Cell‐Active, Irreversible Covalent Inhibitors Targeting a Surface‐Exposed Non‐Catalytic Lysine on Aurora a Kinase by Using Squarate Chemistry

Targeting surface‐exposed lysines in kinases through covalent modification presents a major challenge due to their high p K a and inherently low reactivity. While current research primarily targets more reactive catalytic lysines buried in the ATP‐binding pocket, no systematic rational strategy has yet been developed for selectively engaging surface‐exposed lysines. Herein, we present a versatile strategy for developing cell‐active covalent kinase inhibitors (CKIs) by selectively targeting unique surface‐exposed lysines using squarate chemistry. By using Aurora A (AURKA) as a proof‐of‐concept, we systematically evaluated this approach against other well‐known lysine‐reactive warheads (e.g., EBA, SO 2 F, and OSO 2 F) and demonstrated, for the first time, squarates’ superior efficacy in engaging these challenging low‐reactivity lysines. Amongst various AURKA CKIs, AL8 emerged as the first‐in‐class squarate‐based, cell‐active inhibitor, exhibiting excellent selectivity in both biochemical and cellular assays with long‐residence time in covalently engaging endogenous AURKA. Detailed investigation of effects of leaving groups on squaric esters provided valuable insights for future development of lysine‐reactive CKIs. Our finding has established squarate‐containing ligands as a unique and readily tunable platform for covalent modification of surface‐exposed, non‐catalytic lysines in targeted kinase drug discovery.