Virtual kinetics: using statistical experimental design for rapid analysis of enzyme inhibitor mechanisms

Modern automated drug-screening can generate hundreds of inhibitor leads from diverse chemical sources in a short period of time. Traditional methods of inhibitor analysis are resource intensive and limit the number of inhibitors that can be analyzed for their mechanism of inhibition. This paper presents methods we have developed for rapid estimation of both potency and mechanism of potential inhibitor leads for a biochemically complex screening target (protein kinase C) using commercially available computer programs for statistical experimental design. Our findings indicate that, with careful choice of factor levels, statistical experimental design clearly identifies the various interactions of the assay components with inhibitors. Suitably plotted, the data can be used to examine the competitive nature of the inhibitor and can provide estimates of IC50 and Michaelis constants useful for planning further kinetic work. The techniques used are amenable to automation and should be useful for identifying inhibitors that may have only marginal potency, but exhibit desirable mechanistic profiles suitable for structural analoging efforts.