Microarrays for the Functional Analysis of the Chemical-Kinase Interactome

¹ Reaction Biology Corporation, Malvern, PA.
² Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA.

^* To whom correspondence should be addressed. E-mail: haiching{at}reactionbiology.com.

	Abstract

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 for monitoring 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^ATP_m = 3.3 µM) and IC₅₀ values for staurosporine (210 nM) and PP2 (326 nM) at 10 µM adenosine triphosphate (ATP). Similarly, B-Raf phosphorylation of MEK-coated microarrays was inhibited in the nanoliter reactions by GW5074 at the expected IC₅₀ of 9 nM. Common kinase inhibitors were printed on microarrays, and their inhibitory activities were systematically profiled against B-Raf (V599E), KDR, Met, Flt-3 (D835Y), Lyn, EGFR, PDGFR, and Tie2. All results indicate that this platform is well suited for kinetic analysis, HTS, large-scale IC₅₀ determinations, and selectivity profiling.

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

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