title: High-throughput biochemical kinase selectivity assays: panel development and screening applications.
abstract: Kinases represent attractive targets for drug discovery. Eight small-molecule kinase inhibitors are currently marketed in the area of oncology, and numerous others are in clinical trials. Characterization of the selectivity profiles of these compounds is important to target appropriate patient populations and to reduce the potential of toxicity due to off-target effects. The authors describe the development, validation, and utilization of a biochemical kinase assay panel for the selectivity profiling of inhibitors. The panel was developed as 29 radiometric Flashplate assays, and then an initial 13 were transitioned to a nonradiometric Caliper mobility shift assay format. Generation of high-quality data from the panel is detailed along with a comparison of the assay formats. Both assay technologies were found to be suitable for panel screening, but mobility shift assays yielded higher data quality. The selectivity data generated here should be useful in computational modeling and help facilitate, in conjunction with sequence and structural information, the rational design of inhibitors with well-defined selectivity profiles.
authors.affiliation: Pfizer Research Technology Center, Cambridge, Massachusetts, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Card
authors.forename: Chris
authors.initials: C
authors.lastname: Caldwell
authors.forename: Hyunsuk
authors.initials: H
authors.lastname: Min
authors.forename: Bina
authors.initials: B
authors.lastname: Lokchander
authors.lastname: Hualin Xi
authors.forename: Simone
authors.initials: S
authors.lastname: Sciabola
authors.forename: Ajith V
authors.initials: AV
authors.lastname: Kamath
authors.forename: Susan L
authors.initials: SL
authors.lastname: Clugston
authors.forename: William R
authors.initials: WR
authors.lastname: Tschantz
authors.lastname: Leyu Wang
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Moshinsky
mesh_terms: D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D020128:Inhibitory Concentration 50; D010770:Phosphotransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology*; D015203:Reproducibility of Results
chemical_list: D047428:Protein Kinase Inhibitors; D010770:Phosphotransferases