Screening Kinase Inhibitors with a Microarray-Based Fluorescent and Resonance Light Scattering Assay

Abstract

In this technical note, a microarray-based spectroscopic assay with two readout principles, fluorescence and resonance light scattering (RLS), for screening kinase inhibitors has been reported. In this assay, the phosphorylation and inhibition events are marked by biotinylated antiphosphoserinen/antiphosphotyrosine antibodies, and gold nanoparticles are attached to the antibodies by standard avidin-biotin chemistry followed by silver deposition for RLS signal enhancement. The avidin conjugated fluorescein is used as a fluorescent probe. Assays for both serine kinase, the alpha-catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP) dependent protein kinase (PKA), and tyrosine kinase, leukocyte-specific protein tyrosine kinase (LCK), have been developed. The utility of this assay to high-throughput screening was demonstrated with a commercial inhibitor library, a collection of 80 kinase inhibitors, and satisfactory results were obtained. In addition, quantitative determination of binding strength and the inhibiting type (type I) of these inhibitors are also demonstrated by the adenosine 5'-triphosphate (ATP) competing assays.