Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries

Abstract

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 ...

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“…Compounds from qHTS were classified into four major classes (curve class 1-4) based on quality of curve fit and efficacy using criteria published in ref. [69]. We have the highest confidence in class 1.1 (efficacy > 80% with both upper and lower asymptotes), 1.2 (efficacy 30%-80% with both upper and lower asymptotes), or 2.1 (efficacy > 80% with one asymptote) concentration curves, and have less confidence in class 2.2 (efficacy < 80% with one asymptote) and 3 (single point activity) curves.…”

“…Data were processed as reported previously [69]. Briefly, raw plate reads for each titration point were first normalized relative to control inhibitor (100 %) and DMSO-only wells (basal, 0%), and then corrected by applying a pattern correction algorithm using compound-free control plates (DMSO plates).…”

“…The HTS assay was carried out in a 1536-well plate format using a qHTS platform [69]. Compounds were prepared at eight different concentrations (five-fold dilution) in eight different plates.…”

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