Targeting glutathione peroxidases : identifying environmental modulators, and screening for novel small molecule inhibitors

Glutathione peroxidases (GPXs) are a family of selenoproteins that are critical regulators of reactive oxygen species (ROS) in the cell, specifically hydroperoxides like hydrogen peroxide (H2O2). ROS are important for normal cell signaling and are tightly controlled to promote cell growth, proliferation, and survival. Without the antioxidant activity of enzymes like GPXs, however, the oxidative burden in cells can reach a point that leads to DNA damage, carcinogenesis, and eventually cell death. Although this can be catastrophic in early development, as evidenced by knock-out studies, many cancer therapeutics function through manipulating redox balance, suggesting that targeting these enzymes could have therapeutic potential. The modified metabolism of cancer cells can result in increased hydroperoxide production, and GPXs are often overexpressed to compensate for the increased oxidative stress, as well as in cell lines resistant to chemotherapeutics. The studies comprising this thesis examine several aspects of GPX inhibition to better understand how to utilize GPX-targeting agents as potential anti-cancer therapeutics and to identify novel inhibitors for future development. Paper I addresses the effects of environmental heavy metal exposures on the erythrocyte GPX activity in 9-year-old children from the Matlab region of Bangladesh. Samples from 100 children were initially analyzed for concentrations of selenium, arsenic, mercury, and cadmium, as well as C-reactive protein (CRP) using inductively coupled plasma mass spectrometry (ICPMS). GPX1 expression levels in lysed samples were measured using high-throughput immunoblotting, and total GPX activity was measured using a GPX activity assay in lysates. After finding only a slight positive correlation between GPX1 expression and GPX activity, trace elements and CRP levels were considered, and a multivariable-adjusted linear regression analyses was used to assess predictors of GPX activity. Arsenic and CRP levels were significantly negatively associated with active GPX, while not correlated with each other. These results suggest that independently both arsenic exposure and increased CRP levels due to inflammation can suppress GPX activity in erythrocytes. Paper II characterized the off-target inhibition of selenoprotein thioredoxin reductases (TXNRDs) by the ferroptosis inducers, (1S, 3R)RSL3 and ML162. Identified originally in a synthetic lethal screen for compounds specifically cytotoxic to oncogenic RAS, these compounds were then found to induce an iron-dependent cell death via increased lipid peroxidation associated with GPX4-specific inhibition. However, neither compound showed inhibitory activity in biochemical assays using recombinant GPX4, but both show potent inhibition of TXNRD1 in both biochemical and cellular assays. In three cell lines with varying susceptibility to ferroptosis, the cell death induced by RSL3 differed from the cell death caused by more specific TXNRD1 inhibitors, TRi-1 and TRi-2. Specifically, while RSL3 cytotoxicity could be rescued by co-treatment with the ferroptosis suppressor, Fer-1, the cytotoxicity of the Tri compounds was not rescued. Additionally, selenium supplementation diminished the efficacy of RSL3 while the TRi compounds remained unchanged, or slightly more cytotoxic. In all, these studies indicate that the interconnectedness of TXNRD1 and ferroptosis, and furthermore TXNRD1 and GPX4, is complex, but that this important off-target effect needs to be understood to fully characterize the use of these ferroptosis inducers. Paper III established a discovery pipeline for the identification of novel specific inhibitors of GPX1 and GPX4. GR-coupled activity assays using recombinant GPX1 and GPX4 were optimized and miniaturized to 1536-well formats and screened against 12,000 small molecules with annotated mechanisms of action. A suite of confirmational assays were used to ensure specificity: a GR counter-assay was used to identify false-positives in the primary screens; orthogonal endpoint GPX assays were used to confirm inhibitory activity; GPX2 assay was used to further probe specificity of the confirmed active compounds between isoforms; a TXNRD1 assay was used to differentiate small molecules with broad Sec-targeting activity from GPXspecific inhibition; and nano Differential Scanning Fluorimetry (DSF) was used to confirm direct binding. Interestingly, all GPX1 inhibitors identified showed crossinhibition of GPX2. Ultimately, five novel GPX1/GPX2 inhibitors, 13 GPX4 inhibitors, and 2 novel pan-GPX inhibitors. This series of assays and the resulting compounds identified provide a basis for future development of GPX-specific inhibitors. Paper IV profiled the cytotoxicity of a library of >10,000 small molecules with annotated mechanisms of action (MOA) and >100,000 small molecule scaffolds in a diversity library in both normal and cancer cell lines. These screens revealed a low overall cytotoxicity rate of the diversity library. Importantly, cytotoxicity was assessed in four normal cell lines (HEK293, immortalized human embryonic kidney cells; NIH3T3, an embryonic mouse fibroblast cells; HaCat, immortalized human keratinocytes; and CRL-7250, a primary human foreskin fibroblast cells). The top enriched MOA categories showing broad cell killing in normal cells were proteosome inhibitors, heat shock protein 90 (HSP90) inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, mammalian target of rapamycin (mTOR) inhibitors, and cyclindependent kinase (CDK) inhibitors. Cytotoxic compounds with specific activity against the human adenosarcoma cancer line, KB 3-1, that showed no activity in the normal cell lines were also highlighted. This work will be used as an additional triage step in lead-selection for chemotherapeutic development, removing compounds that show significant cytotoxicity in normal cell lines

Development of therapies for rare genetic disorders of GPX4: roadmap and opportunities

BACKGROUND: Extremely rare progressive diseases like Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD) can be neonatally lethal and therefore go undiagnosed or are difficult to treat. Recent sequencing efforts have linked this disease to mutations in GPX4, with consequences in the resulting enzyme, glutathione peroxidase 4. This offers potential diagnostic and therapeutic avenues for those suffering from this disease, though the steps toward these treatments is often convoluted, expensive, and time-consuming. MAIN BODY: The CureGPX4 organization was developed to promote awareness of GPX4-related diseases like SSMD, as well as support research that could lead to essential therapeutics for patients. We provide an overview of the 21 published SSMD cases and have compiled additional sequencing data for four previously unpublished individuals to illustrate the genetic component of SSMD, and the role of sequencing data in diagnosis. We outline in detail the steps CureGPX4 has taken to reach milestones of team creation, disease understanding, drug repurposing, and design of future studies. CONCLUSION: The primary aim of this review is to provide a roadmap for therapy development for rare, ultra-rare, and difficult to diagnose diseases, as well as increase awareness of the genetic component of SSMD. This work will offer a better understanding of GPx4-related diseases, and help guide researchers, clinicians, and patients interested in other rare diseases find a path towards treatments

Development of therapies for rare genetic disorders of GPX4: roadmap and opportunities

Background Extremely rare progressive diseases like Sedaghatian-type Spondylometaphyseal Dysplasia (SSMD) can be neonatally lethal and therefore go undiagnosed or are difficult to treat. Recent sequencing efforts have linked this disease to mutations in GPX4, with consequences in the resulting enzyme, glutathione peroxidase 4. This offers potential diagnostic and therapeutic avenues for those suffering from this disease, though the steps toward these treatments is often convoluted, expensive, and time-consuming. Main body The CureGPX4 organization was developed to promote awareness of GPX4-related diseases like SSMD, as well as support research that could lead to essential therapeutics for patients. We provide an overview of the 21 published SSMD cases and have compiled additional sequencing data for four previously unpublished individuals to illustrate the genetic component of SSMD, and the role of sequencing data in diagnosis. We outline in detail the steps CureGPX4 has taken to reach milestones of team creation, disease understanding, drug repurposing, and design of future studies. Conclusion The primary aim of this review is to provide a roadmap for therapy development for rare, ultra-rare, and difficult to diagnose diseases, as well as increase awareness of the genetic component of SSMD. This work will offer a better understanding of GPx4-related diseases, and help guide researchers, clinicians, and patients interested in other rare diseases find a path towards treatments

Canvass: A Crowd-Sourced, Natural-Product Screening Library for Exploring Biological Space

Natural products and their derivatives continue to be wellsprings of nascent therapeutic potential. However, many laboratories have limited resources for biological evaluation, leaving their previously isolated or synthesized compounds largely or completely untested. To address this issue, the Canvass library of natural products was assembled, in collaboration with academic and industry researchers, for quantitative high-throughput screening (qHTS) across a diverse set of cell-based and biochemical assays. Characterization of the library in terms of physicochemical properties, structural diversity, and similarity to compounds in publicly available libraries indicates that the Canvass library contains many structural elements in common with approved drugs. The assay data generated were analyzed using a variety of quality control metrics, and the resultant assay profiles were explored using statistical methods, such as clustering and compound promiscuity analyses. Individual compounds were then sorted by structural class and activity profiles. Differential behavior based on these classifications, as well as noteworthy activities, are outlined herein. One such highlight is the activity of (−)-2(S)-cathafoline, which was found to stabilize calcium levels in the endoplasmic reticulum. The workflow described here illustrates a pilot effort to broadly survey the biological potential of natural products by utilizing the power of automation and high-throughput screening

Canvass: a crowd-sourced, natural-product screening library for exploring biological space

NCATS thanks Dingyin Tao for assistance with compound characterization. This research was supported by the Intramural Research Program of the National Center for Advancing Translational Sciences, National Institutes of Health (NIH). R.B.A. acknowledges support from NSF (CHE-1665145) and NIH (GM126221). M.K.B. acknowledges support from NIH (5R01GM110131). N.Z.B. thanks support from NIGMS, NIH (R01GM114061). J.K.C. acknowledges support from NSF (CHE-1665331). J.C. acknowledges support from the Fogarty International Center, NIH (TW009872). P.A.C. acknowledges support from the National Cancer Institute (NCI), NIH (R01 CA158275), and the NIH/National Institute of Aging (P01 AG012411). N.K.G. acknowledges support from NSF (CHE-1464898). B.C.G. thanks the support of NSF (RUI: 213569), the Camille and Henry Dreyfus Foundation, and the Arnold and Mabel Beckman Foundation. C.C.H. thanks the start-up funds from the Scripps Institution of Oceanography for support. J.N.J. acknowledges support from NIH (GM 063557, GM 084333). A.D.K. thanks the support from NCI, NIH (P01CA125066). D.G.I.K. acknowledges support from the National Center for Complementary and Integrative Health (1 R01 AT008088) and the Fogarty International Center, NIH (U01 TW00313), and gratefully acknowledges courtesies extended by the Government of Madagascar (Ministere des Eaux et Forets). O.K. thanks NIH (R01GM071779) for financial support. T.J.M. acknowledges support from NIH (GM116952). S.M. acknowledges support from NIH (DA045884-01, DA046487-01, AA026949-01), the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program (W81XWH-17-1-0256), and NCI, NIH, through a Cancer Center Support Grant (P30 CA008748). K.N.M. thanks the California Department of Food and Agriculture Pierce's Disease and Glassy Winged Sharpshooter Board for support. B.T.M. thanks Michael Mullowney for his contribution in the isolation, elucidation, and submission of the compounds in this work. P.N. acknowledges support from NIH (R01 GM111476). L.E.O. acknowledges support from NIH (R01-HL25854, R01-GM30859, R0-1-NS-12389). L.E.B., J.K.S., and J.A.P. thank the NIH (R35 GM-118173, R24 GM-111625) for research support. F.R. thanks the American Lebanese Syrian Associated Charities (ALSAC) for financial support. I.S. thanks the University of Oklahoma Startup funds for support. J.T.S. acknowledges support from ACS PRF (53767-ND1) and NSF (CHE-1414298), and thanks Drs. Kellan N. Lamb and Michael J. Di Maso for their synthetic contribution. B.S. acknowledges support from NIH (CA78747, CA106150, GM114353, GM115575). W.S. acknowledges support from NIGMS, NIH (R15GM116032, P30 GM103450), and thanks the University of Arkansas for startup funds and the Arkansas Biosciences Institute (ABI) for seed money. C.R.J.S. acknowledges support from NIH (R01GM121656). D.S.T. thanks the support of NIH (T32 CA062948-Gudas) and PhRMA Foundation to A.L.V., NIH (P41 GM076267) to D.S.T., and CCSG NIH (P30 CA008748) to C.B. Thompson. R.E.T. acknowledges support from NIGMS, NIH (GM129465). R.J.T. thanks the American Cancer Society (RSG-12-253-01-CDD) and NSF (CHE1361173) for support. D.A.V. thanks the Camille and Henry Dreyfus Foundation, the National Science Foundation (CHE-0353662, CHE-1005253, and CHE-1725142), the Beckman Foundation, the Sherman Fairchild Foundation, the John Stauffer Charitable Trust, and the Christian Scholars Foundation for support. J.W. acknowledges support from the American Cancer Society through the Research Scholar Grant (RSG-13-011-01-CDD). W.M.W.acknowledges support from NIGMS, NIH (GM119426), and NSF (CHE1755698). A.Z. acknowledges support from NSF (CHE-1463819). (Intramural Research Program of the National Center for Advancing Translational Sciences, National Institutes of Health (NIH); CHE-1665145 - NSF; CHE-1665331 - NSF; CHE-1464898 - NSF; RUI: 213569 - NSF; CHE-1414298 - NSF; CHE1361173 - NSF; CHE1755698 - NSF; CHE-1463819 - NSF; GM126221 - NIH; 5R01GM110131 - NIH; GM 063557 - NIH; GM 084333 - NIH; R01GM071779 - NIH; GM116952 - NIH; DA045884-01 - NIH; DA046487-01 - NIH; AA026949-01 - NIH; R01 GM111476 - NIH; R01-HL25854 - NIH; R01-GM30859 - NIH; R0-1-NS-12389 - NIH; R35 GM-118173 - NIH; R24 GM-111625 - NIH; CA78747 - NIH; CA106150 - NIH; GM114353 - NIH; GM115575 - NIH; R01GM121656 - NIH; T32 CA062948-Gudas - NIH; P41 GM076267 - NIH; R01GM114061 - NIGMS, NIH; R15GM116032 - NIGMS, NIH; P30 GM103450 - NIGMS, NIH; GM129465 - NIGMS, NIH; GM119426 - NIGMS, NIH; TW009872 - Fogarty International Center, NIH; U01 TW00313 - Fogarty International Center, NIH; R01 CA158275 - National Cancer Institute (NCI), NIH; P01 AG012411 - NIH/National Institute of Aging; Camille and Henry Dreyfus Foundation; Arnold and Mabel Beckman Foundation; Scripps Institution of Oceanography; P01CA125066 - NCI, NIH; 1 R01 AT008088 - National Center for Complementary and Integrative Health; W81XWH-17-1-0256 - Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program; P30 CA008748 - NCI, NIH, through a Cancer Center Support Grant; California Department of Food and Agriculture Pierce's Disease and Glassy Winged Sharpshooter Board; American Lebanese Syrian Associated Charities (ALSAC); University of Oklahoma Startup funds; 53767-ND1 - ACS PRF; PhRMA Foundation; P30 CA008748 - CCSG NIH; RSG-12-253-01-CDD - American Cancer Society; RSG-13-011-01-CDD - American Cancer Society; CHE-0353662 - National Science Foundation; CHE-1005253 - National Science Foundation; CHE-1725142 - National Science Foundation; Beckman Foundation; Sherman Fairchild Foundation; John Stauffer Charitable Trust; Christian Scholars Foundation)Published versionSupporting documentatio

Discovery and optimization of piperazine-1-thiourea-based human phosphoglycerate dehydrogenase inhibitors

Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a marked improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling in vitro assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells. This manuscript reports the assay development and medicinal chemistry leading to the development of NCT-502 and -503 reported in Pacold et al. (2016). Keywords: PHGDH; inhibitor; serineNational Institutes of Health (U.S.) (Grant U54MH084681)National Institutes of Health (U.S.) (R37 AI047389)National Institutes of Health (U.S.) (R01 CA103866)National Institutes of Health (U.S.) (R01 CA129105)National Institutes of Health (U.S.) (R37 AI047389)National Institutes of Health (U.S.) (K22 CA212059)Mary Kay Foundation (017-32)V foundation (V2017-004

Resistance to Epigenetic-Targeted Therapy Engenders Tumor Cell Vulnerabilities Associated with Enhancer Remodeling

Drug resistance represents a major challenge to achieving durable responses to cancer therapeutics. Resistance mechanisms to epigenetically targeted drugs remain largely unexplored. We used bromodomain and extra-terminal domain (BET) inhibition in neuroblastoma as a prototype to model resistance to chromatin modulatory therapeutics. Genome-scale, pooled lentiviral open reading frame (ORF) and CRISPR knockout rescue screens nominated the phosphatidylinositol 3-kinase (PI3K) pathway as promoting resistance to BET inhibition. Transcriptomic and chromatin profiling of resistant cells revealed that global enhancer remodeling is associated with upregulation of receptor tyrosine kinases (RTKs), activation of PI3K signaling, and vulnerability to RTK/PI3K inhibition. Large-scale combinatorial screening with BET inhibitors identified PI3K inhibitors among the most synergistic upfront combinations. These studies provide a roadmap to elucidate resistance to epigenetic-targeted therapeutics and inform efficacious combination therapies. Using functional screens, profiling of drug-resistant cells, and drug combination screens in neuroblastoma, Iniguez et al. show that PI3K pathway activation via enhancer remodeling and transcriptional reprogramming confers resistance to BET inhibitors (BETi) and that PI3K inhibitors synergize with BETi

Assessing inhibitors of mutant isocitrate dehydrogenase using a suite of pre-clinical discovery assays

Abstract Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that are mutated in a variety of cancers to confer a gain-of-function activity resulting in the accumulation of an oncometabolite, D-2-hydroxyglutarate (2-HG). Accumulation of 2-HG can result in epigenetic dysregulation and a block in cellular differentiation, suggesting these mutations play a role in neoplasia. Based on its potential as a cancer target, a number of small molecule inhibitors have been developed to specifically inhibit mutant forms of IDH (mIDH1 and mIDH2). We present a comprehensive suite of in vitro preclinical drug development assays that can be used as a tool-box to identify lead compounds for mIDH drug discovery programs, as well as what we believe is the most comprehensive publically available dataset on the top mIDH inhibitors. This involved biochemical, cell-based, and tier-one ADME techniques