Establishment of screening system toward discovery of kinase inhibitors using label-free on-chip phosphorylation assays.
We describe a label-free method for the kinase inhibition assay toward discovery of kinase inhibitors. The surface plasmon resonance (SPR) imaging analysis using zinc(II) compound was adopted on the on-chip phosphorylation analysis. In this study, following three subjects were focused: (1) to monitor the inhibition of three inhibitors supporting by their specific inhibition mechanisms, (2) to quantify the inhibitory activities, and (3) to prove the reliability of the obtained 50% inhibition concentration (IC(50)) value. First, the inhibitory activities of Amide 5-24, H-89 and Gö6983 on PKA and PKCdelta were determined, and specific inhibitions for two kinases could be observed quantitatively. Second, the inhibition curves of Amide 5-24, Amide 14-22 and H-89 were obtained, and the results supported the two previous reports: (1) the inhibition efficiency of Amide 5-24 was much higher than that of Amide 14-22, and (2) the inhibitory activity of H-89 followed ATP-binding site blocking mechanism. Last, the obtained IC(50) values by the SPR imaging were almost corresponded to those by the solution assay, although on-chip phosphorylation efficiency was low (approximately 12%). In conclusion, validation of the on-chip phosphorylation analysis for kinase inhibitors was achieved. This label-free method might be applied for discovery of kinase inhibitors.
Biotechnology Frontier Project, Toyobo Co., Ltd., Tsuruga, Fukui 914-0047, Japan.
Kazuki
K
Inamori
Motoki
M
Kyo
Kazuki
K
Matsukawa
Yusuke
Y
Inoue
Tatsuhiko
T
Sonoda
Takeshi
T
Mori
Takuro
T
Niidome
Yoshiki
Y
Katayama
D000577:Amides / Q000378:metabolism / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D007211:Indoles / Q000378:metabolism / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D007546:Isoquinolines / Q000378:metabolism / Q000494:pharmacology; D008301:Maleimides / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*; D011493:Protein Kinase C / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D015203:Reproducibility of Results; D013449:Sulfonamides / Q000378:metabolism / Q000494:pharmacology; D020349:Surface Plasmon Resonance
C465664:2-(1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl)-3-(1H-indol-3-yl)maleimide; D000577:Amides; D007211:Indoles; D007546:Isoquinolines; D008301:Maleimides; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide