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http://www.nature.com/nchembio
Web End = High-throughput screening assays for the identification of chemical probes
James Inglese, Ronald L Johnson, Anton Simeonov, Menghang Xia, Wei Zheng, Christopher P Austin & Douglas S Auld
High-throughput screening (HTS) assays enable the testing of large numbers of chemical substances for activity in diverse areas of biology. The biological responses measured in HTS assays span isolated biochemical systems containing purified receptors or enzymes to signal transduction pathways and complex networks functioning in cellular environments. This Review addresses factors that need to be considered when implementing assays for HTS and is aimed particularly at investigators new to this field. We discuss assay design strategies, the major detection technologies and examples of HTS assays for common target classes, cellular pathways and simple cellular phenotypes. We conclude with special considerations for configuring sensitive, robust, informative and economically feasible HTS assays.
A variety of strategies (Fig. 1) have been used for HTS assays, including the measurement of catalytic activity from a purified enzyme1, a reconstituted complex2,3, a cellular extract4,5 or a phenotype69 in intact cells. Configuring assays to function within the constraints imposed by HTS differentiates an HTS assay from traditional laboratory assays, as outlined in Table 1. The 96-well microtiter plate, originally designed to facilitate serological studies in virology10, was later recognized as a suitable replacement for cuvette, tube and dish-based assays. Combined with spectrophotometric plate readers, the microtiter plate quickly became the standard for performing parallel assays for rapid preliminary evaluation of compound bioactivity. During the 1990s, the tremendous increase in chemical libraries and assays with sensitivities amenable to miniaturization further drove the parallel processing concept that today is practiced in the 96-, 384- or 1,536-microwell plate formats, among others. High throughput is a relative term, but it is generally defined as the testing of 10,000 to 100,000 compounds per day, accomplished with mechanization that ranges from manually operated workstations to fully automated robotic systems11,12. Assays designed for the purpose of HTS attempt to integrate biological fidelity with enabling assay and screening technologies. Subsequently, understanding the pharmacological impact of the assay design is critical to the identification of biologically relevant compounds from HTS.
Assay design for HTS
An effective HTS strategy considers both the primary and subsequent...