Establishment of screening system toward discovery of kinase inhibitors using label-free on-chip phosphorylation assays

Kazuki Inamori, Motoki Kyo, Kazuki Matsukawa, Yusuke Inoue, Tatsuhiko Sonoda, Takeshi Mori, Takuro Niidome, Yoshiki Katayama

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

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 (IC50) value. First, the inhibitory activities of Amide 5-24, H-89 and Gö6983 on PKA and PKCδ 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 IC50 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.

Original languageEnglish
Pages (from-to)179-185
Number of pages7
JournalBioSystems
Volume97
Issue number3
DOIs
Publication statusPublished - Sep 1 2009

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Phosphorylation
Amides
Inhibitor
Screening
Labels
Assays
Chip
Phosphotransferases
Surface Plasmon Resonance
Surface plasmon resonance
Inhibitory Concentration 50
Surface Plasmon
Zinc Compounds
Imaging techniques
Adenosinetriphosphate
Binding sites
Imaging
Protein Kinase C
Zinc
Adenosine Triphosphate

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Applied Mathematics
  • Modelling and Simulation
  • Statistics and Probability

Cite this

Establishment of screening system toward discovery of kinase inhibitors using label-free on-chip phosphorylation assays. / Inamori, Kazuki; Kyo, Motoki; Matsukawa, Kazuki; Inoue, Yusuke; Sonoda, Tatsuhiko; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki.

In: BioSystems, Vol. 97, No. 3, 01.09.2009, p. 179-185.

Research output: Contribution to journalArticle

Inamori, Kazuki ; Kyo, Motoki ; Matsukawa, Kazuki ; Inoue, Yusuke ; Sonoda, Tatsuhiko ; Mori, Takeshi ; Niidome, Takuro ; Katayama, Yoshiki. / Establishment of screening system toward discovery of kinase inhibitors using label-free on-chip phosphorylation assays. In: BioSystems. 2009 ; Vol. 97, No. 3. pp. 179-185.
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