title: Conjugation-free, visual, and quantitative evaluation of inhibitors on protein tyrosine kinases and phosphatases with a luminescent Tb(III) complex.
abstract: A straightforward and visual method to assess inhibitors on protein tyrosine kinases (PTKs) and phosphatases (PTPs) has been developed. These enzymes play critical roles in a number of diseases and, thus, their inhibitors are important for effective therapy. With the use of the long-life luminescence emitted from a binuclear Tb(III) complex, enzymatic reactions of PTKs and PTPs were monitored in real-time, and the inhibitor activity was quantitatively evaluated in terms of the decrease in the rate of luminescence change. No conjugation of the probe to a substrate peptide was necessary. The IC50 values of four inhibitors on three kinds of PTKs [Src, Fyn, and epidermal growth factor receptor (EGFR)] were determined. For example, gefitinib, which is a selective inhibitor on EGFR, inhibited this PTK with IC50 of 22 nM. Towards Src and Fyn (non-targeted PTK), however, IC50 of this inhibitor was greater than 20 μM as expected. Inhibition of two kinds of PTPs (Shp-1 and PTP1B) by two inhibitors was also assayed, providing completely consistent results on their known selectivity. Furthermore, the system where both PTK and PTP are active was monitored and the reactions were visualized with the present Tb(III) complex-based method. High potential of the present method to a variety of systems has been evidenced.
authors.affiliation: Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Meguro-ku, Tokyo, 153-8904, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Akiba
authors.forename: Jun
authors.initials: J
authors.lastname: Sumaoka
authors.forename: Takao
authors.initials: T
authors.lastname: Hamakubo
authors.forename: Makoto
authors.initials: M
authors.lastname: Komiyama
mesh_terms: D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D049449:Luminescence; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D013725:Terbium / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; D013725:Terbium; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases