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Microarrays for the Functional Analysis of the Chemical-Kinase Interactome

Yuan Wang

Reaction Biology Corporation, Malvern, PA

Scott L. Diamond

Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA

Haiching Ma

Reaction Biology Corporation, Malvern, PA

A central challenge in chemical biology is profiling the activity of a large number of chemical structures against hundreds of biological targets, such as kinases. Conventional 32P-incorporation or immunoassay of phosphorylated residues produces high-quality signals formonitoring kinase reactions but is difficult to use in high-throughput screening (HTS) because of cost and the need for well-plate washing. The authors report a method for densely archiving compounds in nanodroplets on peptide or protein substrate-coated microarrays for subsequent profiling by aerosol deposition of kinases. Each microarray contains over 6000 reaction centers (1.0 nL each) whose phosphorylation progress can be detected by immunofluorescence. For p60^c-src, the microarray produced a signal-to-background ratio of 36.3 and Z' factor of 0.63 for HTS and accurate enzyme kinetic parameters (K _m ATP = 3.3 µ M) and IC50 values for staurosporine (210 nM) and PP2 (326 nM) at 10 µ M adenosine triphosphate (ATP). Similarly, B-Raf phosphorylation of MEK-coatedmicroarrayswas inhibited in the nanoliter reactions by GW5074 at the expected IC ₅₀of 9 nM. Common kinase inhibitors were printed onmicroarrays, and their inhibitory activities were systematically profiled against B-Raf (V599E), KDR, Met, Flt-3 (D835Y), Lyn, EGFR, PDGFRß, and Tie2. All results indicate that this platform is well suited for kinetic analysis, HTS, large-scale IC ⁵⁰ determinations, and selectivity profiling.

Key Words: chemical microarray • kinase profiling • functional assay • high-throughput screening (HTS) • ELISA

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