First Quantum Mechanics/Molecular Mechanics Studies of the Inhibition Mechanism of Cruzain by Peptidyl Halomethyl Ketones
First Quantum Mechanics/Molecular Mechanics Studies of the Inhibition Mechanism of Cruzain by Peptidyl Halomethyl Ketones
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DOI：
10.1021/bi501551g
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发表时间：
2015-06-02
期刊：
影响因子：
2.9
通讯作者：
Moliner, Vicent
中科院分区：
文献类型：
--
作者：
Arafet, Kemel;Ferrer, Silvia;Moliner, Vicent
Cruzain 是原生动物寄生虫克氏锥虫在恰加斯病感染期间表达的主要半胱氨酸蛋白酶，因此，开发该蛋白的抑制剂是设计针对该疾病的有效疗法的有希望的目标。本文通过混合量子力学/分子力学-分子动力学（MD）模拟的方式研究了两种不同的不可逆肽基卤甲基酮（PHK）抑制剂对cruzain的抑制机制，以获得可能的自由能反应路径的完整表示。这些已经根据 AM1d/MM 和 DFT/MM 理论水平计算的平均力势在自由能表面上进行了追踪。对抑制过程可能的反应机制进行的分析表明，活性位点半胱氨酸（Cys25）对抑制剂碳原子的亲核攻击和卤-碳键的裂解在一步中发生。从动力学角度来看，PClK 似乎比 PFK 更有利。该结果与其他木瓜蛋白酶类酶的实验研究一致。对结果的更深入分析表明，PClK 和 PFK 之间差异的根源可能是抑制剂和蛋白质活性位点残基之间建立的不同稳定相互作用。任何通过涉及硫代半缩酮中间体和三元锍中间体形成的逐步机制探索抑制过程可行性的尝试均未成功。然而，尽管表现出更高的自由能垒，但通过质子化硫代半酮缩醛（His159 作为质子供体参与）的机制似乎是可行的。我们的结果表明 PClK 可以作为开发合适的 cruzain 抑制剂的起点
Cruzain is a primary cysteine protease expressed by the protozoan parasite Trypanosoma cruzi during Chagas disease infection, and thus, the development of inhibitors of this protein is a promising target for designing an effective therapy against the disease. In this paper, the mechanism of inhibition of cruzain by two different irreversible peptidyl halomethyl ketones (PHK) inhibitors has been studied by means of hybrid quantum mechanics/molecular mechanics-molecular dynamics (MD) simulations to obtain a complete representation of the possible free energy reaction paths. These have been traced on free energy surfaces in terms of the potential of mean force computed at AM1d/MM and DFT/MM levels of theory. An analysis of the possible reaction mechanisms of the inhibition process has been performed showing that the nucleophilic attack of an active site cysteine, Cys25, on a carbon atom of the inhibitor and the cleavage of the halogen-carbon bond take place in a single step. PClK appears to be much more favorable than PFK from a kinetic point of view. This result would be in agreement with experimental studies in other papain-like enzymes. A deeper analysis of the results suggests that the origin of the differences between PClK and PFK can be the different stabilizing interactions established between the inhibitors and the residues of the active site of the protein. Any attempt to explore the viability of the inhibition process through a stepwise mechanism involving the formation of a thiohemiketal intermediate and a three-membered sulfonium intermediate has been unsuccessful. Nevertheless, a mechanism through a protonated thiohemiketal, with participation of His159 as a proton donor, appears to be feasible despite showing higher free energy barriers. Our results suggest that PClK can be used as a starting point to develop a proper inhibitor of cruzain