Conditional lethality profiling reveals anticancer mechanisms of action and drug-nutrient interactions.

Chemical screens across hundreds of cell lines have shown that the drug sensitivities of human cancers can vary by genotype or lineage. However, most drug discovery studies have relied on culture media that poorly reflect metabolite levels in human blood. Here, we perform drug screens in traditional and Human Plasma-Like Medium (HPLM).

Pan-cancer proteogenomics expands the landscape of therapeutic targets.

Fewer than 200 proteins are targeted by cancer drugs approved by the Food and Drug Administration (FDA). We integrate Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomics data from 1,043 patients across 10 cancer types with additional public datasets to identify potential therapeutic targets. Pan-cancer analysis of 2,863 druggable proteins reveals a wide abundance range and identifies biological factors that affect mRNA-protein correlation.

Conditional lethality profiling reveals anticancer mechanisms of action and drug-nutrient interactions.

Chemical screening studies have identified drug sensitivities across hundreds of cancer cell lines but most putative therapeutics fail to translate. Discovery and development of drug candidates in models that more accurately reflect nutrient availability in human biofluids may help in addressing this major challenge. Here we performed high-throughput screens in conventional versus Human Plasma-Like Medium (HPLM).

SULT1A1-dependent sulfonation of alkylators is a lineage-dependent vulnerability of liver cancers.

Adult liver malignancies, including intrahepatic cholangiocarcinoma and hepatocellular carcinoma, are the second leading cause of cancer-related deaths worldwide. Most individuals are treated with either combination chemotherapy or immunotherapy, respectively, without specific biomarkers for selection. Here using high-throughput screens, proteomics and in vitro resistance models, we identify the small molecule YC-1 as selectively active against a defined subset of cell lines derived from both liver cancer types.

Characterization and tissue localization of zebrafish homologs of the human ABCB1 multidrug transporter.

Capillary endothelial cells of the human blood-brain barrier (BBB) express high levels of P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2). However, little information is available regarding ATP-binding cassette transporters expressed at the zebrafish BBB, which has emerged as a potential model system. We report the characterization and tissue localization of two genes that are similar to ABCB1, zebrafish abcb4 and abcb5.

Structural Characterization of Degrader-Induced Ternary Complexes Using Hydrogen-Deuterium Exchange Mass Spectrometry and Computational Modeling: Implications for Structure-Based Design.

The field of targeted protein degradation (TPD) has grown exponentially over the past decade with the goal of developing therapies that mark proteins for destruction leveraging the ubiquitin-proteasome system. One common approach to achieve TPD is to employ a heterobifunctional molecule, termed as a degrader, to recruit the protein target of interest to the E3 ligase machinery. The resultant generation of an intermediary ternary complex (target-degrader-ligase) is pivotal in the degradation process.

Discovery and Optimization of 2-1λ-Pyridin-2-one Inhibitors of Mutant Isocitrate Dehydrogenase 1 for the Treatment of Cancer.

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are oncogenic for a number of malignancies, primarily low-grade gliomas and acute myeloid leukemia. We report a medicinal chemistry campaign around a 7,7-dimethyl-7,8-dihydro-2-1λ-quinoline-2,5(6)-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 (-, ).

Optimization of ether and aniline based inhibitors of lactate dehydrogenase.

Lactate dehydrogenase (LDH) is a critical enzyme in the glycolytic metabolism pathway that is used by many tumor cells. Inhibitors of LDH may be expected to inhibit the metabolic processes in cancer cells and thus selectively delay or inhibit growth in transformed versus normal cells. We have previously disclosed a pyrazole-based series of potent LDH inhibitors with long residence times on the enzyme.

Pyrazole-Based Lactate Dehydrogenase Inhibitors with Optimized Cell Activity and Pharmacokinetic Properties.

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.

Keeping It Clean: The Cell Culture Quality Control Experience at the National Center for Advancing Translational Sciences.

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) 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.

A High-Throughput Screen of a Library of Therapeutics Identifies Cytotoxic Substrates of P-glycoprotein.

The ATP-binding cassette transporter P-glycoprotein (P-gp) is known to limit both brain penetration and oral bioavailability of many chemotherapy drugs. Although US Food and Drug Administration guidelines require that potential interactions of investigational drugs with P-gp be explored, often this information does not enter the literature. In response, we developed a high-throughput screen to identify substrates of P-gp from a series of chemical libraries, testing a total of 10,804 compounds, most of which have known mechanisms of action.

Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening.

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.

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.

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.

Discovery and Optimization of Potent, Cell-Active Pyrazole-Based Inhibitors of Lactate Dehydrogenase (LDH).

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.

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

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).

Functional analysis of rhomboid proteases during Toxoplasma invasion.

Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in T.

Efficient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/CAS9.

Unlabelled: Toxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excellent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special genetic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms.

Say no to DMSO: dimethylsulfoxide inactivates cisplatin, carboplatin, and other platinum complexes.

The platinum drugs cisplatin, carboplatin, and oxaliplatin are highly utilized in the clinic and as a consequence are extensively studied in the laboratory setting. In this study, we examined the literature and found a significant number of studies (11%-34%) in prominent cancer journals utilizing cisplatin dissolved in DMSO. However, dissolving cisplatin in DMSO for laboratory-based studies results in ligand displacement and changes to the structure of the complex.