The expansion of the target landscape of covalent inhibitors requires the engagement of nucleophiles beyond cysteine. Although the conserved catalytic lysine in protein kinases is an attractive candidate for a covalent approach, selectivity remains an obvious challenge. Moreover, few covalent inhibitors have been shown to engage the kinase catalytic lysine in animals. We hypothesized that reversible, lysine-targeted inhibitors could provide sustained kinase engagement in vivo, with selectivity driven in part by differences in residence time. By strategically linking benzaldehydes to a promiscuous kinase binding scaffold, we developed chemoproteomic probes that reversibly and covalently engage >200 protein kinases in cells and mice. Probe–kinase residence time was dramatically enhanced by a hydroxyl group ortho to the aldehyde. Remarkably, only a few kinases, including Aurora A, showed sustained, quasi-irreversible occupancy in vivo, the structural basis for which was revealed by X-ray crystallography. We anticipate broad application of salicylaldehyde-based probes to proteins that lack a druggable cysteine.

共价抑制剂靶标范围的扩展需要亲核试剂超越半胱氨酸的参与。尽管蛋白激酶中保守的催化赖氨酸是共价方法的一个有吸引力的候选者，但选择性仍然是一个明显的挑战。此外，很少有共价抑制剂被证明可以与动物体内的激酶催化赖氨酸结合。我们假设可逆的赖氨酸靶向抑制剂可以在体内提供持续的激酶参与，选择性部分是由停留时间的差异驱动的。通过战略性地将苯甲醛连接到混杂的激酶结合支架，我们开发了化学蛋白质组学探针，可逆和共价结合细胞和小鼠中的 > 200 种蛋白激酶。邻位羟基显着提高了探针激酶的停留时间到醛。值得注意的是，只有少数激酶（包括 Aurora A）在体内表现出持续的、准不可逆的占据，X 射线晶体学揭示了其结构基础。我们预计基于水杨醛的探针将广泛应用于缺乏可药用半胱氨酸的蛋白质。