Compound activity identification is the primary goal in high throughput screening (HTS) assays. However, assay artifacts including both systematic (e.g., compound autofluorescence) and nonsystematic (e.g., noise) complicate activity interpretation. In addition, other than the traditional potency parameter, half-maximal effect concentration [EC50], additional activity parameters (e.g., point-of-departure [POD] and weighted area-under-the-curve [wAUC]) could be derived from HTS data for activity profiling. A data analysis pipeline has been developed to handle the artifacts, and to provide compound activity characterization with either binary or continuous metrics. This chapter outlines the steps in the pipeline using Tox21 estrogen receptor (ER) β-lactamase assays, including the formats to identify either agonists or antagonists, as well as the counterscreen assays for identifying artifacts as examples. The steps can be applied to other lower throughput assays with concentration-response data.
Keywords: Assay artifacts; Concentration–response data; Data analysis pipeline; HTS; Point-of-departure; Tox21; qHTS.
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