Discovery of a Covalent Inhibitor Selectively Targeting the Autophosphorylation Site of c-Src Kinase.

Nonreceptor tyrosine kinase c-Src plays a crucial role in cell signaling and contributes to tumor progression. However, the development of selective c-Src inhibitors turns out to be challenging. In our previous study, we performed posttranslational modification-inspired drug design (PTMI-DD) to provide a plausible way for designing selective kinase inhibitors. In this study, after identifying a unique pocket comprising a less conserved cysteine and an autophosphorylation site in c-Src as well as a promiscuous covalent inhibitor, chemical optimization was performed to obtain with nearly 75-fold improved potency (IC = 35.83 ± 7.21 nM). Crystallographic studies revealed the critical C-F···C═O interactions that may contribute to tight binding. The and values validated the improved binding affinity and decreased warhead reactivity of for c-Src. Notably, in vitro tyrosine kinase profiling and cellular activity-based protein profiling (ABPP) cooperatively indicated a specific inhibition of c-Src by . Intriguingly, preferentially binds to inactive c-Src with unphosphorylated Y419 both in vitro and in cells, subsequently disrupting the autophosphorylation. Collectively, our study demonstrated the feasibility of developing selective kinase inhibitors by cotargeting a nucleophilic residue and a posttranslational modification site and providing a chemical probe for c-Src functional studies.