title: Kinetic studies of small molecule interactions with protein kinases using biosensor technology.
abstract: Protein kinases are among the most commonly targeted groups of molecules in drug discovery today. Despite this, there are few examples of using surface plasmon resonance (SPR) for kinase inhibitor interaction studies, probably reflecting the need for better developed assays for these proteins. In this article, we present a general methodology that uses biosensor technology to study small molecule binding to eight different serine/threonine and tyrosine kinases. Mild immobilization conditions and a carefully composed assay buffer were identified as key success factors. The methodology package consists of direct binding studies of compounds to immobilized kinases, kinase activity assays to confirm inhibitory effects, detailed kinetic analyses of inhibitor binding, and competition assays with ATP for identification of competitive inhibitors. The kinetic assays resolve affinity into the rates of inhibitor binding and dissociation. Therefore, more detailed information on the relation between inhibitor structure and function is obtained. This might be of key importance for the development of effective kinase inhibitors.
authors.affiliation: Department of Biochemistry and Chemistry, Biacore AB, Rapsgatan 7, SE-754 50 Uppsala, Sweden.
authors.forename: Helena
authors.initials: H
authors.lastname: Nordin
authors.forename: Maria
authors.initials: M
authors.lastname: Jungnelius
authors.forename: Robert
authors.initials: R
authors.lastname: Karlsson
authors.forename: Olof P
authors.initials: OP
authors.lastname: Karlsson
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques; D002021:Buffers; D004800:Enzymes, Immobilized / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D047428:Protein Kinase Inhibitors / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D002021:Buffers; D004800:Enzymes, Immobilized; D007093:Imidazoles; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D011743:Pyrimidines; C110631:SB 220025; D011494:Protein Kinases; C408604:2-(4-nitrophenyl)-4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazole; C093642:SB 203580; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole