title: Microarrays for the functional analysis of the chemical-kinase interactome.
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 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 (KmATP = 3.3 microM) and IC50 values for staurosporine (210 nM) and PP2 (326 nM) at 10 microM 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, PDGFRbeta, and Tie2. All results indicate that this platform is well suited for kinetic analysis, HTS, large-scale IC50 determinations, and selectivity profiling.
authors.affiliation: Reaction Biology Corporation, Malvern, PA 19355, USA.
authors.forename: Kurumi Y
authors.initials: KY
authors.lastname: Horiuchi
authors.forename: Yuan
authors.initials: Y
authors.lastname: Wang
authors.forename: Scott L
authors.initials: SL
authors.lastname: Diamond
authors.forename: Haiching
authors.initials: H
authors.lastname: Ma
mesh_terms: D001681:Biological Assay; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D007700:Kinetics; D008024:Ligands; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D010748:Phosphopeptides / Q000378:metabolism; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism; D019311:Staurosporine; D013379:Substrate Specificity
chemical_list: D008024:Ligands; D010748:Phosphopeptides; D010770:Phosphotransferases; D015688:Oncogene Protein pp60(v-src); D048493:Proto-Oncogene Proteins B-raf; D019311:Staurosporine