High-affinity bisubstrate probe for fluorescence anisotropy binding/displacement assays with protein kinases PKA and ROCK.
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.
Vaasa
Angela
A
Institute of Chemistry, University of Tartu, 2 Jakobi St., 51014 Tartu, Estonia.
Viil
Indrek
I
Enkvist
Erki
E
Viht
Kaido
K
Raidaru
Gerda
G
Lavogina
Darja
D
Uri
Asko
A
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*
D005456:Fluorescent Dyes; D047428:Protein Kinase Inhibitors; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases