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 approximately 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 screen-printed 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[3,4-d]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.
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan, Canada. kagan.kerman@usask.ca
Kagan
K
Kerman
Mun'de
M
Vestergaard
Eiichi
E
Tamiya
D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004563:Electrochemistry; D004583:Electrons; D015394:Molecular Structure; D010084:Oxidation-Reduction; D019000:Phosphotyrosine / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D011743:Pyrimidines / Q000737:chemistry / Q000494:pharmacology*
C412373:AG 1879; D011743:Pyrimidines; D019000:Phosphotyrosine; D016392:Proto-Oncogene Proteins pp60(c-src); D017868:Cyclic AMP-Dependent Protein Kinases