Covalent inhibitors continue to show therapeutic promise. However, off-target reactivity challenges the field. Extensive efforts have been exerted to solve this issue by varying the reactivity attributes of electrophilic warheads, with features such as reversibility or metabolic vulnerability. Here we report the development of a new approach to increase the selectivity of covalent probes and small molecule inhibitors that is independent of warhead reactivity features and can be used in concert with already-existing methods. Using the Bruton’s Tyrosine Kinase (BTK) inhibitor Ibrutinib scaffold for our proof-of-concept, we reasoned that increasing the steric bulk of fumarate-based electrophiles on Ibrutinib should improve selectivity via the steric exclusion of off-targets but ideally retain rates of cysteine reactivity comparable to that of an acrylamide. Using chemical proteomic techniques, we demonstrate that elaboration of the electrophile to a tert-Butyl (t-Bu) fumarate ester significantly decreases time-dependent off-target reactivity and abolishes time-independent off-target reactivity but retains BTK target engagement. While an alkyne-bearing probe analog of Ibrutinib has 247 protein targets, our t-Bu fumarate Ibrutinib probe analog has only 7 protein targets. Of these 7 targets, BTK is the only time-independent target. This 2-order-of-magnitude increase in selectivity is also conferred to the t-Bu inhibitor itself. By shotgun proteomics, we investigated the consequences of treatment with Ibrutinib and our t-Bu analog and discovered that only 8 proteins are downregulated in response to treatment with the t-Bu analog compared to 107 with Ibrutinib. Of these 8 proteins, 7 are also downregulated by Ibrutinib and a majority of these targets are associated with BTK biology. Taken together, these findings reveal a previously-unappreciated opportunity to increase cysteine-reactive covalent inhibitor selectivity through electrophilic structure optimization.

中文翻译：

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改进的亲电子试剂设计，实现出色的共价分子选择性

共价抑制剂继续显示出治疗前景。然而，脱靶反应性对该领域提出了挑战。通过改变亲电弹头的反应属性，具有可逆性或代谢脆弱性等特征，已经做出了广泛的努力来解决这个问题。在这里，我们报告了一种新方法的开发，以提高共价探针和小分子抑制剂的选择性，该方法独立于弹头反应性特征，可与现有方法配合使用。使用 Bruton 的酪氨酸激酶 (BTK) 抑制剂依鲁替尼支架进行我们的概念验证，我们推断，在依鲁替尼上增加基于富马酸盐的亲电子试剂的空间体积应该通过位阻排除脱靶来提高选择性，但理想情况下可以保持与丙烯酰胺相当的半胱氨酸反应率。使用化学蛋白质组学技术，我们证明将亲电试剂加工成叔丁基 (t-Bu) 富马酸酯可显着降低时间依赖性脱靶反应性并消除时间依赖性脱靶反应性，但保留 BTK 目标参与。虽然 Ibrutinib 的带有炔烃的探针类似物有 247 个蛋白质靶标，但我们的 t-Bu 富马酸盐 Ibrutinib 探针类似物只有 7 个蛋白质靶标。在这 7 个目标中，BTK 是唯一一个与时间无关的目标。这种 2 个数量级的选择性增加也归功于 t-Bu 抑制剂本身。通过霰弹枪蛋白质组学，我们研究了依鲁替尼和我们的 t-Bu 类似物治疗的后果，发现只有 8 种蛋白质在使用 t-Bu 类似物治疗时被下调，而依鲁替尼则为 107 种。在这 8 种蛋白质中，有 7 种也被依鲁替尼下调，并且这些靶标中的大多数与 BTK 生物学相关。综上所述，这些发现揭示了通过亲电子结构优化提高半胱氨酸反应性共价抑制剂选择性的一个以前未被重视的机会。