title: High-affinity bisubstrate probe for fluorescence anisotropy binding/displacement assays with protein kinases PKA and ROCK.
abstract: The bisubstrate fluorescent probe ARC-583 (Adc-Ahx-(D-Arg)(6)-d-Lys(5-TAMRA)-NH2) and its application for the characterization of both ATP- and protein/peptide substrate-competitive inhibitors of protein kinases PKA (cyclic AMP-dependent protein kinase) and ROCK (rho kinase) in fluorescence polarization-based assay are described. High affinity of the probe (K(D)=0.48 nM toward PKA) enables its application for the characterization of inhibitors with nanomolar and micromolar potency and determination of the active concentration of the kinase in individual experiments as well as in the high-throughput screening format. The probe can be used for the assessment of protein-protein interactions (e.g., between regulatory and catalytic subunits of PKA) and as a cyclic AMP biosensor.
authors.affiliation: Institute of Chemistry, University of Tartu, 2 Jakobi St., 51014 Tartu, Estonia.
authors.forename: Angela
authors.initials: A
authors.lastname: Vaasa
authors.forename: Indrek
authors.initials: I
authors.lastname: Viil
authors.forename: Erki
authors.initials: E
authors.lastname: Enkvist
authors.forename: Kaido
authors.initials: K
authors.lastname: Viht
authors.forename: Gerda
authors.initials: G
authors.lastname: Raidaru
authors.forename: Darja
authors.initials: D
authors.lastname: Lavogina
authors.forename: Asko
authors.initials: A
authors.lastname: Uri
mesh_terms: D001665:Binding Sites; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D007700:Kinetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D047428:Protein Kinase Inhibitors; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases