Targeted Covalent Inhibitors bind to their targets both covalent and non-covalent modes, providing exceptionally high affinity and selectivity. These inhibitors have been effectively employed as inhibitors of protein kinases, with Taunton and coworkers (Nat. Chem. Biol. 2015, 11 (7), 525–531) reporting a notable example of a TCI with a cyanoacrylamide warhead that forms a covalent thioether linkage to an active-site cysteine (Cys481) of Bruton's tyrosine kinase. The specific mechanism of the binding and the relative importance of the covalent and non-covalent interactions is difficult to determine experimentally, but established simulation methods for calculating the absolute binding affinity of an inhibitor cannot describe the covalent bond forming steps. Here, an integrated approach using alchemical free energy perturbationand QM/MM molecular dynamics methods was employed to model the complete Gibbs energy profile for the covalent inhibition of BTK by a cyanoacrylamide TCI. These calculations provide a rigorous and complete absolute Gibbs energy profile of the covalent modification binding process. The mechanism is ionic, where the target cysteine is deprotonated to form a nucleophilic thiolate, which then undergoes a facile conjugate addition to the electrophilic functional group to form a bond with the non covalently bound ligand. This model predicts that the formation of the covalent linkage makes binding 19.3 kcal/mol more exergonic than the non-covalent binding alone. Nevertheless, non-covalent interactions between the ligand and individual amino acid residues in the binding pocket of the enzyme are also essential for ligand binding,particularly, van der Waals dispersion forces that have a larger contribution to the binding energy than the covalent component in absolute terms. This model also shows that the mechanism of covalent modification of a protein occurs through a complex series of steps and that entropy, conformational flexibility, non-covalent interactions, and the formation of covalent linkage are all significant factors in the ultimatebinding affinity of a covalent drug to its target.

中文翻译：

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计算布鲁顿酪氨酸激酶中可药用半胱氨酸的共价修饰的全自由能谱

靶向共价抑制剂可同时结合其靶标的共价和非共价模式，从而提供极高的亲和力和选择性。这些抑制剂已被有效地用作蛋白激酶的抑制剂，Taunton及其同事（Nat。Chem。Biol。2015，11（7），525–531）报道了TCI与氰基丙烯酰胺弹头形成共价硫醚的著名例子。与布鲁顿酪氨酸激酶活性位点的半胱氨酸（Cys481）连接。结合的具体机制以及共价和非共价相互作用的相对重要性很难通过实验确定，但是用于计算抑制剂的绝对结合亲和力的既定模拟方法无法描述共价键形成步骤。这里，结合了使用炼金术自由能微扰和QM / MM分子动力学方法的综合方法，对氰基丙烯酰胺TCI共价抑制BTK的完整吉布斯能谱进行建模。这些计算提供了共价修饰结合过程的严格和完整的绝对吉布斯能谱。其机理是离子的，其中靶半胱氨酸被去质子化以形成亲核的硫醇盐，然后对亲电子的官能团进行容易的共轭加成以与非共价结合的配体形成键。该模型预测，共价键的形成使结合力比单独的非共价键结合高19.3 kcal / mol。不过，配体与酶结合口袋中单个氨基酸残基之间的非共价相互作用对于配体结合也是必不可少的，特别是，范德华分散力对结合能的贡献比绝对值更大。该模型还表明，蛋白质的共价修饰机制是通过一系列复杂的步骤完成的，并且熵，构象柔韧性，非共价相互作用以及共价键的形成都是共价药物最终结合亲和力的重要因素达到目标