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

Z. Wang, X. Wang, Y. Li, P. Li, S. Huang, P. Chen, G. Tang, X. Ding, Z. Zhang, Z.-M. Zhang, Y. Zhou, S. Q. Yao, X. Lu, 

Angew. Chem. Int. Ed. 2025, e202510763

https://doi.org/10.1002/anie.202510763

Targeting surface-exposed lysines in kinases through covalent modification presents a major challenge due to their high pKa 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, SO2F, and OSO2F) 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.