The perceived and actual burden of false positives in high-throughput screening has received considerable attention; however, few studies exist on the contributions of distinct mechanisms of non-specific effects like chemical reactivity, assay signal interference, and colloidal aggregation. Here, we analyze the outcome of a screen of 197,861 diverse compounds in a concentration-response format against the cysteine protease cruzain, a target expected to be particularly sensitive to reactive compounds and using an assay format with light detection in the short-wavelength region where significant compound autofluorescence is typically encountered. Approximately 1.9% of all compounds screened were detergent-sensitive inhibitors. The contribution from autofluorescence and compounds bearing reactive functionalities was dramatically lower: of all hits, only 1.8% were autofluorescent and 1.48% contained reactive or undesired functional groups. The distribution of false positives was relatively constant across library sources. The simple step of including detergent in the assay buffer suppressed the nonspecific effect of approximately 93% of the original hits.

High-throughput screening (HTS) is increasingly being adopted in academic institutions, where the decoupling of screening and drug development has led to unique challenges, as well as novel uses of instrumentation, assay formulations, and software tools. Advances in technology have made automated unattended screening in the 1536-well plate format broadly accessible and have further facilitated the exploration of new technologies and approaches to screening. A case in point is our recently-developed quantitative high-throughput screening (qHTS) paradigm which tests each library compound at multiple concentrations to construct concentration-response curves (CRCs) generating a comprehensive data set for each assay (Inglese et al, Proc. Natl. Acad. Sci USA 103, 11473–11478). The practical implementation of qHTS for cell-based and biochemical assays across libraries of >100,000 compounds (e.g. between 700,000 –2,000,000 sample wells tested) requires maximal efficiency and miniaturization, and the ability to easily accommodate many different assay formats and screening protocols. Here, we describe the design and utilization of a fully-integrated and automated screening system for qHTS at the NIH Chemical Genomics Center. We report system productivity, reliability, and flexibility, as well as modifications made to increase throughput, add additional capabilities, and address limitations. The combination of this system and qHTS has lead to the generation of over 6 million CRCs from >120 assays in the last three years, and is a technology that can be widely implemented to increase efficiency of screening and lead generation.

The thyroid-stimulating hormone (TSH; thyrotropin) receptor belongs to the glycoprotein hormone receptor subfamily of 7-transmembrane spanning receptors. TSH receptor (TSHR) is expressed mainly in thyroid follicular cells and is activated by TSH, which regulates the growth and function of thyroid follicular cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small-molecule agonists of the TSHR are available. To screen for novel TSHR agonists, the authors miniaturized a commercially available cell-based cyclic adenosine 3',5' monophosphate (cAMP) assay into a 1536-well plate format. This assay uses an HEK293 cell line stably transfected with the TSHR coupled to a cyclic nucleotide gated ion channel as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal homogeneous time-resolved fluorescence cAMP-based assay. Forty-nine compounds in several structural classes have been confirmed as the small-molecule TSHR agonists that will serve as a starting point for chemical optimization and studies of thyroid physiology in health and disease.