Greater than 99% of kinase inhibitors target the highly conserved ATP binding site. These compounds often potently inhibit multiple kinases which complicates their use as biological probes. Recently, attention has turned towards targeting less conserved sites such as the protein substrate binding site. We aim to develop general methodology for the discovery of substrate-competitive kinase inhibitors. Substrate activity screening identifies substrates of an enzyme instead of inhibitors, and then the reactive portion of the substrate is modified to convert it into an inhibitor. Using an ADP-based detection assay, we identified small molecule substrates of the protein tyrosine kinase c-Src. Through the incorporation of multiple fluorines, we have converted a substrate into an inhibitor. Our optimized inhibitor has greater than 60-fold selectivity over the highly similar kinase c-Abl, and Lineweaver-Burk plots have confirmed a substrate-competitive, ATP-noncompetitive binding mode.

Substrate activity screening methodology for protein tyrosine kinases

245th National Meeting (2013)

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