Contemporary chemical biology and drug discovery are increasingly focused on the discovery of inhibitory molecules that interact with enzyme targets in specific ways, such as allosteric or orthosteric binding. Hence, there is increasing interest in evaluating hit compounds from high-throughput diversity screening to determine their mode of interaction with the target. In this work, the common inhibition modalities are reviewed and clarified. The impact of substrate concentration, relative to substrate KM, for each common inhibition modality is also reviewed. The pattern of changes in IC50 that accompany increasing substrate concentration are shown to be diagnostic of specific inhibition modalities. Thus, replots of IC50 as a function of the ratio [S]/KM are recommended as a simple and rapid means of assessing inhibition modality. Finally, specific recommendations are offered for ideal experimental conditions for the determination of inhibition modality through the use of IC50 replots.
Keywords: assay design; enzyme inhibitor; high-throughput screening; inhibition modality.