High-throughput determination of mode of inhibition in lead identification and optimization.

After finishing the primary high-throughput screening, the screening team is often faced with thousands of hits to be evaluated further. Effective filtering of these hits is crucial in identifying leads. Mode of inhibition (MOI) study is extremely useful in validating whether the observed compound activity is specific to the biological target. In this article, the authors describe a high-throughput MOI determination method for evaluating thousands of compounds using an existing screening infrastructure. Based on enzyme or receptor kinetics theory, the authors developed the method by measuring the ratio of IC(50) or percent inhibition at 2 carefully chosen substrate or ligand concentrations to define an inhibitor as competitive, uncompetitive, or noncompetitive. This not only facilitates binning of HTS hits according to their MOI but also greatly expands HTS utility in support of the medicinal chemistry team's lead optimization practice. Three case studies are presented to demonstrate how the method was applied successfully in 3 discovery programs targeting either an enzyme or a G-protein-coupled receptor.