Understanding Enzymes as Reporters or Targets in Assays Using Quantitative High-throughput Screening (qHTS)

The U.S. National Institutes of Health Chemical Genomics Center (NCGC) has established a new screening paradigm, quantitative high-throughput screening (qHTS), wherein concentration-response curves (CRCs) are rapidly recorded on large compound collections (> 300,000). The data is automatically fit to the Hill equation and the CRCs are subjected to a classification scheme. This approach reduces false positive and negative rates compared to the traditional screening approaches where only a single concentration is tested and provides a pharmacological database that can be used to construct large-scale bioactivity profiles. We demonstrate how this approach was used to examine a coupled enzyme assay where the production of ATP by human pyruvate kinase M2 (PykM2) was coupled to the ATP-dependent bioluminescent enzyme, firefly luciferase (FLuc), to produce a luminescent signal. This identified chemical probes which specifically activate PykM2 while also providing a bioactivity profile of FLuc inhibitors. Examining the latter uncovered a counterintuitive phenomenon of great importance to compound discovery efforts wherein FLuc inhibitors specifically produce a non-specific luminescent response in cell-based assays.