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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We provide an overview of the 21 published SSMD cases and have compiled additional sequencing data for four previously unpublished individuals to illustrate the genetic component of SSMD, and the role of sequencing data in diagnosis.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.

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).

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate, with concomitant oxidation of reduced nicotinamide adenine dinucleotide as the final step in the glycolytic pathway. Glycolysis plays an important role in the metabolic plasticity of cancer cells and has long been recognized as a potential therapeutic target. Thus, potent, selective inhibitors of LDH represent an attractive therapeutic approach. However, to date, pharmacological agents have failed to achieve significant target engagement in vivo, possibly because the protein is present in cells at very high concentrations. We report herein a lead optimization campaign focused on a pyrazole-based series of compounds, using structure-based design concepts, coupled with optimization of cellular potency, in vitro drug–target residence times, and in vivo PK properties, to identify first-in-class inhibitors that demonstrate LDH inhibition in vivo. The lead compounds, named NCATS-SM1440 (43) and NCATS-SM1441 (52), possess desirable attributes for further studying the effect of in vivo LDH inhibition.

PURPOSE High grade serous ovarian cancer (“HGSOC”) is the seventh most common cancer worldwide among women. The disease has no unique symptoms and often women are diagnosed in advance stages. The World Health Organization estimates that 225,500 women are diagnosed and 140,200 will die of this disease per year. In the United States, the 5-year survival rate is 43%, and 85% of all HGSOC patients experience recurrence despite aggressive treatment. More efficacious drug combination treatments are needed. Recently it was found that platinum drugs are inactivated in dimethyl sulfoxide (DMSO). Thus drug discovery platforms that use it as a solvent are not able to study platinum drugs in combination with other drugs easily. Advances in Echo technology at the National Center for Advancing Translational Sciences (NCATS) enabled acoustic transfer of platinum drugs in aqueous solutions. This allows us to find novel small molecule/platinum drug combinations that can be tested in clinical trials. METHODS We ran single-agent dose-response experiments to correctly estimate the dose-ranges needed for the matrix screens utilizing the OVCAR8 human carcinoma cell line. Then, we conducted our pilot matrix screen with compounds that are commonly combined with cisplatin for HGSOC treatment. Lastly, we spotted cisplatin against the Mechanism Interrogation PlatE drug library to identify novel combinations for clinical use against HGSOC. Compounds that show synergistic cell killing will give insight into novel drug combinations to treat HGSOC. RESULTS Several synergistic and antagonistic effects were found. These results were refined with smaller-scale screening and tested in other HGSOC cell lines. CONCLUSION The novel combinations discovered will allow chemotherapy patients to receive lower dosages of platinum drugs while maintaining or improving efficacy of cytotoxicity of cancer cells. This will help alleviate the side-effects of platinum drugs experienced by HGSOC patients.

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.

We report the discovery and medicinal chemistry optimization of a novel series of pyrazole-based inhibitors of human lactate dehydrogenase (LDH). Utilization of a quantitative high-throughput screening paradigm facilitated hit identification, while structure-based design and multiparameter optimization enabled the development of compounds with potent enzymatic and cell-based inhibition of LDH enzymatic activity. Lead compounds such as 63 exhibit low nM inhibition of both LDHA and LDHB, submicromolar inhibition of lactate production, and inhibition of glycolysis in MiaPaCa2 pancreatic cancer and A673 sarcoma cells. Moreover, robust target engagement of LDHA by lead compounds was demonstrated using the cellular thermal shift assay (CETSA), and drug-target residence time was determined via SPR. Analysis of these data suggests that drug-target residence time (off-rate) may be an important attribute to consider for obtaining potent cell-based inhibition of this cancer metabolism target.

Quality control monitoring of cell lines utilized in biomedical research is of utmost importance and is critical for the reproducibility of data. Two key pitfalls in tissue culture are 1) cell line authenticity and 2) Mycoplasma contamination. As a collaborative research institute, the National Center for Advancing Translational Sciences (NCATS) receives cell lines from a range of commercial and academic sources, which are adapted for high-throughput screening. Here, we describe the implementation of routine NCATS-wide Mycoplasma testing and short tandem repeat (STR) testing for cell lines. Initial testing identified a >10% Mycoplasma contamination rate. While the implementation of systematic testing has not fully suppressed Mycoplasma contamination rates, clearly defined protocols that include the immediate destruction of contaminated cell lines wherever possible has enabled rapid intervention and removal of compromised cell lines. Data for >2000 cell line samples tested over 3 years, and case studies are provided. STR testing of 186 cell lines with established STR profiles revealed only five misidentified cell lines, all of which were received from external labs. The data collected over the 3 years since implementation of this systematic testing demonstrate the importance of continual vigilance for rapid identification of "problem" cell lines, for ensuring reproducible data in translational science research.

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes, and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed the cell-based cytotoxicity of nearly 10,000 compounds in the National Institutes of Health, National Center for Advancing Translational Sciences annotated libraries and more than 100,000 compounds in a diversity library against four normal cell lines (HEK 293, NIH 3T3, CRL-7250, and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity, and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitute a valuable resource for the scientific community and provide insight into the extent of cytotoxic compounds in screening libraries, allowing for the identification and avoidance of compounds with cytotoxicity during high-throughput screening campaigns.

The platinum-based anticancer agents cisplatin, carboplatin, and oxaliplatin represent a spectacular translational science achievement. The basic research observations that led to the discovery of Pt complexes as DNA-binding agents that elicit cell arrest, the preclinical tumor regression studies, and the inorganic medicinal chemistry that led to clinical implementation of effective platinum complexes in the clinic have fueled multidisciplinary research into platinum-based drugs. While the successes are clear and the research activity continues, a significant window of time has passed since a new Pt drug has been approved for clinical use. Here we assess the current Pt drug landscape and challenges for future Pt development and discuss opportunities for improving our understanding of Pt drugs that utilize contemporary translational science tools such as chemical biology and real-time imaging. The underexplored spaces may reveal new opportunities for Pt drug development.

Cytotoxicity is a critical property in determining the fate of a small molecule in the drug discovery pipeline. Cytotoxic compounds are identified and triaged in both target-based and cell-based phenotypic approaches due to their off-target toxicity or on-target and on-mechanism toxicity for oncology and neurodegenerative targets. It is critical that chemical-induced cytotoxicity be reliably predicted before drug candidates advance to the late stage of development, or more ideally, before compounds are synthesized. In this study, we assessed the cell-based cytotoxicity of nearly 10,000 compounds in NCATS annotated libraries against four 'normal' cell lines (HEK 293, NIH 3T3, CRL-7250 and HaCat) using CellTiter-Glo (CTG) technology and constructed highly predictive models to estimate cytotoxicity from chemical structures. There are 5,241 non-redundant compounds having unambiguous activities in the four different cell lines, among which 11.8% compounds exhibited cytotoxicity in two or more cell lines and are thus labelled cytotoxic. The support vector classification (SVC) models trained with 80% randomly selected molecules achieved the area under the receiver operating characteristic curve (AUC-ROC) of 0.88 on average for the remaining 20% compounds in the test sets in 10 repeating experiments. Application of under-sampling rebalancing method further improved the averaged AUC-ROC to 0.90. Analysis of structural features shared by cytotoxic compounds may offer medicinal chemists heuristic design ideas to eliminate undesirable cytotoxicity. The profiling of cytotoxicity of drug-like molecules with annotated primary mechanism of action (MOA) will inform on the roles played by different targets or pathways in cellular viability. The predictive models for cytotoxicity (accessible at https://tripod.nih.gov/web_adme/cytotox.html) provide the scientific community a fast yet reliable way to prioritize molecules with little or no cytotoxicity for downstream development.

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)

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.

The activity of the histone lysine methyltransferase NSD2 is thought to play a driving role in oncogenesis. Both overexpression of NSD2 and point mutations that increase its catalytic activity are associated with a variety of human cancers. While NSD2 is an attractive therapeutic target, no potent, selective and cell-active inhibitors have been reported to date, possibly due to the challenging nature of developing high-throughput assays for NSD2. To establish a platform for the discovery and development of selective NSD2 inhibitors, multiple assays were optimized and implemented. Quantitative high-throughput screening was performed with full-length wild-type NSD2 and a nucleosome substrate against a diverse collection of known bioactives comprising 16,251 compounds. Actives from the primary screen were further interrogated with orthogonal and counter assays, as well as activity assays with the clinically relevant NSD2 mutants E1099K and T1150A. Five confirmed inhibitors were selected for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. The identification of NSD2 inhibitors that bind the catalytic SET domain and demonstrate activity in cells validates the workflow, providing a template for identifying selective NSD2 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.

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.

Cancer cells have higher basal levels of reactive oxygen species (ROS) production, and altered expression of antioxidant enzymes, when compared to normal cells. At high levels, ROS can cause substantial cellular damage, often leading to cell death, and many cancer therapeutics function through targeting these imbalanced redox systems. Glutathione peroxidases (GPx) protect cells from increased levels of ROS and oxidative stress by coupling the reduction of hydrogen and organic peroxides with the oxidization of glutathione (GSH) to glutathione disulfide (GSSG) [1]. Because of the role of glutathione peroxidases in controlling ROS levels, reducing oxidative stress, and regulating cell death pathways, we are interested in identifying inhibitors of GPx1 and GPx4. Moreover, GPx4 has recently been identified as a unique and important regulator of non-apoptotic cell death pathway, ferroptosis [2]. Using a novel method of recombinant GPx expression [3] we are developing an assay amenable to high-throughput screening of small molecules targeting GPx, and will characterize the identified inhibitors as potential chemotherapeutics for treating Glioblastoma Multiforme.