Covalent inhibitors represent a promising class of therapeutic compounds. Nonetheless, rationally designing covalent inhibitors to achieve a right balance between selectivity and reactivity remains extremely challenging. To better understand the covalent binding mechanism, a computational study is carried out using the irreversible covalent inhibitor of Bruton tyrosine kinase (BTK) ibrutinib as an example. A multi-μs classical molecular dynamics trajectory of the unlinked inhibitor is generated to explore the fluctuations of the compound associated with the kinase binding pocket. Then, the reaction pathway leading to the formation of the covalent bond with the cysteine residue at position 481 via a Michael addition is determined using the string method in collective variables on the basis of hybrid quantum mechanical–molecular mechanical (QM/MM) simulations. The reaction pathway shows a strong correlation between the covalent bond formation and the protonation/deprotonation events taking place sequentially in the covalent inhibition reaction, consistent with a 3-step reaction with transient thiolate and enolates intermediate states. Two possible atomistic mechanisms affecting deprotonation/protonation events from the thiolate to the enolate intermediate were observed: a highly correlated direct pathway involving proton transfer to the C_α of the acrylamide warhead from the cysteine involving one or a few water molecules and a more indirect pathway involving a long-lived enolate intermediate state following the escape of the proton to the bulk solution. The results are compared with experiments by simulating the long-time kinetics of the reaction using kinetic modeling.

中文翻译：

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依鲁替尼对布鲁顿酪氨酸激酶共价抑制机制的计算研究

共价抑制剂代表了一类有前途的治疗化合物。尽管如此，合理设计共价抑制剂以实现选择性和反应性之间的适当平衡仍然极具挑战性。为了更好地理解共价结合机制，以布鲁顿酪氨酸激酶（BTK）不可逆共价抑制剂伊布替尼为例进行了计算研究。生成未连接抑制剂的多μs经典分子动力学轨迹，以探索与激酶结合袋相关的化合物的波动。然后，基于混合量子力学-分子力学（QM/MM）模拟，使用集合变量中的弦方法确定通过迈克尔加成与481位半胱氨酸残基形成共价键的反应途径。反应途径显示共价键形成与共价抑制反应中顺序发生的质子化/去质子化事件之间存在很强的相关性，这与具有瞬时硫醇盐和烯醇化物中间态的三步反应一致。观察到影响从硫醇盐到烯醇化物中间体的去质子化/质子化事件的两种可能的原子机制：高度相关的直接途径，涉及质子从半胱氨酸转移到丙烯酰胺弹头的Cα，_涉及一个或几个水分子；以及更间接的途径涉及质子逃逸到本体溶液后的长寿命烯醇中间态。通过使用动力学模型模拟反应的长时间动力学，将结果与实验进行比较。