). NRF2 and the Hallmarks of Cancer. Cancer cell, 34(1), 21-43.
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  The transcription factor NRF2 is the master regulator of the cellular antioxidant response. Though recognized originally as a target of chemopreventive compounds that help prevent cancer and other maladies, accumulating evidence has established the NRF2 pathway as a driver of cancer progression, metastasis, and resistance to therapy. Recent studies have identified new functions for NRF2 in the regulation of metabolism and other essential cellular functions, establishing NRF2 as a truly pleiotropic transcription factor. In this review, we explore the roles of NRF2 in the hallmarks of cancer, indicating both tumor suppressive and tumor-promoting effects.
• Tao, S., Rojo de la Vega, M., Chapman, E., Ooi, A., & Zhang, D. D. (2018). The effects of NRF2 modulation on the initiation and progression of chemically and genetically induced lung cancer. Molecular carcinogenesis, 57(2), 182-192.
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  Targeting the transcription factor NRF2 has been recognized as a feasible strategy for cancer prevention and treatment, but many of the mechanistic details underlying its role in cancer development and progression are lacking. Therefore, careful mechanistic studies of the NRF2 pathway in cancer initiation and progression are needed to identify which therapeutic avenue-activation or inhibition-is appropriate in a given context. Moreover, while numerous reports confirm the protective effect of NRF2 activation against chemical carcinogenesis little is known of its role in cancer arising from spontaneous mutations. Here, we tested the effects of NRF2 modulation (activation by sulforaphane or inhibition by brusatol) in lung carcinogenesis using a chemical (vinyl carbamate) model in A/J mice and a genetic (conditional KrasG12D oncogene expression, to simulate spontaneous oncogene mutation) model in C57BL/6J mice. Mice were treated with NRF2 modulators before carcinogen exposure or KrasG12D expression to test the role of NRF2 in cancer initiation, or treated after tumor development to test the role of NRF2 in cancer progression. Lung tissues were analyzed to determine tumor burden, as well as status of NRF2 and KRAS pathways. Additionally, proliferation, apoptosis, and oxidative DNA damage were assessed. Overall, NRF2 activation prevents initiation of chemically induced cancer, but promotes progression of pre-existing tumors regardless of chemical or genetic etiology. Once tumors are initiated, NRF2 inhibition is effective against the progression of chemically and spontaneously induced tumors. These results have important implications for NRF2-targeted cancer prevention and intervention strategies.
• Ambrose, A. J., Santos, E. A., Jimenez, P. C., Rocha, D. D., Wilke, D. V., Beuzer, P., Axelrod, J., Kumar Kanduluru, A., Fuchs, P. L., Cang, H., Costa-Lotufo, L. V., Chapman, E., & La Clair, J. J. (2017). Ritterostatin GN 1N , a Cephalostatin-Ritterazine Bis-steroidal Pyrazine Hybrid, Selectively Targets GRP78. Chembiochem : a European journal of chemical biology, 18(6), 506-510.
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  Natural products discovered by using agnostic approaches, unlike rationally designed leads or those obtained through high-throughput screening, offer the ability to reveal new biological pathways and, hence, serve as an important vehicle to unveil new avenues in drug discovery. The ritterazine-cephalostatin family of natural products displays robust and potent antitumor activities, with sub-nanomolar growth inhibition against multiple cell lines and potent activity in xenograft models. Herein, we used comparative cellular and molecular biological methods to uncover the ritterazine-cephalostatin cytotoxic mode of action (MOA) in human tumor cells. Our findings indicated that, whereas ritterostatin GN 1N , a cephalostatin-ritterazine hybrid, binds to multiple HSP70s, its cellular trafficking confines activity to the endoplasmic reticulum (ER)-based HSP70 isoform, GRP78. This targeting results in activation of the unfolding protein response (UPR) and subsequent apoptotic cell death.
• Bashyal, B. P., Wijeratne, E. M., Tillotson, J., Arnold, A. E., Chapman, E., & Gunatilaka, A. A. (2017). Chlorinated Dehydrocurvularins and Alterperylenepoxide A from Alternaria sp. AST0039, a Fungal Endophyte of Astragalus lentiginosus. Journal of natural products, 80(2), 427-433.
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  Investigation of Alternaria sp. AST0039, an endophytic fungus obtained from the leaf tissue of Astragalus lentiginosus, led to the isolation of (-)-(10E,15S)-4,6-dichloro-10(11)-dehydrocurvularin (1), (-)-(10E,15S)-6-chloro-10(11)-dehydrocurvularin (2), (-)-(10E,15S)-10(11)-dehydrocurvularin (3), and alterperylenepoxide A (4) together with scytalone and α-acetylorcinol. Structures of 1 and 4 were established from their spectroscopic data, and the relative configuration of 4 was determined with the help of nuclear Overhauser effect difference data. All metabolites were evaluated for their cytotoxic activity and ability to induce heat-shock and unfolded protein responses. Compounds 2 and 3 exhibited cytotoxicity to all five cancer cell lines tested and increased the level of the pro-apoptotic transcription factor CHOP, but only 3 induced the heat-shock response and caused a strong unfolded protein response.
• Gubiani, J. R., Wijeratne, E. M., Shi, T., Araujo, A. R., Arnold, A. E., Chapman, E., & Gunatilaka, A. A. (2017). An epigenetic modifier induces production of (10'S)-verruculide B, an inhibitor of protein tyrosine phosphatases by Phoma sp. nov. LG0217, a fungal endophyte of Parkinsonia microphylla. Bioorganic & medicinal chemistry, 25(6), 1860-1866.
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  Incorporation of the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), to a culture broth of the endophytic fungus Phoma sp. nov. LG0217 isolated from Parkinsonia microphylla changed its metabolite profile and resulted in the production of (10'S)-verruculide B (1), vermistatin (2) and dihydrovermistatin (3). When cultured in the absence of the epigenetic modifier, it produced a new metabolite, (S,Z)-5-(3',4'-dihydroxybutyldiene)-3-propylfuran-2(5H)-one (4) together with nafuredin (5). The structure of 4 was elucidated by spectroscopic analyses and its absolute configuration was determined by application of the modified Mosher's ester method. The absolute structure of (10'S)-verruculide B was determined as 5-[(10'S,2'E,6'E)-10',11'-dihydroxy-3',7',11'-trimethyldodeca-2',6'-dien-1'-yl]-(3R)-6,8-dihydroxy-3-methylisochroman-1-one (1) with the help of CD and NOE data. Compound 1 inhibited the activity of protein tyrosine phosphatases (PTPs) 1B (PTP1B), Src homology 2-containing PTP 1 (SHP1) and T-cell PTP (TCPTP) with IC50 values of 13.7±3.4, 8.8±0.6, and 16.6±3.8μM, respectively. Significance of these activities and observed modest selectivity of 1 for SHP1 over PTP1B and TCPTP is discussed.
• Harder, B., Tian, W., La Clair, J. J., Tan, A. C., Ooi, A., Chapman, E., & Zhang, D. D. (2017). Brusatol overcomes chemoresistance through inhibition of protein translation. Molecular carcinogenesis, 56(5), 1493-1500.
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  The NRF2 pathway activates a cell survival response when cells are exposed to xenobiotics or are under oxidative stress. Therapeutic activation of NRF2 can also be used prior to insult as a means of disease prevention. However, prolonged expression of NRF2 has been shown to protect cancer cells by inducing the metabolism and efflux of chemotherapeutics, leading to both intrinsic and acquired chemoresistance to cancer drugs. This effect has been termed the "dark side" of NRF2. In an effort to combat this chemoresistance, our group discovered the first NRF2 inhibitor, the natural product brusatol, however the mechanism of inhibition was previously unknown. In this report, we show that brusatol's mode of action is not through direct inhibition of the NRF2 pathway, but through the inhibition of both cap-dependent and cap-independent protein translation, which has an impact on many short-lived proteins, including NRF2. Therefore, there is still a need to develop a new generation of specific NRF2 inhibitors with limited toxicity and off-target effects that could be used as adjuvant therapies to sensitize cancers with high expression of NRF2.
• Tao, S., Liu, P., Luo, G., Rojo de la Vega, M., Chen, H., Wu, T., Tillotson, J., Chapman, E., & Zhang, D. D. (2017). p97 Negatively Regulates NRF2 by Extracting Ubiquitylated NRF2 from the KEAP1-CUL3 E3 Complex. Molecular and cellular biology, 37(8).
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  Activation of the stress-responsive transcription factor NRF2 is the major line of defense to combat oxidative or electrophilic insults. Under basal conditions, NRF2 is continuously ubiquitylated by the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex and is targeted to the proteasome for degradation (the canonical mechanism). However, the path from the CUL3 complex to ultimate proteasomal degradation was previously unknown. p97 is a ubiquitin-targeted ATP-dependent segregase that extracts ubiquitylated client proteins from membranes, protein complexes, or chromatin and has an essential role in autophagy and the ubiquitin proteasome system (UPS). In this study, we show that p97 negatively regulates NRF2 through the canonical pathway by extracting ubiquitylated NRF2 from the KEAP1-CUL3 E3 complex, with the aid of the heterodimeric cofactor UFD1/NPL4 and the UBA-UBX-containing protein UBXN7, for efficient proteasomal degradation. Given the role of NRF2 in chemoresistance and the surging interest in p97 inhibitors to treat cancers, our results indicate that dual p97/NRF2 inhibitors may offer a more potent and long-term avenue of p97-targeted treatment.
• Tillotson, J., Kedzior, M., Guimarães, L., Ross, A. B., Peters, T. L., Ambrose, A. J., Schmidlin, C. J., Zhang, D. D., Costa-Lotufo, L. V., Rodríguez, A. D., Schatz, J. H., & Chapman, E. (2017). ATP-competitive, marine derived natural products that target the DEAD box helicase, eIF4A. Bioorganic & medicinal chemistry letters, 27(17), 4082-4085.
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  Activation of translation initiation is a common trait of cancer cells. Formation of the heterotrimeric eukaryotic initiation factor F (eIF4F) complex is the rate-limiting step in 5' m7GpppN cap-dependent translation. This trimeric complex includes the eIF4E cap binding protein, the eIF4G scaffolding protein, and the DEAD box RNA helicase eIF4A. eIF4A is an ATP-dependent helicase and because it is the only enzyme in the eIF4F complex, it has been shown to be a potential therapeutic target for a variety of malignancies. To this end, we have used a simple ATPase biochemical screen to survey several hundred marine and terrestrial derived natural products. Herein, we report the discovery of two natural products from marine sources, elisabatin A (1) and allolaurinterol (2), which show low µM inhibition of eIF4A ATPase activity. Enzymological analyses revealed 1 and 2 to be ATP-competitive, and cellular evaluations showed reasonable cytotoxicity against A549 (lung cancer) and MDA-MA-468 (breast cancer) cell lines. However, only compound 2 showed potent inhibition of helicase activity congruent with its ATPase inhibitory activity.
• Tillotson, J., Zerio, C. J., Harder, B., Ambrose, A. J., Jung, K. S., Kang, M., Zhang, D. D., & Chapman, E. (2017). Arsenic Compromises Both p97 and Proteasome Functions. Chemical research in toxicology, 30(7), 1508-1514.
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  Exposure to arsenic is a worldwide problem that affects more than 200 million people. The underlying mechanisms of arsenic toxicity have been difficult to ascertain due to arsenic's pleotropic effects. A number of recent investigations have shown that arsenic can compromise protein quality control through the ubiquitin proteasome system (UPS) or the endoplasmic reticulum associated protein degradation (ERAD) pathway. In this article, a link between arsenic and protein quality control is reported. Biochemical and cellular data demonstrate a misregulation of the ATPase cycle of the ATPase associated with various cellular activities (AAA+) chaperone, p97. Interestingly, the loss of p97 activity is due to the increased rate of ATP hydrolysis, which mimics a collection of pathogenic genetic p97 lesions. Cellular studies, using a well characterized reporter of both the proteasome and p97, show the proteasome to also be compromised. This loss of both p97 and proteasome functions can explain the catastrophic protein quality control issues observed in acute, high level arsenic exposures.
• Abdeen, S., Salim, N., Mammadova, N., Summers, C. M., Frankson, R., Ambrose, A. J., Anderson, G. G., Schultz, P. G., Horwich, A. L., Chapman, E., & Johnson, S. M. (2016). GroEL/ES inhibitors as potential antibiotics. Bioorganic & medicinal chemistry letters, 26(13), 3127-3134.
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  We recently reported results from a high-throughput screening effort that identified 235 inhibitors of the Escherichia coli GroEL/ES chaperonin system [Bioorg. Med. Chem. Lett.2014, 24, 786]. As the GroEL/ES chaperonin system is essential for growth under all conditions, we reasoned that targeting GroEL/ES with small molecule inhibitors could be a viable antibacterial strategy. Extending from our initial screen, we report here the antibacterial activities of 22 GroEL/ES inhibitors against a panel of Gram-positive and Gram-negative bacteria, including E. coli, Bacillus subtilis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. GroEL/ES inhibitors were more effective at blocking the proliferation of Gram-positive bacteria, in particular S. aureus, where lead compounds exhibited antibiotic effects from the low-μM to mid-nM range. While several compounds inhibited the human HSP60/10 refolding cycle, some were able to selectively target the bacterial GroEL/ES system. Despite inhibiting HSP60/10, many compounds exhibited low to no cytotoxicity against human liver and kidney cell lines. Two lead candidates emerged from the panel, compounds 8 and 18, that exhibit >50-fold selectivity for inhibiting S. aureus growth compared to liver or kidney cell cytotoxicity. Compounds 8 and 18 inhibited drug-sensitive and methicillin-resistant S. aureus strains with potencies comparable to vancomycin, daptomycin, and streptomycin, and are promising candidates to explore for validating the GroEL/ES chaperonin system as a viable antibiotic target.
• Abdeen, S., Salim, N., Mammadova, N., Summers, C. M., Goldsmith-Pestana, K., McMahon-Pratt, D., Schultz, P. G., Horwich, A. L., Chapman, E., & Johnson, S. M. (2016). Targeting the HSP60/10 chaperonin systems of Trypanosoma brucei as a strategy for treating African sleeping sickness. Bioorganic & medicinal chemistry letters, 26(21), 5247-5253.
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  Trypanosoma brucei are protozoan parasites that cause African sleeping sickness in humans (also known as Human African Trypanosomiasis-HAT). Without treatment, T. brucei infections are fatal. There is an urgent need for new therapeutic strategies as current drugs are toxic, have complex treatment regimens, and are becoming less effective owing to rising antibiotic resistance in parasites. We hypothesize that targeting the HSP60/10 chaperonin systems in T. brucei is a viable anti-trypanosomal strategy as parasites rely on these stress response elements for their development and survival. We recently discovered several hundred inhibitors of the prototypical HSP60/10 chaperonin system from Escherichia coli, termed GroEL/ES. One of the most potent GroEL/ES inhibitors we discovered was compound 1. While examining the PubChem database, we found that a related analog, 2e-p, exhibited cytotoxicity to Leishmania major promastigotes, which are trypanosomatids highly related to Trypanosoma brucei. Through initial counter-screening, we found that compounds 1 and 2e-p were also cytotoxic to Trypanosoma brucei parasites (EC50=7.9 and 3.1μM, respectively). These encouraging initial results prompted us to develop a library of inhibitor analogs and examine their anti-parasitic potential in vitro. Of the 49 new chaperonin inhibitors developed, 39% exhibit greater cytotoxicity to T. brucei parasites than parent compound 1. While many analogs exhibit moderate cytotoxicity to human liver and kidney cells, we identified molecular substructures to pursue for further medicinal chemistry optimization to increase the therapeutic windows of this novel class of chaperonin-targeting anti-parasitic candidates. An intriguing finding from this study is that suramin, the first-line drug for treating early stage T. brucei infections, is also a potent inhibitor of GroEL/ES and HSP60/10 chaperonin systems.
• Lee, T. C., Kang, M., Kim, C. H., Schultz, P. G., Chapman, E., & Deniz, A. A. (2016). Dual Unnatural Amino Acid Incorporation and Click-Chemistry Labeling to Enable Single-Molecule FRET Studies of p97 Folding. Chembiochem : a European journal of chemical biology, 17(11), 981-4.
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  Many cellular functions are critically dependent on the folding of complex multimeric proteins, such as p97, a hexameric multidomain AAA+ chaperone. Given the complex architecture of p97, single-molecule (sm) FRET would be a powerful tool for studying folding while avoiding ensemble averaging. However, dual site-specific labeling of such a large protein for smFRET is a significant challenge. Here, we address this issue by using bioorthogonal azide-alkyne chemistry to attach an smFRET dye pair to site-specifically incorporated unnatural amino acids, allowing us to generate p97 variants reporting on inter- or intradomain structural features. An initial proof-of-principle set of smFRET results demonstrated the strengths of this labeling method. Our results highlight this as a powerful tool for structural studies of p97 and other large protein machines.
• Tillotson, J., Bashyal, B. P., Kang, M., Shi, T., De La Cruz, F., Gunatilaka, A. A., & Chapman, E. (2016). Selective inhibition of p97 by chlorinated analogues of dehydrocurvularin. Organic & biomolecular chemistry, 14(25), 5918-21.
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  The ATPase p97 is a ubiquitin targeted segregase that uses the energy of ATP binding and hydrolysis to extract ubiquitylated substrates from biological membranes, from other proteins, or from protein complexes to carry out myriad tasks in eukaryotes. Increased p97 activity has been linked to a poor prognosis in cancer patients, making p97 an anti-neoplastic target. In the present study, we show that dehydrocurvularin (DHC) and its chlorinated variants are covalent inhibitors of p97, interfering with its ATPase activity. Interestingly, cellular studies revealed both DHC and its monochloro analogue interfere with both the proteasome and p97, whereas its dichloro analogue showed p97 specificity.
• Wijeratne, E. M., Gunaherath, G. M., Chapla, V. M., Tillotson, J., de la Cruz, F., Kang, M., U Ren, J. M., Araujo, A. R., Arnold, A. E., Chapman, E., & Gunatilaka, A. A. (2016). Oxaspirol B with p97 Inhibitory Activity and Other Oxaspirols from Lecythophora sp. FL1375 and FL1031, Endolichenic Fungi Inhabiting Parmotrema tinctorum and Cladonia evansii. Journal of natural products.
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  A new metabolite, oxaspirol D (4), together with oxaspirols B (2) and C (3) were isolated from Lecythophora sp. FL1375, an endolichenic fungus isolated from Parmotrema tinctorum, whereas Lecythophora sp. FL1031 inhabiting the lichen Cladonia evansii afforded oxaspirols A (1), B (2), and C (3). Of these, oxaspirol B (2) showed moderate p97 ATPase inhibitory activity. A detailed characterization of all oxaspirols was undertaken because structures proposed for known oxaspirols have involved incomplete assignments of NMR spectroscopic data leading only to their planar structures. Thus, the naturally occurring isomeric mixture (2a and 2b) of oxaspirol B was separated as their diacetates (5a and 5b) and the structures and absolute configurations of 1, 2a, 2b, 3, and 4 were determined by the application of spectroscopic techniques including two-dimensional NMR and the modified Mosher's ester method. Oxaspirol B (2) and its diacetates 5a and 5b were evaluated for their ATPase inhibitory activities of p97, p97 mutants, and other ATP-utilizing enzymes, and only 2 was found to be active, indicating the requirement of some structural features in oxaspirols for their activity. Additional biochemical and cellular assays suggested that 2 was a reversible, non-ATP competitive, and specific inhibitor of p97.
• de la Vega, M. R., Dodson, M., Chapman, E., & Zhang, D. D. (2016). NRF2-targeted therapeutics: New targets and modes of NRF2 regulation. Current opinion in toxicology, 1, 62-70.
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  Pharmacological activation of the transcription factor nuclear factor-erythroid derived 2-like 2 (NRF2), the key regulator of the cellular antioxidant response, has been recognized as a feasible strategy to reduce oxidative/electrophilic stress and prevent carcinogenesis or other chronic illnesses, such as diabetes and chronic kidney disease. In contrast, due to the discovery of the "dark side" of NRF2, where prolonged activation of NRF2 causes tissue damage, cancer progression, or chemoresistance, efforts have been devoted to identify inhibitors. Currently, only one NRF2 activator has been approved for use in the clinic, while no specific NRF2 inhibitors have been discovered. Future development of NRF2-targeted therapeutics should be based on our current understanding of the regulatory mechanisms of this protein. In addition to the KEAP1-dependent mechanisms, the recent discovery of other pathways involved in the degradation of NRF2 have opened up new possibilities for the development of safe and specific therapeutics. Here, we review available and putative NRF2-targeted therapeutics and discuss their modes of action as well as their potential for disease prevention and treatment.
• de la Vega, M. R., Dodson, M., Gross, C., Manzour, H., Lantz, R. C., Chapman, E., Wang, T., Black, S. M., Garcia, J. G., & Zhang, D. D. (2016). Role of Nrf2 and Autophagy in Acute Lung Injury. Current pharmacology reports, 2(2), 91-101.
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  Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are the clinical manifestations of severe lung damage and respiratory failure. Characterized by severe inflammation and compromised lung function, ALI/ARDS result in very high mortality of affected individuals. Currently, there are no effective treatments for ALI/ARDS, and ironically, therapies intended to aid patients (specifically mechanical ventilation, MV) may aggravate the symptoms. Key events contributing to the development of ALI/ARDS are: increased oxidative and proteotoxic stresses, unresolved inflammation, and compromised alveolar-capillary barrier function. Since the airways and lung tissues are constantly exposed to gaseous oxygen and airborne toxicants, the bronchial and alveolar epithelial cells are under higher oxidative stress than other tissues. Cellular protection against oxidative stress and xenobiotics is mainly conferred by Nrf2, a transcription factor that promotes the expression of genes that regulate oxidative stress, xenobiotic metabolism and excretion, inflammation, apoptosis, autophagy, and cellular bioenergetics. Numerous studies have demonstrated the importance of Nrf2 activation in the protection against ALI/ARDS, as pharmacological activation of Nrf2 prevents the occurrence or mitigates the severity of ALI/ARDS. Another promising new therapeutic strategy in the prevention and treatment of ALI/ARDS is the activation of autophagy, a bulk protein and organelle degradation pathway. In this review, we will discuss the strategy of concerted activation of Nrf2 and autophagy as a preventive and therapeutic intervention to ameliorate ALI/ARDS.
• Ambrose, A. J., Fenton, W., Mason, D. J., Chapman, E., & Horwich, A. L. (2015). Unfolded DapA forms aggregates when diluted into free solution, confounding comparison with folding by the GroEL/GroES chaperonin system. FEBS letters, 589(4), 497-9.
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  A recent hydrogen-deuterium exchange study of folding of the GroEL/GroES-dependent bacterial enzyme DapA has suggested that the DapA folding pathway when free in solution may differ from the folding pathway used in the presence of the GroEL/GroES chaperonin. Here, we have investigated whether DapA aggregation might be occurring in free solution under the conditions of the exchange experiment, as this would confound interpretation of the pathway predictions. Dynamic light scattering (DLS) data, sedimentation analysis and refolding yield indicate that significant aggregation occurs upon dilution of DapA from denaturant, bringing into question the earlier conclusion that different folding pathways occur in the absence and presence of the chaperonin system.
• Chapman, E., Maksim, N., de la Cruz, F., & La Clair, J. J. (2015). Inhibitors of the AAA+ chaperone p97. Molecules (Basel, Switzerland), 20(2), 3027-49.
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  It is remarkable that a pathway as ubiquitous as protein quality control can be targeted to treat cancer. Bortezomib, an inhibitor of the proteasome, was first approved by the US Food and Drug Administration (FDA) more than 10 years ago to treat refractory myeloma and later extended to lymphoma. Its use has increased the survival rate of myeloma patients by as much as three years. This success was followed with the recent accelerated approval of the natural product derived proteasome inhibitor carfilzomib (Kyprolis®), which is used to treat patients with bortezomib-resistant multiple myeloma. The success of these two drugs has validated protein quality control as a viable target to fight select cancers, but begs the question why are proteasome inhibitors limited to lymphoma and myeloma? More recently, these limitations have encouraged the search for additional targets within the protein quality control system that might offer heightened cancer cell specificity, enhanced clinical utility, a lower rate of resistance, reduced toxicity, and mitigated side effects. One promising target is p97, an ATPase associated with various cellular activities (AAA+) chaperone. p97 figures prominently in protein quality control as well as serving a variety of other cellular functions associated with cancer. More than a decade ago, it was determined that up-regulation of p97 in many forms of cancer correlates with a poor clinical outcome. Since these initial discoveries, a mechanistic explanation for this observation has been partially illuminated, but details are lacking. Understandably, given this clinical correlation, myriad roles within the cell, and its importance in protein quality control, p97 has emerged as a potential therapeutic target. This review provides an overview of efforts towards the discovery of small molecule inhibitors of p97, offering a synopsis of efforts that parallel the excellent reviews that currently exist on p97 structure, function, and physiology.
• Harder, B., Jiang, T., Wu, T., Tao, S., de la Vega, M. R., Tian, W., Chapman, E., & Zhang, D. D. (2015). Molecular mechanisms of Nrf2 regulation and how these influence chemical modulation for disease intervention. Biochemical Society transactions, 43(4), 680-6.
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  Nrf2 (nuclear factor erytheroid-derived-2-like 2) transcriptional programmes are activated by a variety of cellular stress conditions to maintain cellular homoeostasis. Under non-stress conditions, Nrf2 is under tight regulation by the ubiquitin proteasome system (UPS). Detailed mechanistic investigations have shown the Kelch-like ECH-associated protein 1 (Keap1)-cullin3 (Cul3)-ring-box1 (Rbx1) E3-ligase to be the primary Nrf2 regulatory system. Recently, both beta-transducin repeat-containing E3 ubiquitin protein ligase (β-TrCP) and E3 ubiquitin-protein ligase synoviolin (Hrd1) have been identified as novel E3 ubiquitin ligases that negatively regulate Nrf2 through Keap1-independent mechanisms. In addition to UPS-mediated regulation of Nrf2, investigations have revealed a cross-talk between Nrf2 and the autophagic pathway resulting in activation of Nrf2 in a non-canonical manner. In addition to regulation at the protein level, Nrf2 was recently shown to be regulated at the transcriptional level by oncogenic K-rat sarcoma (Ras). A consequence of these differential regulatory mechanisms is the dual role of Nrf2 in cancer: the canonical, protective role and the non-canonical 'dark-side' of Nrf2. Based on the protective role of Nrf2, a vast effort has been dedicated towards identifying novel chemical inducers of Nrf2 for the purpose of chemoprevention. On the other hand, upon malignant transformation, some cancer cells have a constitutively high level of Nrf2 offering a growth advantage, as well as rendering cancer cells resistant to chemotherapeutics. This discovery has led to a new paradigm in cancer treatment; the initially counterintuitive use of Nrf2 inhibitors as adjuvants in chemotherapy. Herein, we will discuss the mechanisms of Nrf2 regulation and how this detailed molecular understanding can be leveraged to develop Nrf2 modulators to prevent diseases, mitigate disease progression or overcome chemoresistance.
• Jiang, T., Harder, B., Rojo de la Vega, M., Wong, P. K., Chapman, E., & Zhang, D. D. (2015). p62 links autophagy and Nrf2 signaling. Free radical biology & medicine, 88(Pt B), 199-204.
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  The Nrf2-Keap1-ARE pathway is a redox and xenobiotic sensitive signaling axis that functions to protect cells against oxidative stress, environmental toxicants, and harmful chemicals through the induction of cytoprotective genes. To enforce strict regulation, cells invest a great deal of energy into the maintenance of the Nrf2 pathway to ensure rapid induction upon cellular insult and rapid return to basal levels once the insult is mitigated. Because of the protective role of Nrf2 transcriptional programs, controlled activation of the pathway has been recognized as a means for chemoprevention. On the other hand, constitutive activation of Nrf2, due to somatic mutations of genes that control Nrf2 degradation, promotes carcinogenesis and imparts chemoresistance to cancer cells. Autophagy, a bulk protein degradation process, is another tightly regulated complex cellular process that functions as a cellular quality control system to remove damaged proteins or organelles. Low cellular nutrient levels can also activate autophagy, which acts to restore metabolic homeostasis through the degradation of macromolecules to provide nutrients. Recently, these two cellular pathways were shown to intersect through the direct interaction between p62 (an autophagy adaptor protein) and Keap1 (the Nrf2 substrate adaptor for the Cul3 E3 ubiquitin ligase). Dysregulation of autophagy was shown to result in prolonged Nrf2 activation in a p62-dependent manner. In this review, we will discuss the progress that has been made in dissecting the intersection of these two pathways and the potential tumor-promoting role of prolonged Nrf2 activation.
• Lau, E. C., Mason, D. J., Eichhorst, N., Engelder, P., Mesa, C., Kithsiri Wijeratne, E. M., Gunaherath, G. M., Gunatilaka, A. A., La Clair, J. J., & Chapman, E. (2015). Functional chromatographic technique for natural product isolation. Organic & biomolecular chemistry, 13(8), 2255-9.
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  Natural product discovery arises through a unique interplay between chromatographic purification and biological assays. Currently, most techniques used for natural product purification deliver leads without a defined biological action. We now describe a technique, referred to herein as functional chromatography, that deploys biological affinity as the matrix for compound isolation.
• Shen, T., Jiang, T., Long, M., Chen, J., Ren, D., Wong, P. K., Chapman, E., Zhou, B., & Zhang, D. D. (2015). A Curcumin Derivative That Inhibits Vinyl Carbamate-Induced Lung Carcinogenesis via Activation of the Nrf2 Protective Response. Antioxidants & redox signaling, 23(8), 651-64.
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  Lung cancer has a high worldwide morbidity and mortality. The employment of chemopreventive agents is effective to reduce lung cancer. Nuclear factor erythroid 2-related factor 2 (Nrf2) mitigates insults from both exogenous and endogenous sources and thus has been verified as a target for chemoprevention. Curcumin has long been recognized as a chemopreventive agent, but poor bioavailability and weak Nrf2 induction have prohibited clinical application. Thus, we have developed new curcumin derivatives and tested their Nrf2 induction.
• Tao, S., Tillotson, J., Wijeratne, E. M., Xu, Y., Kang, M., Wu, T., Lau, E. C., Mesa, C., Mason, D. J., Brown, R. V., La Clair, J. J., Gunatilaka, A. A., Zhang, D. D., & Chapman, E. (2015). Withaferin A Analogs That Target the AAA+ Chaperone p97. ACS chemical biology, 10(8), 1916-24.
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  Understanding the mode of action (MOA) of many natural products can be puzzling with mechanistic clues that seem to lack a common thread. One such puzzle lies in the evaluation of the antitumor properties of the natural product withaferin A (WFA). A variety of seemingly unrelated pathways have been identified to explain its activity, suggesting a lack of selectivity. We now show that WFA acts as an inhibitor of the chaperone, p97, both in vitro and in cell models in addition to inhibiting the proteasome in vitro. Through medicinal chemistry, we have refined the activity of WFA toward p97 and away from the proteasome. Subsequent studies indicated that these WFA analogs retained p97 activity and cytostatic activity in cell models, suggesting that the modes of action reported for WFA could be connected by proteostasis modulation. Through this endeavor, we highlight how the parallel integration of medicinal chemistry with chemical biology offers a potent solution to one of natures' intriguing molecular puzzles.
• Álvarez-Micó, X., Rocha, D. D., Guimarães, L. A., Ambrose, A., Chapman, E., Costa-Lotufo, L. V., La Clair, J. J., & Fenical, W. (2015). The Hybrid Pyrroloisoindolone-Dehydropyrrolizine Alkaloid (-)-Chlorizidine A Targets Proteins within the Glycolytic Pathway. Chembiochem : a European journal of chemical biology, 16(14), 2002-6.
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  The cytotoxic activity of (-)-chlorizidine A, a marine alkaloid containing a unique fusion between a pyrroloisoindolone and dehydropyrrolizine, was explored by using a combination of cellular and molecular methods. Our studies began by applying preliminary SAR evidence gathered from semisynthetic bioactivity evaluations to prepare an active immunoaffinity fluorescent (IAF) probe. This probe was then used to identify two cytosolic proteins, GAPDH and hENO1, as the targets of (-)-chlorizidine A.
• Johnson, S. M., Sharif, O., Mak, P. A., Wang, H., Engels, I. H., Brinker, A., Schultz, P. G., Horwich, A. L., & Chapman, E. (2014). A biochemical screen for GroEL/GroES inhibitors. Bioorganic & medicinal chemistry letters, 24(3), 786-9.
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  High-throughput screening of 700,000 small molecules has identified 235 inhibitors of the GroEL/GroES-mediated refolding cycle. Dose-response analysis of a subset of these hits revealed that 21 compounds are potent inhibitors of GroEL/GroES-mediated refolding (IC50
• Johnson, S. M., Sharif, O., Mak, P. A., Wang, H., Engels, I. H., Brinker, A., Schultz, P. G., Horwich, A. L., & Chapman, E. (2014). A biochemical screen for GroEL/GroES inhibitors. Bioorganic and Medicinal Chemistry Letters, 24(3), 786-789.
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  Abstract: High-throughput screening of 700,000 small molecules has identified 235 inhibitors of the GroEL/GroES-mediated refolding cycle. Dose-response analysis of a subset of these hits revealed that 21 compounds are potent inhibitors of GroEL/GroES-mediated refolding (IC50
• Kang, M. J., Wu, T., Wijeratne, E. M., Lau, E. C., Mason, D. J., Mesa, C., Tillotson, J., Zhang, D. D., Gunatilaka, A. A., La Clair, J. J., & Chapman, E. (2014). Functional chromatography reveals three natural products that target the same protein with distinct mechanisms of action. Chembiochem : a European journal of chemical biology, 15(14), 2125-31.
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  Access to lead compounds with defined molecular targets continues to be a barrier to the translation of natural product resources. As a solution, we developed a system that uses discrete, recombinant proteins as the vehicles for natural product isolation. Here, we describe the use of this functional chromatographic method to identify natural products that bind to the AAA+ chaperone, p97, a promising cancer target. Application of this method to a panel of fungal and plant extracts identified rheoemodin, 1-hydroxydehydroherbarin, and phomapyrrolidone A as distinct p97 modulators. Excitingly, each of these molecules displayed a unique mechanism of p97 modulation. This discovery provides strong support for the application of functional chromatography to the discovery of protein modulators that would likely escape traditional high-throughput or phenotypic screening platforms.
• La Clair, J. J., Loveridge, S. T., Tenney, K., O'Neil-Johnson, M., Chapman, E., & Crews, P. (2014). In situ natural product discovery via an artificial marine sponge. PloS one, 9(7), e100474.
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  There is continuing international interest in exploring and developing the therapeutic potential of marine-derived small molecules. Balancing the strategies for ocean based sampling of source organisms versus the potential to endanger fragile ecosystems poses a substantial challenge. In order to mitigate such environmental impacts, we have developed a deployable artificial sponge. This report provides details on its design followed by evidence that it faithfully recapitulates traditional natural product collection protocols. Retrieving this artificial sponge from a tropical ecosystem after deployment for 320 hours afforded three actin-targeting jasplakinolide depsipeptides that had been discovered two decades earlier using traditional sponge specimen collection and isolation procedures. The successful outcome achieved here could reinvigorate marine natural products research, by producing new environmentally innocuous sources of natural products and providing a means to probe the true biosynthetic origins of complex marine-derived scaffolds.
• Tao, S., Wang, S., Moghaddam, S. J., Ooi, A., Chapman, E., Wong, P. K., & Zhang, D. D. (2014). Oncogenic KRAS confers chemoresistance by upregulating NRF2. Cancer research, 74(24), 7430-41.
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  Oncogenic KRAS mutations found in 20% to 30% of all non-small cell lung cancers (NSCLC) are associated with chemoresistance and poor prognosis. Here we demonstrate that activation of the cell protective stress response gene NRF2 by KRAS is responsible for its ability to promote drug resistance. RNAi-mediated silencing of NRF2 was sufficient to reverse resistance to cisplatin elicited by ectopic expression of oncogenic KRAS in NSCLC cells. Mechanistically, KRAS increased NRF2 gene transcription through a TPA response element (TRE) located in a regulatory region in exon 1 of NRF2. In a mouse model of mutant KrasG12D-induced lung cancer, we found that suppressing the NRF2 pathway with the chemical inhibitor brusatol enhanced the antitumor efficacy of cisplatin. Cotreatment reduced tumor burden and improved survival. Our findings illuminate the mechanistic details of KRAS-mediated drug resistance and provide a preclinical rationale to improve the management of lung tumors harboring KRAS mutations with NRF2 pathway inhibitors.
• Wu, T., Zhao, F., Gao, B., Tan, C., Yagishita, N., Nakajima, T., Wong, P. K., Chapman, E., Fang, D., & Zhang, D. D. (2014). Hrd1 suppresses Nrf2-mediated cellular protection during liver cirrhosis. Genes & development, 28(7), 708-22.
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  Increased endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) are the salient features of end-stage liver diseases. Using liver tissues from liver cirrhosis patients, we observed up-regulation of the XBP1-Hrd1 arm of the ER stress response pathway and down-regulation of the Nrf2-mediated antioxidant response pathway. We further confirmed this negative regulation of Nrf2 by Hrd1 using Hrd1 conditional knockout mice. Down-regulation of Nrf2 was a surprising result, since the high levels of ROS should have inactivated Keap1, the primary ubiquitin ligase regulating Nrf2 levels. Here, we identified Hrd1 as a novel E3 ubiquitin ligase responsible for compromised Nrf2 response during liver cirrhosis. In cirrhotic livers, activation of the XBP1-Hrd1 arm of ER stress transcriptionally up-regulated Hrd1, resulting in enhanced Nrf2 ubiquitylation and degradation and attenuation of the Nrf2 signaling pathway. Our study reveals not only the convergence of ER and oxidative stress response pathways but also the pathological importance of this cross-talk in liver cirrhosis. Finally, we showed the therapeutic importance of targeting Hrd1, rather than Keap1, to prevent Nrf2 loss and suppress liver cirrhosis.
• Rothmann, M., Kang, M., Villa, R., Ntai, I., J., J., Kelleher, N. L., Chapman, E., & Burkart, M. D. (2013). Metabolic perturbation of an essential pathway: Evaluation of a glycine precursor of coenzyme A. Journal of the American Chemical Society, 135(16), 5962-5965.
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  PMID: 23550886;PMCID: PMC3657554;Abstract: Pantetheine and its corresponding disulfide pantethine play a key role in metabolism as building blocks of coenzyme A (CoA), an essential cofactor utilized in ∼4% of primary metabolism and central to fatty acid, polyketide, and nonribosomal peptide synthases. Using a combination of recombinant engineering and chemical synthesis, we show that the disulfide of N-pantoylglycyl-2-aminoethanethiol (GlyPan), with one fewer carbon than pantetheine, can rescue a mutant E. coli strain MG1655ΔpanC lacking a functional pantothenate synthetase. Using mass spectrometry, we show that the GlyPan variant is accepted by the downstream CoA biosynthetic machinery, ultimately being incorporated into essential acyl carrier proteins. These findings point to further flexibility in CoA-dependent pathways and offer the opportunity to incorporate orthogonal analogues. © 2013 American Chemical Society.
• Villeneuve, N. F., Tian, W., Tongde, W. u., Sun, Z., Lau, A., Chapman, E., Fang, D., & Zhang, D. D. (2013). USP15 negatively regulates Nrf2 through Deubiquitination of Keap1. Molecular Cell, 51(1), 68-79.
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  PMID: 23727018;Abstract: Nrf2 is a master regulator of the antioxidant response. Under basal conditions, Nrf2 is polyubiquitinated by the Keap1-Cul3 E3 ligase and degraded by the 26S proteasome. In response to Nrf2 inducers there is a switch in polyubiquitination from Nrf2 to Keap1. Currently, regulation of the Nrf2-Keap1 pathway by ubiquitination is largely understood. However, the mechanism responsible for removal of ubiquitin conjugated to Nrf2 or Keap1 remains unknown. Here we report that the deubiquitinating enzyme, USP15, specifically deubiquitinates Keap1, which suppresses the Nrf2 pathway. We demonstrated that deubiquitinated Keap1 incorporates into the Keap1-Cul3-E3 ligase complex more efficiently, enhancing the complex stability and enzymatic activity. Consequently, there is an increase in Nrf2 protein degradation and a reduction in Nrf2 target gene expression. Furthermore, USP15-siRNA enhances chemoresistance of cells through upregulation of Nrf2. These findings further our understanding of how the Nrf2-Keap1 pathway is regulated, which is imperative in targeting this pathway for chemoprevention or chemotherapy. © 2013 Elsevier Inc.
• Villeneuve, N. F., Tian, W., Wu, T., Sun, Z., Lau, A., Chapman, E., Fang, D., & Zhang, D. D. (2013). USP15 negatively regulates Nrf2 through deubiquitination of Keap1. Molecular cell, 51(1), 68-79.
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  Nrf2 is a master regulator of the antioxidant response. Under basal conditions, Nrf2 is polyubiquitinated by the Keap1-Cul3 E3 ligase and degraded by the 26S proteasome. In response to Nrf2 inducers there is a switch in polyubiquitination from Nrf2 to Keap1. Currently, regulation of the Nrf2-Keap1 pathway by ubiquitination is largely understood. However, the mechanism responsible for removal of ubiquitin conjugated to Nrf2 or Keap1 remains unknown. Here we report that the deubiquitinating enzyme, USP15, specifically deubiquitinates Keap1, which suppresses the Nrf2 pathway. We demonstrated that deubiquitinated Keap1 incorporates into the Keap1-Cul3-E3 ligase complex more efficiently, enhancing the complex stability and enzymatic activity. Consequently, there is an increase in Nrf2 protein degradation and a reduction in Nrf2 target gene expression. Furthermore, USP15-siRNA enhances chemoresistance of cells through upregulation of Nrf2. These findings further our understanding of how the Nrf2-Keap1 pathway is regulated, which is imperative in targeting this pathway for chemoprevention or chemotherapy.
• Chapman, E., & Hanson, S. R. (2011). Sulfotransferases and sulfatases: Sulfate modification of carbohydrates. Carbohydrate-Modifying Biocatalysts, 329-396.
• Chapman, E., Fry, A. N., & Kang, M. (2011). The complexities of p97 function in health and disease. Molecular BioSystems, 7(3), 700-710.
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  PMID: 21152665;PMCID: PMC3050576;Abstract: p97 is a homohexameric, toroidal machine that harnesses the energy of ATP binding and hydrolysis to effect structural reorganization of a diverse and primarily uncharacterized set of substrate proteins. This action has been linked to endoplasmic reticulum associated degradation (ERAD), homotypic membrane fusion, transcription factor control, cell cycle progression, DNA repair, and post-mitotic spindle disassembly. Exactly how these diverse processes use p97 is not fully understood, but it is clear that binding sites, primarily on the N-and C-domains of p97, facilitate this diversity by coordinating a growing collection of cofactors. These cofactors act at the levels of mechanism, sub-cellular localization, and substrate modification. Another unifying theme is the use of ubiquitylation. Both p97 and many of the associated cofactors have demonstrable ubiquitin-binding competence. The present review will discuss some of the current mechanistic studies and controversies and how these relate to cofactors as well as discussing potential therapeutic targeting of p97. © The Royal Society of Chemistry 2011.
• Chapman, E., Fry, A. N., & Kang, M. (2011). The complexities of p97 function in health and disease. Molecular bioSystems, 7(3), 700-10.
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  p97 is a homohexameric, toroidal machine that harnesses the energy of ATP binding and hydrolysis to effect structural reorganization of a diverse and primarily uncharacterized set of substrate proteins. This action has been linked to endoplasmic reticulum associated degradation (ERAD), homotypic membrane fusion, transcription factor control, cell cycle progression, DNA repair, and post-mitotic spindle disassembly. Exactly how these diverse processes use p97 is not fully understood, but it is clear that binding sites, primarily on the N- and C-domains of p97, facilitate this diversity by coordinating a growing collection of cofactors. These cofactors act at the levels of mechanism, sub-cellular localization, and substrate modification. Another unifying theme is the use of ubiquitylation. Both p97 and many of the associated cofactors have demonstrable ubiquitin-binding competence. The present review will discuss some of the current mechanistic studies and controversies and how these relate to cofactors as well as discussing potential therapeutic targeting of p97.
• Charbon, G., Brustad, E., Scott, K. A., Wang, J., Løbner-Olesen, A., Schultz, P. G., Jacobs-Wagner, C., & Chapman, E. (2011). Subcellular Protein Localization by Using a Genetically Encoded Fluorescent Amino Acid. ChemBioChem, 12(12), 1818-1821.
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  PMID: 21681882;PMCID: PMC3175735;
• Charbon, G., Brustad, E., Scott, K. A., Wang, J., Løbner-Olesen, A., Schultz, P. G., Jacobs-Wagner, C., & Chapman, E. (2011). Subcellular protein localization by using a genetically encoded fluorescent amino acid. Chembiochem : a European journal of chemical biology, 12(12), 1818-21.
• Charbon, G., Wang, J., Brustad, E., Schultz, P. G., Horwich, A. L., Jacobs-Wagner, C., & Chapman, E. (2011). Localization of GroEL determined by in vivo incorporation of a fluorescent amino acid. Bioorganic & medicinal chemistry letters, 21(20), 6067-70.
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  The molecular chaperone GroEL is required for bacterial growth under all conditions, mediating folding assistance, via its central cavity, to a diverse set of cytosolic proteins; yet the subcellular localization of GroEL remains unresolved. An earlier study, using antibody probing of fixed Escherichia coli cells, indicated colocalization with the cell division protein FtsZ at the cleavage furrow, while a second E. coli study of fixed cells indicated more even distribution throughout the cytoplasm. Here, for the first time, we have examined the spatial distribution of GroEL in living cells using incorporation of a fluorescent unnatural amino acid into the chaperone. Fluorescence microscopy indicated that GroEL is diffusely distributed, both under normal and stress conditions. Importantly, the present procedure uses a small, fluorescent unnatural amino acid to visualize GroEL in vivo, avoiding the steric demands of a fluorescent protein fusion, which compromises proper GroEL assembly. Further, this unnatural amino acid incorporation avoids artifacts that can occur with fixation and antibody staining.
• Charbon, G., Wang, J., Brustad, E., Schultz, P. G., Horwich, A. L., Jacobs-Wagner, C., & Chapman, E. (2011). Localization of GroEL determined by in vivo incorporation of a fluorescent amino acid. Bioorganic and Medicinal Chemistry Letters, 21(20), 6067-6070.
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  PMID: 21890355;PMCID: PMC3177974;Abstract: The molecular chaperone GroEL is required for bacterial growth under all conditions, mediating folding assistance, via its central cavity, to a diverse set of cytosolic proteins; yet the subcellular localization of GroEL remains unresolved. An earlier study, using antibody probing of fixed Escherichia coli cells, indicated colocalization with the cell division protein FtsZ at the cleavage furrow, while a second E. coli study of fixed cells indicated more even distribution throughout the cytoplasm. Here, for the first time, we have examined the spatial distribution of GroEL in living cells using incorporation of a fluorescent unnatural amino acid into the chaperone. Fluorescence microscopy indicated that GroEL is diffusely distributed, both under normal and stress conditions. Importantly, the present procedure uses a small, fluorescent unnatural amino acid to visualize GroEL in vivo, avoiding the steric demands of a fluorescent protein fusion, which compromises proper GroEL assembly. Further, this unnatural amino acid incorporation avoids artifacts that can occur with fixation and antibody staining. © 2011 Elsevier Ltd. All rights reserved.
• Chapman, E., Farr, G. W., Furtak, K., & Horwich, A. L. (2009). A small molecule inhibitor selective for a variant ATP-binding site of the chaperonin GroEL. Bioorganic & medicinal chemistry letters, 19(3), 811-3.
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  The chaperonin GroEL is a megadalton-sized molecular machine that plays an essential role in the bacterial cell assisting protein folding to the native state through actions requiring ATP binding and hydrolysis. A combination of medicinal chemistry and genetics has been employed to generate an orthogonal pair, a small molecule that selectively inhibits ATPase activity of a GroEL ATP-binding pocket variant. An initial screen of kinase-directed inhibitors identified an active pyrazolo-pyrimidine scaffold that was iteratively modified and screened against a collective of GroEL nucleotide pocket variants to identify a cyclopentyl carboxamide derivative, EC3016, that specifically inhibits ATPase activity and protein folding by the GroEL mutant, I493C, involving a side chain positioned near the base of ATP. This orthogonal pair will enable in vitro studies of the action of ATP in triggering activation of GroEL-mediated protein folding and might enable further studies of GroEL action in vivo. The approach originated for studying kinases by Shokat and his colleagues may thus also be used to study large macromolecular machines.
• Chapman, E., Farr, G. W., Furtak, K., & Horwich, A. L. (2009). A small molecule inhibitor selective for a variant ATP-binding site of the chaperonin GroEL. Bioorganic and Medicinal Chemistry Letters, 19(3), 811-813.
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  PMID: 19110421;PMCID: PMC2633924;Abstract: The chaperonin GroEL is a megadalton-sized molecular machine that plays an essential role in the bacterial cell assisting protein folding to the native state through actions requiring ATP binding and hydrolysis. A combination of medicinal chemistry and genetics has been employed to generate an orthogonal pair, a small molecule that selectively inhibits ATPase activity of a GroEL ATP-binding pocket variant. An initial screen of kinase-directed inhibitors identified an active pyrazolo-pyrimidine scaffold that was iteratively modified and screened against a collective of GroEL nucleotide pocket variants to identify a cyclopentyl carboxamide derivative, EC3016, that specifically inhibits ATPase activity and protein folding by the GroEL mutant, I493C, involving a side chain positioned near the base of ATP. This orthogonal pair will enable in vitro studies of the action of ATP in triggering activation of GroEL-mediated protein folding and might enable further studies of GroEL action in vivo. The approach originated for studying kinases by Shokat and his colleagues may thus also be used to study large macromolecular machines. © 2008 Elsevier Ltd. All rights reserved.
• Chapman, E., Farr, G. W., Fenton, W. A., Johnson, S. M., & Horwich, A. L. (2008). Requirement for binding multiple ATPs to convert a GroEL ring to the folding-active state. Proceedings of the National Academy of Sciences of the United States of America, 105(49), 19205-10.
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  Production of the folding-active state of a GroEL ring involves initial cooperative binding of ATP, recruiting GroES, followed by large rigid body movements that are associated with ejection of bound substrate protein into the encapsulated hydrophilic chamber where folding commences. Here, we have addressed how many of the 7 subunits of a GroEL ring are required to bind ATP to drive these events, by using mixed rings with different numbers of wild-type and variant subunits, the latter bearing a substitution in the nucleotide pocket that allows specific block of ATP binding and turnover by a pyrazolol pyrimidine inhibitor. We observed that at least 2 wild-type subunits were required to bind GroES. By contrast, the triggering of polypeptide release and folding required a minimum of 4 wild-type subunits, with the greatest extent of refolding observed when all 7 subunits were wild type. This is consistent with the requirement for a "power stroke" of forceful apical movement to eject polypeptide into the chamber.
• Chapman, E., Farr, G. W., Fenton, W. A., Johnson, S. M., & Horwich, A. L. (2008). Requirement for binding multiple ATPs to convert a GroEL ring to the folding-active state. Proceedings of the National Academy of Sciences of the United States of America, 105(49), 19205-19210.
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  PMID: 19050077;PMCID: PMC2592988;Abstract: Production of the folding-active state of a GroEL ring involves initial cooperative binding of ATP, recruiting GroES, followed by large rigid body movements that are associated with ejection of bound substrate protein into the encapsulated hydrophilic chamber where folding commences. Here, we have addressed how many of the 7 subunits of a GroEL ring are required to bind ATP to drive these events, by using mixed rings with different numbers of wild-type and variant subunits, the latter bearing a substitution in the nucleotide pocket that allows specific block of ATP binding and turnover by a pyrazolol pyrimidine inhibitor. We observed that at least 2 wild-type subunits were required to bind GroES. By contrast, the triggering of polypeptide release and folding required a minimum of 4 wild-type subunits, with the greatest extent of refolding observed when all 7 subunits were wild type. This is consistent with the requirement for a "power stroke" of forceful apical movement to eject polypeptide into the chamber. © 2008 by The National Academy of Sciences of the USA.
• Horwich, A. L., Fenton, W. A., Chapman, E., & Farr, G. W. (2007). Two families of chaperonin: Physiology and mechanism. Annual Review of Cell and Developmental Biology, 23, 115-145.
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  PMID: 17489689;Abstract: Chaperonins are large ring assemblies that assist protein folding to the native state by binding nonnative proteins in their central cavities and then, upon binding ATP, release the substrate protein into a now-encapsulated cavity to fold productively. Two families of such components have been identified: type I in mitochondria, chloroplasts, and the bacterial cytosol, which rely on a detachable "lid" structure for encapsulation, and type II in archaea and the eukaryotic cytosol, which contain a built-in protrusion structure. We discuss here a number of issues under current study. What is the range of substrates acted on by the two classes of chaperonin, in particular by GroEL in the bacterial cytoplasm and CCT in the eukaryotic cytosol, and are all these substrates subject to encapsulation? What are the determinants for substrate binding by the type II chaperonins? And is the encapsulated chaperonin cavity a passive container that prevents aggregation, or could it be playing an active role in polypeptide folding? Copyright © 2007 by Annual Reviews. All rights reserved.
• Chapman, E., Farr, G. W., Usaite, R., Furtak, K., Fenton, W. A., Chaudhuri, T. K., Hondorp, E. R., Matthews, R. G., Wolf, S. G., Yates, J. R., Pypaert, M., & Horwich, A. L. (2006). Global aggregation of newly translated proteins in an Escherichia coli strain deficient of the chaperonin GroEL. Proceedings of the National Academy of Sciences of the United States of America, 103(43), 15800-15805.
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  PMID: 17043235;PMCID: PMC1613232;Abstract: In a newly isolated temperature-sensitive lethal Escherichia coli mutant affecting the chaperonin GroEL, we observed wholesale aggregation of newly translated proteins. After temperature shift, transcription, translation, and growth slowed over two to three generations, accompanied by filamentation and accretion (in ≈2% of cells) of paracrystalline arrays containing mutant chaperonin complex. A biochemically isolated inclusion body fraction contained the collective of abundant proteins of the bacterial cytoplasm as determined by SDS/PAGE and proteolysis/MS analyses. Pulse-chase experiments revealed that newly made proteins, but not preexistent ones, were recruited to this insoluble fraction. Although aggregation of "stringent" GroEL/GroES-dependent substrates may secondarily produce an "avalanche" of aggregation, the observations raise the possibility, supported by in vitro refolding experiments, that the widespread aggregation reflects that GroEL function supports the proper folding of a majority of newly translated polypeptides, not just the limited number indicated by interaction studies and in vitro experiments. © 2006 by The National Academy of Sciences of the USA.
• Best, M. D., Brik, A., Chapman, E., Lee, L. V., Cheng, W., & Wong, C. (2004). Rapid discovery of potent sulfotransferase inhibitors by diversity-oriented reaction in microplates followed by in situ screening. ChemBioChem, 5(6), 811-819.
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  PMID: 15174164;Abstract: Rapid diversity-oriented microplate library synthesis and in situ screening with a high-throughput fluorescence-based assay were used to develop potent inhibitors of β-arylsulfotransferase IV (β-AST-IV). This strategy leads to facile inhibitor synthesis and study as it allows protecting-group manipulation and product isolation from other library components to be avoided. Through repeated library formation, three aspects of inhibitor makeup, the identities of the two binding groups and the length of the linker between AKthem, were independently optimized. Several potent inhibitors were obtained, one of which was determined to have an inhibition constant K; of 5 nm. This compound is the most potent β-AST-IV inhibitor developed to date, with a K; value more than five orders of magnitude lower than the Michaelis constant Km for the substrate whose binding it inhibits.
• Chapman, E., Best, M. D., Hanson, S. R., & Wong, C. (2004). Sulfotransferases: Structure, mechanism, biological activity, inhibition, and synthetic utility. Angewandte Chemie - International Edition, 43(27), 3526-3548.
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  PMID: 15293241;Abstract: The sulfonation (also known as sulfurylation) of biomolecules has long been known to take place in a variety of organisms, from prokaryotes to multicellular species, and new biological functions continue to be uncovered in connection with this important transformation. Early studies of sulfotransferases (STs), the enzymes that catalyze sulfonation, focused primarily on the cytosolic STs, which are involved in detoxification, hormone regulation, and drug metabolism. Although known to exist, the membrane-associated STs were not studied as extensively until more recently. Involved in the sulfonation of complex carbohydrates and proteins, they have emerged as central players in a number of molecular-recognition events and biochemical signaling pathways. STs have also been implicated in many pathophysiological processes. As a result, much interest in the complex roles of STs and in their targeting for therapeutic intervention has been generated. Progress in the elucidation of the structures and mechanisms of sulfotransferases, as well as their biological activity, inhibition, and synthetic utility, are discussed in this Review.
• Chapman, E., Bryan, M. C., & Wong, C. (2003). Mechanistic studies of β-arylsulfotransferase IV. Proceedings of the National Academy of Sciences of the United States of America, 100(3), 910-915.
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  PMID: 12547913;PMCID: PMC298700;Abstract: Sulfotransferases are an important class of enzymes that catalyze the transfer of a sulfuryl group to a hydroxyl or amine moiety on various molecules including small-molecule drugs, steroids, hormones, carbohydrates, and proteins. They have been implicated in a number of disease states but remain poorly understood, complicating the design of specific, small-molecule inhibitors. A linear free-energy analysis in both the forward and reverse directions indicates that the transfer of a sulfuryl group to an aryl hydroxyl group catalyzed by β-arylsulfotransferase IV likely proceeds by a dissociative (sulfotrioxide-like) mechanism. Values for the Broønsted coefficients (βnuc and βlg) are +0.33 and -0.45, giving Leffler α values of 0.19 and 0.61 for the forward and reverse reactions, respectively.
• Wong, C., Bryan, M. C., Nyffeler, P. T., Liu, H., & Chapman, E. (2003). Synthesis of carbohydrate-based antibiotics. Pure and Applied Chemistry, 75(2-3), 179-186.
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  Abstract: Tackling the problem of carbohydrate recognition is a subject of current interest. Our strategies in this regard include development of new methods for the synthesis of saccharides and sugar arrays for screening, design of D-peptides to target bacterial cell-surface sugars, preparation of mechanism-based inhibitors of the enzymes involved in the biosynthesis of saccharides and the synthesis of aminoglycoside mimetics to target RNA.
• Chapman, E., & Wong, C. (2002). A pH sensitive colorometric assay for the high-throughput screening of enzyme inhibitors and substrates: A case study using kinases. Bioorganic and Medicinal Chemistry, 10(3), 551-555.
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  PMID: 11814841;Abstract: We have developed an uncoupled, pH sensitive kinase assay that can be used for high-throughput screening of potential inhibitors or for determining substrate specificity. Kinases catalyze the transfer of a γ-phosphoryl group from ATP to an appropriate hydroxyl acceptor with the release of a proton. This assay is based on the detection of this proton using an appropriately matched buffer/indicator system. The assay was used to measure the activity of four readily available kinases, including hexokinase, glucokinase, glycerokinase, and pyruvate kinase, which was run in the reverse direction. We also went on to screen a small series of mono- and diphosphonucleotides for inhibition of hexokinase as well as a modest set of potential hexokinase substrates. We determined sucrose to be a modest substrate for hexokinase with a Km) of 1.8±0.2 mM, a kcat of 142±3 min-1, and a Vmax that is 15% of that for glucose. Given the importance of kinases in a diverse array of biological functions and disease states, there is a need for a simple, rapid assay system. We feel this assay will lend itself well to meet this end. This method should be applicable to many other enzymatic reactions which involve a change in pH. © 2002 Elsevier Science Ltd. All rights reserved.
• Chapman, E., Ding, S., Schultz, P. G., & Wong, C. (2002). A potent and highly selective sulfotransferase inhibitor. Journal of the American Chemical Society, 124(49), 14524-14525.
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  PMID: 12465948;Abstract: In the present work, we have used a newly developed, fluorescence-based assay to screen a library of >30000 compounds as potential β-arylsulfotransferase-IV inhibitors. A total of 11 inhibitors were discovered. Most of the compounds discovered showed low micromolar inhibition, but one of the compounds showed potent inhibition (Ki = 96 nM). The most potent of these inhibitors was tested against a variety of other purine binding enzymes and showed remarkable specificity. Copyright © 2002 American Chemical Society.
• Burkart, M. D., Izumi, M., Chapman, E., Lin, C., & Wong, C. (2000). Regeneration of PAPS for the enzymatic synthesis of sulfated oligosaccharides. Journal of Organic Chemistry, 65(18), 5565-5574.
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  PMID: 10970295;Abstract: This paper describes the study of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) regeneration from 3'-phosphoadenosine-5'-phosphate (PAP) for use in practical syntheses of carbohydrate sulfates which are catalyzed by sulfotransferases. Among the regeneration systems, the one with recombinant aryl sulfotransferase proved to be the most practical. This regeneration system was coupled with a sulfotransferase-catalyzed reaction, using a recombinant Nod factor sulfotransferase, for the synthesis of various oligosaccharide sulfates that were further glycosylated using glycosyl-transferases.
• Chapman, E., Thorson, J. S., & Schultz, P. G. (1997). Mutational analysis of backbone hydrogen bonds in staphylococcal nuclease [2]. Journal of the American Chemical Society, 119(30), 7151-7152.
• Thorson, J. S., Chapman, E., & Schultz, P. G. (1995). Analysis of hydrogen bonding strengths in proteins using unnatural amino acids. Journal of the American Chemical Society, 117(36), 9361-9362.
• Thorson, J. S., Chapman, E., Murphy, E. C., Schultz, P. G., & Judice, J. K. (1995). Linear free energy analysis of hydrogen bonding in proteins. Journal of the American Chemical Society, 117(3), 1157-1158.

Presentations

• Chapman, E. (2019, September). Modulation of NRF2 in Chemoprevention and Cancer Therapy. Heidelberger Symposium on Cancer Research. Stintino, Sardinia, Italy.
• Chapman, E. (2019, June). Modulation of NRF2 in Chemoprevention and Cancer Therapy. Gordon Research Conference - High Throughput Chemistry and Chemical Biology. New London, New Hampshire: Gordon Research Conference.
• Chapman, E. (2019, September). Modulation of NRF2 in Chemoprevention and Cancer Therapy. The Environmental Response V. Sendai, Japan.

Poster Presentations

• Chapman, E. (2019, June). Modulation of NRF2 in Chemoprevention and Cancer Therapy. Gordon Research Conference - High Throughput Chemistry and Chemical Biology. New London, New Hampshire: Gordon Research Conference.

Others

• Chapman, E., Maksim, N., de la Cruz, F., & La Clair, J. J. (2015). Correction: Chapman, E.; et al. Inhibitors of the AAA+ chaperone p97. Molecules 2015, 20, 3027-3049. Molecules (Basel, Switzerland).