Completing the Screen: Biochemical Cascades to Prioritize HTS Output

Conducting a high‐throughput screen (HTS) of a medically interesting enzyme reaction against a library of 10 5 –10 6 compounds is a common way to identify ligands that may be chemical starting points for drug‐discovery programs. Although the use of HTS has been widely applied for this purpose for several decades, the approach still presents challenges and difficulties that must be navigated uniquely for individual targets. A framework for applying biochemical assays and tools for efficiently progressing through the various stages of a HTS screening campaign from primary screen completion through hit validation has been developed. Specific topics covered include the criteria for primary and secondary (orthogonal) assays, intentional vs. uncertain screen bias, tools for efficient non‐specific inhibitor removal, and inhibitor‐target binding confirmation.