Analysis of protein-peptide interaction by a miniaturized fluorescence polarization assay using cyclin-dependent kinase 2/cyclin E as a model system

As a result of the increasing size of chemical libraries, more rapid and hi ghly sensitive strategies are needed to accelerate the process of drug disc overy without increasing the cost. One means of accomplishing this is to mi niaturize the assays that enter high-throughput screening (HTS). Miniaturiz ation requires an assay design that has few steps, has a large degree of se paration between the signal and background, and has a low well to well sign al variation. Fluorescence polarization (FP) is an assay type that, in many cases, meets all of the above requirements. FP is a homogenous method that allows interactions between molecules to be measured directly in solution. This article demonstrates the application of FP in a miniaturized HTS form at, using 1536-well plates, to measure direct binding between cyclin-depend ent kinase 2/cyclin E complex (CDK2/E) and an 8-mer-peptide kinase inhibito r. The data indicate that low variability and high specificity allow rapid and precise identification of antagonist compounds affecting CDK2/E-peptide interactions. (C) 1999 Academic Press.