Using IMAP technology to identify kinase inhibitors: comparison with a substrate depletion approach and analysis of the nature of false positives.
IMAP is a fluorescence polarisation-based assay method which can be applied to the measurement of protein kinase activity. Using a model serine/threonine kinase we found that IMAP generated a good assay window (Z' > 0.8), was very tolerant of DMSO, and was flexible with respect to sample processing (stopped reactions were stable over a period of several days). Using a set of six low molecular weight inhibitors of the kinase, we found a good correlation between IMAP and scintillation proximity assay (SPA) potency data. IMAP, which measures product accumulation, was compared in an HTS setting with a substrate depletion method (luminescence-based measurement of ATP concentration). There was a reasonable (approximately 50%) overlap in primary hits from a 17,000 compound set, but more apparent false positives were generated from the IMAP method. We followed up the compounds that showed activity in the IMAP method but not in the luminescence assay. Approximately 10% of these compounds displayed intrinsic fluorescence, suggesting that they were false actives by virtue of intrinsic spectroscopic properties. Compound activity by competition of phosphopeptide binding to IMAP beads can occur with high concentrations of chelating compounds, but did not occur with any of the false actives, suggesting that this form of interference is rare.
Singh
P
P
AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK. pirthipal.singh@astrazeneca.com
Lillywhite
B
B
Bannaghan
C
C
Broad
P
P
D000255:Adenosine Triphosphate / Q000032:analysis / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005189:False Positive Reactions; D005454:Fluorescence Polarization / Q000379:methods*; D008163:Luminescent Measurements; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000187:drug effects* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D019311:Staurosporine / Q000494:pharmacology
D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine; D004121:Dimethyl Sulfoxide