Screening assay for identifying agents that modulate pak kinase or cd45 tyrosine phosphatase activity

摘要

Screening assays for identifying an agent that modulates the activity of a phosphate catalyzing enzyme, i.e., a PAK Kinase or a CD45 tyrosine Kinase, comprising the following steps: a) providing a phosphate catalyzing enzyme, b) providing a peptide, c) optionally providing a phosphate containing donor, d) providing a candidate compound, e) contacting the enzyme of step a) with a peptide of step b) and optionally the phosphate containing donor of step c) in the absence of d) and in the presence of d) for a predetermined period of time so that an enzymatic product can be for med, f) transferring at least an aliquot of the enzymatic product formed in step e) to a solid phase which is capable to bind to the peptide in the enzymatic product, g) detecting the enzymatic product bound to the solid phase, h) determining whether there is a difference in the amount of enzymatic product in the absence and in the presence of a candidate compound in step e), and i) choosing an agent that modulated the activity of an enzyme, which method is characterized in that I) the enzyme is a PAK kinase, - the peptide comprises the amico acid sequence S-S-L-R-A-S-T, - the phosphate containing donor of step c) is present in step e), and - the enzymatic product detected in step g) is the amount of phosphoserine and/or phosphothreonine in the bound peptide; OR II) the enzyme is CD45 tyrosine phosphatase, - the peptide comprises the amino acid sequence R-N-Q-E-T-Y-E-T-L-K-H or A-E-N-T-I-T-Y-S-L-L-M-H-P, wherein Y is phosphorylated tyrosine, - the phosphate containing donor of step c) is absent in step e), and - the enzymatic product detected in step g) is the amount of phosphotyrosine in the bound peptide.