Minhee Lee, PhD
Columbia University
Substrate-Derived Peptides for Selective Covalent Inhibition of Protein Tyrosine Kinases
Protein tyrosine kinases are critical regulators of cell signaling, and aberrant kinase activity contributes to many diseases. Most kinase inhibitors are ATP-competitive small molecules with strong potency, but achieving selectivity across kinases and disease mutants remains challenging due to the highly conserved ATP-binding site. By contrast, the substrate-binding site recognizes a diverse set of substrates and offers an opportunity for selective inhibition. Here, we present a strategy to design selective, covalent peptide inhibitors of tyrosine kinases with a distinct binding mode from existing inhibitors. Our peptides simultaneously target the substrate-binding site and react with non-conserved cysteines near the active site. The peptides are designed using a broadly applicable pipeline consisting of high-throughput substrate profiling, structural modeling, and non-canonical amino acids to improve potency. In a proof-of-principle study with Src kinase, we demonstrate that optimizing sequence and strategically positioning electrophiles achieves selective reactivity. We apply this strategy to design a selective inhibitor for an oncogenic mutant, K656E, in fibroblast growth factor receptor 1 (FGFR1) kinase. We also show these peptides can bind simultaneously alongside small molecule inhibitors, potentially suppressing the emergence of drug resistant mutations. The presented workflow provides a promising framework for future drug development targeting tyrosine kinases.
Protein tyrosine kinases are critical regulators of cell signaling, and aberrant kinase activity contributes to many diseases. Most kinase inhibitors are ATP-competitive small molecules with strong potency, but achieving selectivity across kinases and disease mutants remains challenging due to the highly conserved ATP-binding site. By contrast, the substrate-binding site recognizes a diverse set of substrates and offers an opportunity for selective inhibition. Here, we present a strategy to design selective, covalent peptide inhibitors of tyrosine kinases with a distinct binding mode from existing inhibitors. Our peptides simultaneously target the substrate-binding site and react with non-conserved cysteines near the active site. The peptides are designed using a broadly applicable pipeline consisting of high-throughput substrate profiling, structural modeling, and non-canonical amino acids to improve potency. In a proof-of-principle study with Src kinase, we demonstrate that optimizing sequence and strategically positioning electrophiles achieves selective reactivity. We apply this strategy to design a selective inhibitor for an oncogenic mutant, K656E, in fibroblast growth factor receptor 1 (FGFR1) kinase. We also show these peptides can bind simultaneously alongside small molecule inhibitors, potentially suppressing the emergence of drug resistant mutations. The presented workflow provides a promising framework for future drug development targeting tyrosine kinases.
