High-throughput screening (HTS) of chemical compounds to identify modulators of molecular targets is a mainstay of pharmaceutical development. Increasingly, HTS is being used to identify chemical probes of gene, pathway, and cell functions, with the ultimate goal of comprehensively delineating relationships between chemical structures and biological activities. Achieving this goal will require methodologies that efficiently generate pharmacological data from the primary screen and reliably profile the range of biological activities associated with large chemical libraries. Traditional HTS, which tests compounds at a single concentration, is not suited to this task, because HTS is burdened by frequent false positives and false negatives and requires extensive follow-up testing. We have developed a paradigm, quantitative HTS (qHTS), tested with the enzyme pyruvate kinase, to generate concentration-response curves for >60,000 compounds in a single experiment. We show that this method is precise, refractory to variations in sample preparation, and identifies compounds with a wide range of activities. Concentration-response curves were classified to rapidly identify pyruvate kinase activators and inhibitors with a variety of potencies and efficacies and elucidate structure-activity relationships directly from the primary screen. Comparison of qHTS with traditional single-concentration HTS revealed a high prevalence of false negatives in the single-point screen. This study demonstrates the feasibility of qHTS for accurately profiling every compound in large chemical libraries (>10 5 compounds). qHTS produces rich data sets that can be immediately mined for reliable biological activities, thereby providing a platform for chemical genomics and accelerating the identification of leads for drug discovery.1,536-well ͉ chemical genomics ͉ enzyme assay ͉ PubChem ͉ pyruvate kinase T he first description of biological effect versus chemical compound concentration was made by Paracelsus ca. 1534 and quantified by A. V. Hill in 1910 (1). The basis of these observations is that ligands affecting biological systems have optimal ranges of activity (EC 50 ), and give rise to concentration-effect relationships that can be complex, varying in potency, efficacy, and steepness of response. Far below an EC 50 , no effect may be seen (referred to as the no observable effect level or NOEL), and much above it, toxic or ''off-target'' effects may be observed. This well known behavior of chemical compounds in biological systems requires a specific dose of a compound to achieve a desired biological effect, whether in basic or clinical applications (2, 3).Historically, new compounds with medicinal qualities were discovered through laborious testing of samples using low-throughput assays including animal and isolated tissue models. In the early 1990s, the advent of combinatorial chemistry and commercial consolidation of small molecule collections resulted in a tremendous increase in compound numbers, requiring the development of high-throughput ...

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.

ABSTRACT:Thirty-two structurally diverse drugs used for the treatment of various conditions of the central nervous system (CNS), along with two active metabolites, and eight non-CNS drugs were measured in brain, plasma, and cerebrospinal fluid in the P-glycoprotein (P-gp) knockout mouse model after subcutaneous administration, and the data were compared with corresponding data obtained in wild-type mice. Total brain-to-plasma (B/P) ratios for the CNS agents ranged from 0.060 to 24. Of the 34 CNS-active agents, only 7 demonstrated B/P area under the plasma concentration curve ratios between P-gp knockout and wild-type mice that did not differ significantly from unity. Most of the remaining drugs demonstrated 1.1-to 2.6-fold greater B/P ratios in P-gp knockout mice versus wild-type mice. Three, risperidone, its active metabolite 9-hydroxyrisperidone, and metoclopramide, showed marked differences in B/P ratios between knockout and wild-type mice (6.6-to 17-fold). Differences in B/P ratios and cerebrospinal fluid/ plasma ratios between wild-type and knockout animals were correlated. Through the use of this model, it appears that most CNSactive agents demonstrate at least some P-gp-mediated transport that can affect brain concentrations. However, the impact for the majority of agents is probably minor. The example of risperidone illustrates that even good P-gp substrates can still be clinically useful CNS-active agents. However, for such agents, unbound plasma concentrations may need to be greater than values projected using receptor affinity data to achieve adequate receptor occupancy for effect.Active transport mechanisms as determinants of drug absorption, distribution, and clearance have been the focus of considerable research effort over the past decade. Of the numerous transporter proteins recently investigated, the one for which the greatest amount of knowledge exists is P-glycoprotein (MDR1). Originally described as a transporter involved in imparting drug resistance to tumor cells, P-glycoprotein has been demonstrated to be important in reducing absorption of drugs from the intestinal lumen, in active secretion of drugs into urine and bile, and in extrusion of drugs from vital organs such as the brain and reproductive tissues (Troutman et al., 2002). As such, P-glycoprotein-mediated transport has become an important issue in the discovery and development of new drugs. For example, new compounds that are promising with regard to target receptor/ enzyme activity can be severely hampered in their ability to elicit pharmacological effects in vivo should they be good substrates for P-glycoprotein, especially if the route of administration is intended to be oral or the target tissues is one rich in P-glycoprotein activity. Furthermore, the potential for drug-drug interactions arises in the event that the P-glycoprotein substrate is coadministered with another agent that can inhibit P-glycoprotein.Several models have been developed to assess drugs as P-glycoprotein substrates. In vitro models have included the Caco...

Small-molecule compounds approved for use as drugs may be “repurposed” for new indications and studied to determine the mechanisms of their beneficial and adverse effects. A comprehensive collection of all small-molecule drugs approved for human use would be invaluable for systematic repurposing across human diseases, particularly for rare and neglected diseases, for which the cost and time required for development of a new chemical entity are often prohibitive. Previous efforts to build such a comprehensive collection have been limited by the complexities, redundancies, and semantic inconsistencies of drug naming within and among regulatory agencies worldwide; a lack of clear conceptualization of what constitutes a drug; and a lack of access to physical samples. We report here the creation of a definitive, complete, and nonredundant list of all approved molecular entities as a freely available electronic resource and a physical collection of small molecules amenable to high-throughput screening.

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.translational research | PCI-32765 | Imbruvica

Since 2009, the Tox21 project has screened ∼8500 chemicals in more than 70 high-throughput assays, generating upward of 100 million data points, with all data publicly available through partner websites at the United States Environmental Protection Agency (EPA), National Center for Advancing Translational Sciences (NCATS), and National Toxicology Program (NTP). Underpinning this public effort is the largest compound library ever constructed specifically for improving understanding of the chemical basis of toxicity across research and regulatory domains. Each Tox21 federal partner brought specialized resources and capabilities to the partnership, including three approximately equal-sized compound libraries. All Tox21 data generated to date have resulted from a confluence of ideas, technologies, and expertise used to design, screen, and analyze the Tox21 10K library. The different programmatic objectives of the partners led to three distinct, overlapping compound libraries that, when combined, not only covered a diversity of chemical structures, use-categories, and properties but also incorporated many types of compound replicates. The history of development of the Tox21 "10K" chemical library and data workflows implemented to ensure quality chemical annotations and allow for various reproducibility assessments are described. Cheminformatics profiling demonstrates how the three partner libraries complement one another to expand the reach of each individual library, as reflected in coverage of regulatory lists, predicted toxicity end points, and physicochemical properties. ToxPrint chemotypes (CTs) and enrichment approaches further demonstrate how the combined partner libraries amplify structure−activity patterns that would otherwise not be detected. Finally, CT enrichments are used to probe global patterns of activity in combined ToxCast and Tox21 activity data sets relative to test-set size and chemical versus biological end point diversity, illustrating the power of CT approaches to discern patterns in chemical− activity data sets. These results support a central premise of the Tox21 program: A collaborative merging of programmatically distinct compound libraries would yield greater rewards than could be achieved separately.

An efficient and versatile Compound Management operation is essential for the success of all downstream processes in high-throughput screening (HTS) and small molecule lead development. Staff, equipment, and processes need to be not only reliable, but remain flexible and prepared to incorporate paradigm changes. In the present report, we describe a system and associated processes which enable handling of compounds for both screening and follow-up purposes at the NIH Chemical Genomics Center (NCGC), a recently-established HTS and probe development center within the Molecular Libraries Initiative of the NIH Roadmap. Our screening process, termed quantitative HTS (qHTS), involves assaying the complete compound library, currently containing >200,000 members, at a series of dilutions to construct a full concentration-response profile. As such, Compound Management at the NCGC has been uniquely tasked to prepare, store, register, and track a verticallydeveloped plate dilution series (i.e., inter-plate titrations) in the 384-well format. These are compressed into a series of 1,536-well plates and are registered to track all subsequent plate storage. Here, we present details on the selection of equipment to enable automated, reliable and parallel compound manipulation in 384-and 1,536-well formats, protocols for preparation of inter-plate dilution series for qHTS, as well as qHTS-specific processes and issues.

Plasmodium falciparumresistance to artemisinin derivatives, the first-line anti-malarial drug, drives the search for new classes of chemotherapeutic agents. Current discovery is primarily directed against the intracellular forms of the parasite. However, late schizont-infected red blood cells (RBCs) may still rupture and cause disease by sequestration; consequently targeting invasion may reduce disease severity. Merozoite invasion of RBCs requires interaction between two parasite proteins AMA1 and RON2. Here we identify the first inhibitor of this interaction that also blocks merozoite invasion in genetically distinct parasites by screening a library of over 21,000 compounds. We demonstrate that this inhibition is mediated by the small molecule binding to AMA1 and blocking the formation of AMA1-RON complex. Electron microscopy confirms that the inhibitor prevents junction formation, a critical step in invasion that results from AMA1-RON2 binding. This study uncovers a strategy that will allow for highly effective combination therapies alongside existing anti-malarial drugs.