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This version was published on February 1, 2006
Journal of Biomolecular Screening, Vol. 11, No. 1, 48-56 (2006)
DOI: 10.1177/1087057105282097
© 2006 Society for Biomolecular Sciences

Microarrays for the Functional Analysis of the Chemical-Kinase Interactome

Kurumi Y. Horiuchi

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 p60c-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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This Article
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