Surface-plasmon-resonance-based biosensor with immobilized bisubstrate analog inhibitor for the determination of affinities of ATP- and protein-competitive ligands of cAMP-dependent protein kinase.
Interactions between adenosine-oligoarginine conjugates (ARC), bisubstrate analog inhibitors of protein kinases, and catalytic subunits of cAMP-dependent protein kinase (cAPK Calpha) were characterized with surface-plasmon-resonance-based biosensors. ARC-704 bound to the immobilized kinase with subnanomolar affinity. The immobilization of ARC-704 to the chip surface via streptavidin-biotin complex yielded a high-affinity surface (K(D)=16nM). The bisubstrate character of ARC-704 was demonstrated with various ligands targeted to ATP-binding pocket (ATP and inhibitors H89 and H1152P) and protein-substrate-binding domain of Calpha (RIIalpha and GST-PKIalpha) in competition assays. The experiments performed on surfaces with different immobilization levels of ARC-704 produced similar results. The closeness of the obtained affinities of the tested compounds to the inhibitory potencies and affinities of the compounds measured with other methods demonstrates the applicability of the chip with the immobilized biligand inhibitor for the characterization of both ATP- and substrate protein-competitive ligands of basophilic protein kinases.
Viht
Kaido
K
Institute of Organic and Bioorganic Chemistry, University of Tartu, 51014 Tartu, Estonia.
Schweinsberg
Sonja
S
Lust
Marje
M
Vaasa
Angela
A
Raidaru
Gerda
G
Lavogina
Darja
D
Uri
Asko
A
Herberg
Friedrich W
FW
D000255:Adenosine Triphosphate / Q000378:metabolism; D001667:Binding, Competitive; D015374:Biosensing Techniques / Q000379:methods*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000378:metabolism; D008024:Ligands; D008954:Models, Biological; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*
D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D008024:Ligands; D011506:Proteins; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases