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

doi:10.1073/pnas.0604348103

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

NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3370

Communicated by Francis S. Collins, National Institutes of Health, Bethesda, MD, May 31, 2006 (received for review April 12, 2006)

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 (>10⁵ 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.

Footnotes

*To whom correspondence should be addressed at:
NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, Bethesda, MD 20892-3370.
E-mail: jinglese{at}mail.nih.gov
Author contributions: J.I. and D.S.A. designed research; D.S.A., R.L.J., A.S., A.Y., and W.Z. performed research; A.S. and W.Z. contributed new reagents/analytic tools; J.I., D.S.A., A.J., and R.L.J. analyzed data; and J.I., D.S.A., R.L.J., and C.P.A. wrote the paper.
Conflict of interest statement: No conflicts declared.
Data deposition: The bioassays reported in this paper have been deposited in the PubChem database, http://pubchem.ncbi.nlm.nih.gov (ID codes 361, 410, and 411).
↵ † Calculations are based on 30- and 4-μl well volumes for 384- and 1,536-well plates, respectively.
↵ ‡ Additional analysis comparing qHTS with the maximal concentrations screened is given in supporting information.
Abbreviations:

AC50,

half-maximal activity concentration;

HTS,

high-throughput screening;

qHTS,

quantitative HTS;

PK,

pyruvate kinase;

SAR,

structure–activity relationship
Freely available online through the PNAS open access option.