Label-free electrical sensing of small-molecule inhibition on tyrosine phosphorylation

Protein tyrosine kinases (PTKs) play a central role in human carcinogenesis and have emerged as the promising new targets. Small-molecule inhibitors of PTKs have shown impressive anticancer effects and are rapidly entering the clinic. PTK assays allow for high-throughput identification of small-molecule inhibitors. However, current methods of detecting kinase activity require the use of radioisotopes or expensive reagents; such as fluorescently labeled antibodies. We have developed a novel label-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) cannot be oxidized at similar to 0.65 V. However, when the phosphorylation is successfully inhibited using a small molecule, Tyr can be oxidized and result in a high current response on a multiwalled carbon nanotube-modified screenprinted carbon electrode. We determined the activity of cellular-sarcoma (c-Src) nonreceptor PTK, p60(c-Src), in combination with its highly specific substrate peptide, Raytide. Tyr kinase reactions were also performed in the presence of a well-defined small-molecule inhibitor, 4-amino-5-(4-chlorophenyl)-7- (tert-butyl)pyrazolo[3,4d]pyrimidine (PP2). Based on the dependency of Tyr oxidation signal on inhibitor concentration, IC50 value, half-maximal inhibition of the inhibitor, was estimated as 5 nM for PP2. Our label-free electrochemical method is a promising candidate for pharmaceutical research and development in screening small-molecule inhibitors of PTKs.