| Abstract
| - Protein tyrosine kinases (PTKs) play a central role inhuman carcinogenesis and have emerged as the promising new targets. Small-molecule inhibitors of PTKs haveshown impressive anticancer effects and are rapidlyentering the clinic. PTK assays allow for high-throughputidentification of small-molecule inhibitors. However, current methods of detecting kinase activity require the useof radioisotopes or expensive reagents; such as fluorescently labeled antibodies. We have developed a novellabel-free approach for the quantitative detection of peptide tyrosine (Tyr) phosphorylation using the electrochemical oxidation current signal of Tyr. When the phosphorylation is achieved, the phosphorylated Tyr (Tyr-P) cannotbe oxidized at ∼0.65 V. However, when the phosphorylation is successfully inhibited using a small molecule,Tyr can be oxidized and result in a high current responseon a multiwalled carbon nanotube-modified screen-printed carbon electrode. We determined the activity ofcellular-sarcoma (c-Src) nonreceptor PTK, p60c-Src, incombination with its highly specific substrate peptide,Raytide. Tyr kinase reactions were also performed in thepresence of a well-defined small-molecule inhibitor,4-amino-5-(4-chlorophenyl)-7- (tert-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Based on the dependency of Tyroxidation signal on inhibitor concentration, IC50 value,half-maximal inhibition of the inhibitor, was estimated as5 nM for PP2. Our label-free electrochemical method isa promising candidate for pharmaceutical research anddevelopment in screening small-molecule inhibitors ofPTKs.
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