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Supplementary Figure S1. PHGDH is highly expressed in Ewing sarcoma cell lines; Supplementary Figure S2. Measurement of PHGDH protein expression. Immunoblot analysis of PHGDH in the specified cell lines expressing lentiviral control shRNA (shControl) or no shRNA (Parental); Supplementary Figure S3. PHGDH knockdown and ES proliferation in vitro; Supplementary Figure S4. Effect of GNE-618 on ES cellular proliferation; Supplementary Figure S5. Dose-effect Curves for GNE-618 and NCT-503; Supplementary Figure S6. Mouse body weight is unaffected by inhibitor treatments; Supplementary Figure S7. EWS-FLI1 is important for sarcoma cellular proliferation in the absence of serine; Supplementary Figure S8. Mouse body weight is unaffected by serine/glycine deficient diet

Supplementary Table S1. Evaluation of drug combinations in TC32 and TC71 Ewing sarcoma cells; Supplementary Table S2. Evaluation of drug combinations in EW8 Ewing sarcoma cells.

Purpose:Ewing sarcoma is an aggressive solid tumor malignancy of childhood. Although current treatment regimens cure approximately 70% of patients with localized disease, they are ineffective for most patients with metastases or relapse. New treatment combinations are necessary for these patients.Experimental Design:Ewing sarcoma cells are dependent on focal adhesion kinase (FAK) for growth. To identify candidate treatment combinations for Ewing sarcoma, we performed a small-molecule library screen to identify compounds synergistic with FAK inhibitors in impairing Ewing cell growth. The activity of a top-scoring class of compounds was then validated across multiple Ewing cell lines in vitro and in multiple xenograft models of Ewing sarcoma.Results:Numerous Aurora kinase inhibitors scored as synergistic with FAK inhibition in this screen. We found that Aurora kinase B inhibitors were synergistic across a larger range of concentrations than Aurora kinase A inhibitors when combined with FAK inhibitors in multiple Ewing cell lines. The combination of AZD-1152, an Aurora kinase B–selective inhibitor, and PF-562271 or VS-4718, FAK-selective inhibitors, induced apoptosis in Ewing sarcoma cells at concentrations that had minimal effects on survival when cells were treated with either drug alone. We also found that the combination significantly impaired tumor progression in multiple xenograft models of Ewing sarcoma.Conclusions:FAK and Aurora kinase B inhibitors synergistically impair Ewing sarcoma cell viability and significantly inhibit tumor progression. This study provides preclinical support for the consideration of a clinical trial testing the safety and efficacy of this combination for patients with Ewing sarcoma.

Supplemental Table S7. Reverse Phase Protein Array (RPPA) data. Total protein and phosphorylation levels in untreated and treated A673 and TC32 cells are normalized to total protein levels in each sample.

Supplemental Methods and Figures Supplemental Figure S1. Aurora kinase expression in Ewing sarcoma Supplemental Figure S2. Effects of cell growth on response to AZD-1152 as a function of duration of treatment. Supplemental Figure S3. Aurora kinase and FAK inhibitor combinations are synergistic in Ewing sarcoma cell lines Supplemental Figure S4. Response of Ewing cell lines treated with combinations of Aurora kinase and FAK inhibitors Supplemental Figure S5. Cell cycle and apoptotic effects of Aurora kinase B knock out in Ewing sarcoma cell lines Supplemental Figure S6. PYK2 is poorly expressed in Ewing sarcoma cells Supplemental Figure S7. Zebrafish and Murine studies

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are oncogenic for a number of malignancies, primarily low-grade gliomas and acute myeloid leukemia (AML). We report a medicinal chemistry campaign around a 7,7-dimethyl-7,8-dihydro-2H-1λ(2)-quinoline-2,5(6H)-dione screening hit against the R132H and R132C mutant forms of isocitrate dehydrogenase (IDH1). Systematic SAR efforts produced a series of potent pyrid-2-one mIDH1 inhibitors, including the atropisomer (+)-119 (NCATS-SM5637, NSC 791985). In an engineered mIDH1-U87-xenograft mouse model; after a single oral dose of 30 mg/kg, 16 h post dose, between 16-48 h, (+)-119 showed higher tumoral concentrations that corresponded to lower 2-HG concentrations, when compared with the approved drug AG-120 (ivosidenib).

[Image: see text] Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are important molecular players in a variety of diseases, such as cancer. Currently available PI5P4K inhibitors are reversible small molecules, which may lack selectivity and sufficient cellular on-target activity. In this study, we present a new class of covalent pan-PI5P4K inhibitors with potent biochemical and cellular activity. Our designs are based on THZ-P1-2, a covalent PI5P4K inhibitor previously developed in our lab. Here, we report further structure-guided optimization and structure–activity relationship (SAR) study of this scaffold, resulting in compound 30, which retained biochemical and cellular potency, while demonstrating a significantly improved selectivity profile. Furthermore, we confirm that the inhibitors show efficient binding affinity in the context of HEK 293T cells using isothermal CETSA methods. Taken together, compound 30 represents a highly selective pan-PI5P4K covalent lead molecule.

Classic galactosemia is a rare disease caused by inherited deficiency of galactose-1 phosphate uridylyltransferase (GALT). Accumulation of galactose-1 phosphate (gal-1P) is thought to be the major cause of the chronic complications associated with this disease, which currently has no treatment. Inhibiting galactokinase (GALK1), the enzyme that generates galactose-1 phosphate, has been proposed as a novel strategy for treating classic galactosemia. Our previous work identified a highly selective unique dihydropyrimidine inhibitor against GALK1. With the determination of a co-crystal structure of this inhibitor with human GALK1, we initiated a structure-based structure–activity relationship (SAR) optimization campaign that yielded novel analogs with potent biochemical inhibition (IC(50) < 100 nM). Lead compounds were also able to prevent gal-1P accumulation in patient-derived cells at low micromolar concentrations and have pharmacokinetic properties suitable for evaluation in rodent models of galactosemia.

Dystonias are a group of chronic movement-disabling disorders for which highly effective oral medications or disease-modifying therapies are lacking. The most effective treatments require invasive procedures such as deep brain stimulation (DBS). In this study, we used a high-throughput assay based on a monogenic form of dystonia, DYT1 (DYT-TOR1A), to screen a library of compounds approved for use in humans, the NCATS Pharmaceutical Collection (NPC; 2816 compounds) and identify drugs able to correct mislocalization of the disease-causing protein variant, ΔE302/3 hTorsinA. The HIV protease inhibitor, ritonavir, was among 18 compounds found to normalize hTorsinA mislocalization. Using a DYT1 knock-in mouse model to test efficacy on brain pathologies, we found that ritonavir restored multiple brain abnormalities across development. Ritonavir acutely corrected striatal cholinergic interneuron physiology in the mature brain and yielded sustained correction of diffusion tensor magnetic resonance imaging (MRI) signals when delivered during a discrete early developmental window. Mechanistically, we found that across the family of HIV protease inhibitors, efficacy correlated with integrated stress response (ISR) activation. These preclinical results identify ritonavir as a drug candidate for dystonia with disease-modifying potential.

Classic Galactosemia is a potentially lethal autosomal recessive metabolic disorder caused by deficient galactose-1-phosphate uridyltransferase (GALT) that results in the buildup of galactose-1-phosphate (gal-1-p) in cells. Galactokinase (GALK1) is the enzyme responsible for converting galactose into gal-1-p. A pharmacological inhibitor of GALK1 is hypothesized to be therapeutic strategy for treating galactosemia by reducing production of gal-1-p. In this study, we report the discovery of novel series of GALK1 inhibitors by structure-based virtual screening (VS). Followed by an extensive structural modeling and binding mode analysis of the active compounds identified from quantitative high-throughput screen (qHTS), we developed an efficient pharmacophore-based VS approach and applied for a large-scale in silico database screening. Out of 230,000 compounds virtually screened, 350 compounds were cherry-picked based on multi-factor prioritization procedure, and 75 representing a diversity of chemotypes exhibited inhibitory activity in GALK1 biochemical assay. Furthermore, a phenylsulfonamide series with excellent in vitro ADME properties was selected for downstream characterization and demonstrated its ability to lower gal-1-p in primary patient fibroblasts. The compounds described herein should provide a starting point for further development of drug candidates for the GALK1 modulation in the Classic Galactosemia.

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 library of natural products, was evaluated through HTS in a spectrum of disease-relevant assays to survey the broad biological potential of natural products in drug discovery.

Summary: Therapy of advanced melanoma is changing dramatically. Following mutational and biological subclassification of this heterogeneous cancer, several targeted and immune therapies were approved and increased survival significantly. To facilitate further advancements through pre-clinical in vivo modeling, we have established 459 patient-derived xenografts (PDX) and live tissue samples from 384 patients representing the full spectrum of clinical, therapeutic, mutational, and biological heterogeneity of melanoma. PDX have been characterized using targeted sequencing and protein arrays and are clinically annotated. This exhaustive live tissue resource includes PDX from 57 samples resistant to targeted therapy, 61 samples from responders and non-responders to immune checkpoint blockade, and 31 samples from brain metastasis. Uveal, mucosal, and acral subtypes are represented as well. We show examples of pre-clinical trials that highlight how the PDX collection can be used to develop and optimize precision therapies, biomarkers of response, and the targeting of rare genetic subgroups. : Krepler et al. have established a collection of melanoma patient-derived xenografts (PDX). Melanoma is a very heterogeneous cancer, and this large collection includes even rare subtypes and genetic aberrations in sufficient numbers. Multiple PDX from therapy-resistant patients are characterized and tested in pre-clinical trials for second line therapies. Keywords: melanoma, patient-derived xenografts, targeted therapy, immune checkpoint blockade, melanoma brain metastasis, in vivo models, BRAF inhibitor resistance, ERK inhibitor, MDM2 inhibitor, PI3K beta inhibitor

// Sui Seng Tee 1, 5, * , Jae Mo Park 1, 6, 7, * , Ralph E. Hurd 2 , Kyle R. Brimacombe 3, 4 , Matthew B. Boxer 3 , Tarik F. Massoud 1 , Brian K. Rutt 1 and Daniel M. Spielman 1 1 Department of Radiology, Stanford University, Stanford, CA, USA 2 Applied Sciences Laboratory, GE Healthcare, Menlo Park, CA, USA 3 National Center for Advancing Translational Sciences, NIH, Bethesda, MD, USA 4 NIH Chemical Genomics Center, Bethesda, MD, USA 5 Current/Present address: Memorial Sloan Kettering Cancer Center, New York, NY, USA 6 Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, TX, USA 7 Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX, USA * These authors have contributed equally to this work Correspondence to: Sui Seng Tee, email: tees@mskcc.org Keywords: molecular imaging, metabolic imaging, hyperpolarized MRI, cancer metabolism, PKM2 Received: October 19, 2016 Accepted: July 06, 2017 Published: July 26, 2017 ABSTRACT Cancer metabolism has emerged as an increasingly attractive target for interfering with tumor growth. Small molecule activators of pyruvate kinase isozyme M2 (PKM2) suppress tumor formation but have an unknown effect on established tumors. We demonstrate that TEPP-46, a PKM2 activator, results in increased glucose consumption, providing the rationale for combining PKM2 activators with the toxic glucose analog, 2-deoxy-D-glucose (2-DG). Combination treatment resulted in reduced viability of a range of cell lines in standard cell culture conditions at concentrations of drugs that had no effect when used alone. This effect was replicated in vivo on established subcutaneous tumors. We further demonstrated the ability to detect acute metabolic differences in combination treatment using hyperpolarized magnetic resonance spectroscopy (MRS). Combination treated tumors displayed a higher pyruvate to lactate 13 C-label exchange 2 hr post-treatment. This ability to assess the effect of drugs non-invasively may accelerate the implementation and clinical translation of drugs that target cancer metabolism.

Despite a growing body of knowledge about the genomic landscape of Ewing sarcoma, translation of basic discoveries into targeted therapies and significant clinical gains has remained elusive. Recent insights have revealed that the oncogenic transcription factor EWS-FLI1 can impact Ewing sarcoma cellular metabolism, regulating expression of 3-phosphoglycerate dehydrogenase (PHGDH), the first enzyme in de novo serine synthesis. Here, we have examined the importance of serine metabolism in Ewing sarcoma tumorigenesis and evaluated the therapeutic potential of targeting serine metabolism in preclinical models of Ewing sarcoma. We show that PHGDH knockdown resulted in decreased Ewing sarcoma cell proliferation, especially under serine limitation, and significantly inhibited xenograft tumorigenesis in preclinical orthotopic models of Ewing sarcoma. In addition, the PHGDH inhibitor NCT-503 caused a dose-dependent decrease in cellular proliferation. Moreover, we report a novel drug combination in which nicotinamide phosphoribosyltransferase (NAMPT) inhibition, which blocks production of the PHGDH substrate NAD+, synergized with NCT-503 to abolish Ewing sarcoma cell proliferation and tumor growth. Furthermore, we show that serine deprivation inhibited Ewing sarcoma cell proliferation and tumorigenesis, indicating that Ewing sarcoma cells depend on exogenous serine in addition to de novo serine synthesis. Our findings suggest that serine metabolism is critical for Ewing sarcoma tumorigenesis, and that targeting metabolic dependencies should be further investigated as a potential therapeutic strategy for Ewing sarcoma. In addition, the combination strategy presented herein may have broader clinical applications in other PHGDH-overexpressing cancers as well.

Loss of function mutations in Kelch Like ECH Associated Protein 1 (KEAP1) or gain-of-function mutations in nuclear factor erythroid 2-related factor 2 (NRF2) are common in non-small cell lung cancer (NSCLC) and is associated with therapeutic resistance. To discover novel NRF2 inhibitors for targeted therapy, we conducted a quantitative high-throughput screen using a diverse set of ~400,000 small molecules (Molecular Libraries Small Molecule Repository Library, MLSMR) at the National Center for Advancing Translational Sciences. We identified ML385 as a probe molecule that binds to NRF2 and inhibits its downstream target gene expression. Specifically, ML385 binds to the Neh1, the Cap ‘N’ Collar Basic Leucine Zipper (CNC-bZIP) domain of NRF2, and interferes with the binding of the V-Maf Avian Musculoaponeurotic Fibrosarcoma Oncogene Homolog G (MAFG)-NRF2 protein complex to regulatory DNA binding sequences. In clonogenic assays, when used in combination with platinum-based drugs such as doxorubicin or taxol, ML385 substantially enhances cytotoxicity in NSCLC cells compared to single agents alone. ML385 shows specificity and selectivity for NSCLC cells with KEAP1 mutation leading to gain of NRF2 function. In preclinical models of NSCLC with gain of NRF2 function, ML385 in combination with carboplatin showed significant anti-tumor activity. We demonstrate the discovery and validation of ML385 as a novel and specific NRF2 inhibitor and conclude that targeting NRF2 may represent a promising strategy for the treatment of advanced NSCLC.

Recent advances in single-particle cryoelecton microscopy (cryo-EM) are enabling generation of numerous near-atomic-resolution structures for well-ordered protein complexes with sizes ≥ ~200 kDa. Whether cryo-EM methods are equally useful for high-resolution structural analysis of smaller, dynamic protein complexes such as those involved in cellular metabolism remains an important question. Here, we present 3.8 Å resolution cryo-EM structures of isocitrate dehydrogenase (93 kDa) and identify the nature of conformational changes induced by binding of the allosteric small-molecule inhibitor ML309. We also report 2.8-Å- and 1.8-Å-resolution structures of the cancer targets lactate dehydrogenase (145 kDa) and glutamate dehydrogenase (334 kDa), respectively. With these results, two perceived barriers in single-particle cryo-EM are overcome: our tests demonstrated crossing 2 Å resolution and obtaining maps of proteins with sizes < 100 kDa, demonstrating that cryo-EM can be used to investigate a broad spectrum of drug-target interactions and dynamic conformational states.

Excess lipid storage is an epidemic problem in human populations. Thus, the identification of small molecules to treat or prevent lipid storage-related metabolic complications is of great interest. Here we screened > 320.000 compounds for their ability to prevent a cellular lipid accumulation phenotype. We used fly cells because the multifarious tools available for this organism should facilitate unraveling the mechanism-of-action of active small molecules. Of the several hundred lipid storage inhibitors identified in the primary screen we concentrated on three structurally diverse and potent compound classes active in cells of multiple species (including human) and negligible cytotoxicity. Together with Drosophila in vivo epistasis experiments, RNA-Seq expression profiles suggested that the target of one of the small molecules was diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in the production of triacylglycerols and prominent human drug target. We confirmed this prediction by biochemical and enzymatic activity tests., Highlights • We identified > 600 potent small molecule inhibitors of cellular lipid storage deposition. • RNA-Seq expression profiling discriminated the activity of three lead scaffolds and guided subsequent functional studies. • We discovered a class of DGAT1 inhibitors, which is active in fly and mammalian cell lines as well as whole flies. Obesity and other lipid storage associated diseases are a growing health threat of human populations. In an undirected phenotypic screen, we identified pharmacologically active small molecules that reduce or enhance lipid storage. Our work focuses on three lead structures that prevent lipid storage in diverse cellular systems including cells from a diabetes patient. In order to elucidate the compound mechanisms-of-action and cellular targets, we used a combination of RNA-Seq transcriptional profiling and diverse functional assays. Our results strongly suggest that one of our lead structures represents a class of inhibitors targeting the key lipogenic enzyme diacylglycerol acyltransferase 1.

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; serine, National 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.) (K22 CA212059), Mary Kay Foundation (017-32), V foundation (V2017-004)

// Lisa Zhang 1 , Yaqin Zhang 2 , Amit Mehta 1, 3 , Myriem Boufraqech 1 , Sean Davis 4 , Jing Wang 5 , Ze Tian 5 , Zhiya Yu 6 , Matthew B. Boxer 2 , Jeffrey A. Kiefer 7 , John A. Copland 8 , Robert C. Smallridge 8, 9 , Zhuyin Li 2 , Min Shen 2 , Electron Kebebew 1 1 Endocrine Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 2 Division of Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA 3 Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA 4 Cancer Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA 5 Curis, Inc., Translational Science MA, USA 6 Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 7 Division of Information Sciences, Translational Genomics Research Institute, Phoenix, AZ, USA 8 Department of Cancer Biology, Mayo Clinic, Jacksonville, FL, USA 9 Endocrinology Division, Internal Medicine Department, Mayo Clinic, Jacksonville, FL, USA Correspondence to: Electron Kebebew, e-mail: kebebewe@mail.nih.gov Keywords: CUDC-101, anaplastic thyroid cancer, quantitative high-throughput screening, EGFR, HDAC Received: January 26, 2015 Accepted: January 31, 2015 Published: April 13, 2015 ABSTRACT Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies that currently has no effective therapy. We performed quantitative high-throughput screening (qHTS) in three ATC cell lines using 3,282 clinically approved drugs and drug candidates, and identified 100 active agents. Enrichment analysis of active compounds showed that inhibitors of EGFR and histone deacetylase (HDAC) were most active. Of these, the first-in-class dual inhibitor of EGFR, HER2 and HDACs, CUDC-101, had higher efficacy and lower IC 50 than established drugs. We validated that CUDC-101 inhibited cellular proliferation and resulted in cell death by inducing cell cycle arrest and caspase-dependent apoptosis. CUDC-101 also inhibited cellular migration in vitro . Mechanistically, CUDC-101 inhibited MAPK signaling and histone deacetylation in ATC cell lines with multiple driver mutations present in human ATC. The anticancer effect of CUDC-101 was associated with increased expression of p21 and E-cadherin, and reduced expression of survivin, XIAP, β-catenin, N-cadherin, and vimentin. In an in vivo mouse model of metastatic ATC, CUDC-101 inhibited tumor growth and metastases, and significantly prolonged survival. Response to CUDC-101 treatment in vivo was associated with increased histone 3 acetylation and reduced survivin nuclear expression. Our findings provide a preclinical basis to evaluate CUDC-101 therapy in ATC.

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 and secretion 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). Here, a panel of mIDH inhibitors were systematically profiled using biochemical, cell-based, and tier-one ADME techniques. We quantified the biochemical effect of each inhibitor on mIDH1 (R132H and R132C) and mIDH2 (R172Q). The effect of these inhibitors on 2-HG concentrations in seven cell lines representing five different IDH1 mutations in both 2D and 3D cell cultures was assessed. Target engagement of these inhibitors was analyzed utilizing cellular thermal shift assays (CETSA), the effects of inhibitors on reversing 2- HG-induced block on leukemic cellular differentiation. We conclude from our mIDH1 assay panel that AG-120 and a Novartis inhibitor exhibited excellent activity in all biochemical and most cellular assays. While AG-120 has superior DMPK properties, it lacks efficacy a leukemic differentiation model. In conclusion, 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.

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 and secretion 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). Here, a panel of mIDH inhibitors were systematically profiled using biochemical, cell-based, and tier-one ADME techniques. We quantified the biochemical effect of each inhibitor on mIDH1 (R132H and R132C) and mIDH2 (R172Q). The effect of these inhibitors on 2-HG concentrations in seven cell lines representing five different IDH1 mutations in both 2D and 3D cell cultures was assessed. Target engagement of these inhibitors was analyzed utilizing cellular thermal shift assays (CETSA), the effects of inhibitors on reversing 2-HG-induced block on leukemic cellular differentiation. We conclude from our mIDH1 assay panel that AG-120 and a Novartis inhibitor exhibited excellent activity in all biochemical and most cellular assays. While AG-120 has superior DMPK properties, it lacks efficacy a leukemic differentiation model. In conclusion, 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.

Purpose: Ewing sarcoma is an aggressive solid tumor malignancy of childhood. Although current treatment regimens cure approximately 70% of patients with localized disease, they are ineffective for most patients with metastases or relapse. New treatment combinations are necessary for these patients. Experimental Design: Ewing sarcoma cells are dependent on focal adhesion kinase (FAK) for growth. To identify candidate treatment combinations for Ewing sarcoma, we performed a small-molecule library screen to identify compounds synergistic with FAK inhibitors in impairing Ewing cell growth. The activity of a top-scoring class of compounds was then validated across multiple Ewing cell lines in vitro and in multiple xenograft models of Ewing sarcoma. Results: Numerous Aurora kinase inhibitors scored as synergistic with FAK inhibition in this screen. We found that Aurora kinase B inhibitors were synergistic across a larger range of concentrations than Aurora kinase A inhibitors when combined with FAK inhibitors in multiple Ewing cell lines. The combination of AZD-1152, an Aurora kinase B–selective inhibitor, and PF-562271 or VS-4718, FAK-selective inhibitors, induced apoptosis in Ewing sarcoma cells at concentrations that had minimal effects on survival when cells were treated with either drug alone. We also found that the combination significantly impaired tumor progression in multiple xenograft models of Ewing sarcoma. Conclusions: FAK and Aurora kinase B inhibitors synergistically impair Ewing sarcoma cell viability and significantly inhibit tumor progression. This study provides preclinical support for the consideration of a clinical trial testing the safety and efficacy of this combination for patients with Ewing sarcoma.

Classic galactosemia is an inherited metabolic disease for which, at present, no therapy is available apart from galactose-restricted diet. However, the efficacy of the diet is questionable, since it is not able to prevent the insurgence of chronic complications later in life. In addition, it is possible that dietary restriction itself could induce negative side effects. Therefore, there is a need for an alternative therapeutic approach that can avert the manifestation of chronic complications in the patients. In this review, the authors describe the development of a novel class of pharmaceutical agents that target the production of a toxic metabolite, galactose-1-phosphate, considered as the main culprit for the cause of the complications, in the patients.

Two mutant forms (R132H and R132C) of isocitrate dehydrogenase 1 (IDH1) have been associated with a number of cancers including glioblastoma and acute myeloid leukemia. These mutations confer a neomorphic activity of 2-hydroxyglutarate (2-HG) production, and 2-HG has previously been implicated as an oncometabolite. Inhibitors of mutant IDH1 can potentially be used to treat these diseases. In this study, we investigated the mechanism of action of a newly discovered inhibitor, ML309, using biochemical, cellular, and biophysical approaches. Substrate binding and product inhibition studies helped to further elucidate the IDH1 R132H catalytic cycle. This rapidly equilibrating inhibitor is active in both biochemical and cellular assays. The (+) isomer is active (IC50 = 68 nm), whereas the (−) isomer is over 400-fold less active (IC50 = 29 μm) for IDH1 R132H inhibition. IDH1 R132C was similarly inhibited by (+)-ML309. WT IDH1 was largely unaffected by (+)-ML309 (IC50 >36 μm). Kinetic analyses combined with microscale thermophoresis and surface plasmon resonance indicate that this reversible inhibitor binds to IDH1 R132H competitively with respect to α-ketoglutarate and uncompetitively with respect to NADPH. A reaction scheme for IDH1 R132H inhibition by ML309 is proposed in which ML309 binds to IDH1 R132H after formation of the IDH1 R132H NADPH complex. ML309 was also able to inhibit 2-HG production in a glioblastoma cell line (IC50 = 250 nm) and had minimal cytotoxicity. In the presence of racemic ML309, 2-HG levels drop rapidly. This drop was sustained until 48 h, at which point the compound was washed out and 2-HG levels recovered.

The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug-drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell-like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton's tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL.

Glutaminase catalyzes the hydrolysis of glutamine to glutamate and plays a central role in the proliferation of neoplastic cells via glutaminolysis, as well as in the generation of excitotoxic glutamate in central nervous system disorders such as HIV-associated dementia (HAD) and multiple sclerosis. Both glutaminase siRNA and glutaminase inhibition have been shown to be effective in in vitro models of cancer and HAD, suggesting a potential role for small molecule glutaminase inhibitors. However, there are no potent, selective inhibitors of glutaminase currently available. The two prototypical glutaminase inhibitors, BPTES and DON, are either insoluble or non-specific. In a search for more drug-like glutaminase inhibitors, we conducted a screen of 1280 in vivo active drugs (Library of Pharmacologically Active Compounds (LOPAC(1280))) and identified ebselen, chelerythrine and (R)-apomorphine. The newly identified inhibitors exhibited 10 to 1500-fold greater affinities than DON and BPTES and over 100-fold increased efficiency of inhibition. Although non-selective, it is noteworthy that the affinity of ebselen for glutaminase is more potent than any other activity yet described. It is possible that the previously reported biological activity seen with these compounds is due, in part, to glutaminase inhibition. Ebselen, chelerythrine and apomorphine complement the armamentarium of compounds to explore the role of glutaminase in disease.

The importance of the trifluoromethyl group in the polypharmacological profile of nilotinib was investigated. Molecular editing of nilotinib led to the design, synthesis and biological evaluation of analogues where the trifluoromethyl group was replaced by a proton, fluorine and a methyl group. While these analogues were less active than nilotinib toward Abl, their activity toward Kit was comparable, with the monofluorinated analogue being the most active. Docking of nilotinib and of analogues 2a-c to the binding pocket of Abl and of Kit showed that the lack of shape complementarity in Kit is compensated by the stabilizing effect from its juxtamembrane region.

Plasmodium falciparum , the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small-molecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ± 1.2, a coefficient of variation of 6.8% ± 2.9%, and a Z′-factor of 0.75 ± 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as ∼1 μM, and several were found to have low-micromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development.

The aggregation of α-synuclein (aSyn) leading to the formation of Lewy bodies is the defining pathological hallmark of Parkinson's disease (PD). Rare familial PD-associated mutations in aSyn render it aggregation-prone; however, PD patients carrying wild type (WT) aSyn also have aggregated aSyn in Lewy bodies. The mechanisms by which WT aSyn aggregates are unclear. Here, we report that inflammation can play a role in causing the aggregation of WT aSyn. We show that activation of the inflammasome with known stimuli results in the aggregation of aSyn in a neuronal cell model of PD. The insoluble aggregates are enriched with truncated aSyn as found in Lewy bodies of the PD brain. Inhibition of the inflammasome enzyme caspase-1 by chemical inhibition or genetic knockdown with shRNA abated aSyn truncation. In vitro characterization confirmed that caspase-1 directly cleaves aSyn, generating a highly aggregation-prone species. The truncation-induced aggregation of aSyn is toxic to neuronal culture, and inhibition of caspase-1 by shRNA or a specific chemical inhibitor improved the survival of a neuronal PD cell model. This study provides a molecular link for the role of inflammation in aSyn aggregation, and perhaps in the pathogenesis of sporadic PD as well.

Serine is a both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical glucose-derived serine synthesis pathway, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic towards PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we use a quantitative high-throughput screen to identify small molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and suggest that one-carbon unit wasting may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.

Deubiquitinases are important components of the protein degradation regulatory network. We report the discovery of ML364, a small molecule inhibitor of the deubiquitinase USP2 and its use to interrogate the biology of USP2 and its putative substrate cyclin D1. ML364 has an IC50 of 1.1 μm in a biochemical assay using an internally quenched fluorescent di-ubiquitin substrate. Direct binding of ML364 to USP2 was demonstrated using microscale thermophoresis. ML364 induced an increase in cellular cyclin D1 degradation and caused cell cycle arrest as shown in Western blottings and flow cytometry assays utilizing both Mino and HCT116 cancer cell lines. ML364, and not the inactive analog 2, was antiproliferative in cancer cell lines. Consistent with the role of cyclin D1 in DNA damage response, ML364 also caused a decrease in homologous recombination-mediated DNA repair. These effects by a small molecule inhibitor support a key role for USP2 as a regulator of cell cycle, DNA repair, and tumor cell growth.

PYK (pyruvate kinase) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small-molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit LmPYK (Leishmania mexicana PYK) activity in a time- (and dose-) dependent manner, a characteristic of irreversible inhibition. The crystal structure of DBS {4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid} complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS-binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors.

Serine is both a proteinogenic amino acid and the source of one-carbon units essential for de novo purine and deoxythymidine synthesis. In the canonical pathway of glucose-derived serine synthesis, Homo sapiens phosphoglycerate dehydrogenase (PHGDH) catalyzes the first, rate-limiting step. Genetic loss of PHGDH is toxic toward PHGDH-overexpressing breast cancer cell lines even in the presence of exogenous serine. Here, we used a quantitative high-throughput screen to identify small-molecule PHGDH inhibitors. These compounds reduce the production of glucose-derived serine in cells and suppress the growth of PHGDH-dependent cancer cells in culture and in orthotopic xenograft tumors. Surprisingly, PHGDH inhibition reduced the incorporation into nucleotides of one-carbon units from glucose-derived and exogenous serine. We conclude that glycolytic serine synthesis coordinates the use of one-carbon units from endogenous and exogenous serine in nucleotide synthesis, and we suggest that one-carbon unit wasting thus may contribute to the efficacy of PHGDH inhibitors in vitro and in vivo.

Control of intracellular reactive oxygen species (ROS) concentrations is critical for cancer cell survival. We show that, in human lung cancer cells, acute increases in intracellular concentrations of ROS caused inhibition of the glycolytic enzyme pyruvate kinase M2 (PKM2) through oxidation of Cys[superscript 358]. This inhibition of PKM2 is required to divert glucose flux into the pentose phosphate pathway and thereby generate sufficient reducing potential for detoxification of ROS. Lung cancer cells in which endogenous PKM2 was replaced with the Cys[superscript 358] to Ser[superscript 358] oxidation-resistant mutant exhibited increased sensitivity to oxidative stress and impaired tumor formation in a xenograft model. Besides promoting metabolic changes required for proliferation, the regulatory properties of PKM2 may confer an additional advantage to cancer cells by allowing them to withstand oxidative stress., National Institutes of Health (U.S.) (R03MH085679), National Institutes of Health (U.S.) (1P30CA147882), Burroughs Wellcome Fund, Damon Runyon Cancer Research Foundation, Smith Family Foundation, Starr Cancer Consortium

Compared to normal differentiated cells, cancer cells have altered metabolic regulation to support biosynthesis and the expression of the M2 isozyme of pyruvate kinase (PKM2) plays an important role in this anabolic metabolism. While the M1 isoform is a highly active enzyme, the alternatively spliced M2 variant is considerably less active and expressed in tumors. While the exact mechanism by which decreased pyruvate kinase activity contributes to anabolic metabolism remains unclear, it is hypothesized that activation of PKM2 to levels seen with PKM1 may promote a metabolic program that is not conducive to cell proliferation. Here we report the third chemotype in a series of PKM2 activators based on the 2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamide scaffold. The synthesis, structure activity relationships, selectivity and notable physiochemical properties are described., National Human Genome Research Institute (U.S.) (Molecular Libraries Initiative of the NIH Roadmap for Medical Research)

Cancer multidrug resistance (MDR) mediated by ATP-binding cassette (ABC) transporters presents a significant unresolved clinical challenge. One strategy to resolve MDR is to develop compounds that selectively kill cells overexpressing the efflux transporter P-glycoprotein (MDR1, P-gp, ABCB1). We have previously reported structure-activity studies based around the lead compound NSC73306 (1, 1-isatin-4-(4'-methoxyphenyl)-3-thiosemicarbazone, 4.3-fold selective). Here we sought to extend this work on MDR1-selective analogues by establishing whether 1 showed "robust" activity against a range of cell lines expressing P-gp. We further aimed to synthesize and test analogues with varied substitution at the N4-position, and substitution around the N4-phenyl ring of isatin-β-thiosemicarbazones (IBTs), to identify compounds with increased MDR1-selectivity. Compound 1 demonstrated MDR1-selectivity against all P-gp-expressing cell lines examined. This selectivity was reversed by inhibitors of P-gp ATPase activity. Structural variation at the 4'-phenyl position of 1 yielded compounds of greater MDR1-selectivity. Two of these analogues, 1-isatin-4-(4'-nitrophenyl)-3-thiosemicarbazone (22, 8.3-fold selective) and 1-isatin-4-(4'-tert-butyl phenyl)-3-thiosemicarbazone (32, 14.8-fold selective), were selected for further testing and were found to retain the activity profile of 1. These compounds are the most active IBTs identified to date.

Nature Communications 5: Article number: 4608 (2014); Published: 3 April 2014; Updated: 2 November 2016 During the preparation of the PCR gel panels in Fig. 1b,c, duplicate or otherwise irrelevant lanes were excised; these excisions were not noted in the published figure as per the Nature journal policy.

We recently developed a new use of the super silyl group in organic reactions, and showed that it has unique reactivity. One of the reactions using this reagent is diastereoselective Mukaiyama aldehyde cross-aldol reaction catalyzed by a highly active Bronsted acid. This reaction produced syn-1,3-diol derivatives with high yield and high stereoselectivity. Certain features and outcomes of the reaction were not clear, so we decided to investigate the reaction mechanism through a theoretical study. In this paper, structures of super silyl species were observed as the subject of study. First 1 and 2 were investigated so that the effect of a TMS group adjacent to a silicenium ion could be determined. Interestingly, it was found that the TMS group stabilized the silicenium ion. It is presumed that the stability of the tris(trimethylsilyl) silicenium ion (TTMSS+) is related to the unique reactivity of super silyl group in theses reactions. We also investigated equivalent structures of silyl enol ethers (SEE) and found an unusual structure of 5. This structure is a result of unusual interactions between the silyl portion and enol ether (EE) portion; 1) C=C and Si-Si bonds, 2) lone pair of oxygen and Si-Si bond.

The tumour microenvironment contributes to cancer metastasis and drug resistance. However, most high throughput screening (HTS) assays for drug discovery use cancer cells grown in monolayers. Here we show that a multilayered culture containing primary human fibroblasts, mesothelial cells and extracellular matrix can be adapted into a reliable 384- and 1,536-multi-well HTS assay that reproduces the human ovarian cancer (OvCa) metastatic microenvironment. We validate the identified inhibitors in secondary in vitro and in vivo biological assays using three OvCa cell lines: HeyA8, SKOV3ip1 and Tyk-nu. The active compounds directly inhibit at least two of the three OvCa functions: adhesion, invasion and growth. In vivo, these compounds prevent OvCa adhesion, invasion and metastasis, and improve survival in mouse models. Collectively, these data indicate that a complex three-dimensional culture of the tumour microenvironment can be adapted for quantitative HTS and may improve the disease relevance of assays used for drug screening.

The synthesis of the acetaldehyde-derived tris(trimethylsilyl)silyl (super silyl) enol ether is described, as well as its use in the high-yielding aldehyde cross-aldol reaction. The super silyl enol ether shows unprecedented reactivity in giving the 1:1 adduct in very high yield. This reaction is catalyzed by 0.05 mol% of the Brønsted acid triflimide (HNTf2) and is complete within 15 min, making the protocol very attractive for large-scale synthesis.

Glioblastoma multiforme (GBM) is the most malignant brain tumor and resistant to cell senescence. Through proteomic screening we have identified a novel type of kinase, tentatively named, SSK1 (Super Signaling Kinase-1) that could connect cellular metabolism to cell growth. Depletion of SSK1 induced cellular senescence and diminished GBM cell growth in vitro and in a xenograft tumor model. The senescence caused by SSK1 depletion was accompanied by the induction of p21WAF1/CIP1, a critical inducer of cell senescence. Importantly, p21WAF1/CIP1 induction was independent of p53, reactive oxygen species (ROS) status, and DNA damage response pathways. Critically, the kinase activity of SSK1 is indispensable for GBM to suppress the induction of p21WAF1/CIP1 and escape senescence. Through chemical library screening, we identified a small molecule, which inhibited SSK1 activity in vitro and in vivo. Treatment of SSK1 inhibitor increased p21WAF1/CIP1 and triggered GBM senescence. Our data reveal a novel p21WAF1/CIP1 regulatory pathway mediated by SSK1, which is essential for GBM to suppress senescence, and suggest a novel therapy for their treatment, pharmacologically targeting SSK1.