Substrate-Derived Peptides for Selective Covalent Inhibition of Protein Tyrosine Kinases.

Protein tyrosine kinases are important regulators of cell signaling, and aberrant kinase activity contributes to many human diseases, including cancers. All protein tyrosine kinases share a highly conserved ATP-binding pocket but diverge in their substrate-binding sites in order to mediate distinct signaling events. Many potent and efficacious ATP-competitive tyrosine kinase inhibitors have been developed; however, it remains challenging to achieve on-target selectivity across different kinases and target-specific disease mutants, given the high degree of conservation in the ATP-binding pocket. By contrast, the variable substrate-binding site offers an opportunity for selective inhibition, provided molecules can be targeted to this site. Here, we present a modular strategy to design selective, peptide-based covalent inhibitors of tyrosine kinases with a distinct binding mode from existing ATP-competitive inhibitors. Using Src kinase as a model system, we demonstrate that Src-selective reactivity can be achieved by first designing an optimized substrate peptide and then strategically positioning an electrophile on the peptide to target a nonconserved cysteine on the kinase. We show that substrate-derived covalent peptides can inhibit kinase activity, bind simultaneously with an ATP-competitive inhibitor, and even inhibit the activity of kinases bearing a common drug resistance mutation. We further explore the application of this approach to develop an inhibitor of the cancer-relevant fibroblast growth factor receptor 1 kinase that shows selectivity for an oncogenic mutant over the wild-type enzyme. Our modular strategy to generate selective covalent peptides targeting protein tyrosine kinases provides a promising framework for future chemical probe and drug development efforts.