Targeted Covalent Inhibition of Pyk2 via a Paralog-Specific Cysteine for Selective Antiplatelet Action

Focal adhesion kinase 2 (FAK2, also known as Pyk2), a focal adhesion kinase (FAK) family member, has emerged as a promising antiplatelet target with minimal impact on normal hemostasis. However, the development of Pyk2-selective inhibitors has been constrained by limited paralog selectivity and suboptimal in vivo pharmacokinetics. Here, we report compound 2-1, a first-in-class covalent inhibitor that selectively targets Pyk2 by hijacking a unique cysteine residue (Cys545) absent in the sister kinase FAK1. Using structure-guided design, we modified the reversible inhibitor PF-4594755 with an acrylamide electrophile and a methylazetidine spacer to enable covalent engagement of Cys545. Intracellular NanoBRET assays and an alkyne-tagged analogue of 2-1 validated its covalent mechanism and kinome-wide selectivity. Functionally, 2-1 impaired platelet responses to the thromboxane A2 analog U46619, while showing moderate-to-minimal effects on adenosine diphosphate- and thrombin-induced aggregation. Under microfluidic simulated arterial and venous flow conditions in human whole blood, 2-1 reduced platelet adhesion comparably to aspirin. In an electrolytic carotid artery thrombosis model, 2-1 tripled the thrombolytic efficacy of recombinant tissue plasminogen activator without increasing bleeding risks. This work provides a foundation for the development of selective Pyk2 inhibitors as a new class of safe, bleeding-sparing antiplatelets for thrombotic disorders.