Calculating the probability of detection for inhibitors in enzymatic or binding reactions in high-throughput screening

In high-throughput screening (HTS) for drug candidates from a library containing tens of thousands to millions of chemical compounds. one problem is assessing the sensitivity of an assay for detecting compounds with a particular potency. For example, when looking for inhibitors of an enzyme, what is the potency of an inhibitor that will be readily detected by an enzyme inhibition assay? Similarly. when assessing compounds that inhibit binding between receptors and ligands or similar molecule-to-molecule interactions, what potency of an inhibitor will be readily detected? In this article, the well-established concepts of Michaelis-Menten kinetics and Langmuir binding isotherms are combined with fundamental statistical principles to yield a measure of assay sensitivity. The approach is general and can be modified to accommodate situations where the reaction kinetics is known to be more complicated than situations described by, the Michaelis-Menten and Langmuir equations. The calculations presented take into account the concentration Of inhibitor used. the variability of the assay, the relationship between the K-m or K-d of the reaction and the substrate or ligand concentration used, the threshold or cutoff value used for determining "hits," and the number of replicates used in screening. (c) 2005 Elsevier Inc. All rights reserved.