Division of Medicinal and Process Chemistry, Central Drug Research Institute, CSIR, Lucknow, India.
J Chem Inf Model. 2011 Aug 22;51(8):1966-85. doi: 10.1021/ci200185q. Epub 2011 Aug 1.
The current study deciphers the combined ligand- and structure-based computational insights to profile structural determinants for the selectivity of representative diverse classes of FXa-selective and thrombin-selective as well as dual FXa-thrombin high affinity inhibitors. The thrombin-exclusive insertion 60-loop (D-pocket) was observed to be one of the most notable recognition sites for the known thrombin-selective inhibitors. Based on the topological comparison of four common active-site pockets (S1-S4) of FXa and thrombin, the greater structural disparity was observed in the S4-pocket, which was more symmetrical (U-shaped) in FXa as compared to thrombin mainly due to the presence of L99 and I174 residues in latter in place of Y99 and F174 respectively in former protease. The S2 pocket forming partial roof at the entry of 12 Å deep S1-pocket, with two extended β-sheets running antiparallel to each other by undergoing U-turn (∼180̊), has two conserved glycine residues forming H-bonds with the bound ligand for governing ligand binding affinity. The docking, scoring, and binding pose comparison of the representative high-affinity and selective inhibitors into the active sites of FXa and thrombin revealed critical residues (S214, Y99, W60D) mediating selectivity through direct- and long-range electrostatic interactions. Interestingly, most of the thrombin-selective inhibitors attained S-shaped conformation in thrombin, while FXa-selective inhibitors attained L-shaped conformations in FXa. The role of residue at 99th position of FXa and thrombin toward governing protease selectivity was further substantiated using molecular dynamics simulations on the wild-type and mutated Y99L FXa bound to thrombin-selective inhibitor 2. Furthermore, predictive CoMFA (FXa q² = 0.814; thrombin q² = 0.667) and CoMSIA (FXa q² = 0.807; thrombin q² = 0.624) models were developed and validated (FXa r²(test) = 0.823; thrombin r(2)(test) = 0.816) to feature molecular determinants of ligand binding affinity using the docking-based conformational alignments (DBCA) of 141 (88(train)+53(test)) and 39 (27(train)+11(test)) nonamidine class of potent FXa (0.004 ≤ K(i) (nM) ≤ 4700) and thrombin (0.001 ≤ K(i) (nM) ≤ 940) inhibitors, respectively. Interestingly, the ligand-based insights well corroborated with the structure-based insights in terms of the role of steric, electrostatic, and hydrophobic parameters for governing the selectivity for the two proteases. The new computational insights presented in this study are expected to be valuable for understanding and designing potent and selective antithrombotic agents.
当前的研究揭示了基于配体和结构的综合计算见解，以描绘代表性的多种 FXa 选择性、凝血酶选择性以及双重 FXa-凝血酶高亲和力抑制剂的选择性结构决定因素。观察到凝血酶专有的插入 60 环（D 口袋）是已知的凝血酶选择性抑制剂的最显著识别位点之一。基于 FXa 和凝血酶的四个常见活性口袋（S1-S4）的拓扑比较，观察到 S4 口袋的结构差异更大，该口袋在 FXa 中更为对称（U 形），而在凝血酶中则不太对称，主要是由于后者中的 L99 和 I174 残基取代了前者中的 Y99 和 F174 残基。S2 口袋在 12 Å 深的 S1 口袋入口处形成部分屋顶，两个延伸的 β-片层通过 U 形转弯（约 180°）彼此反向平行运行，具有两个保守的甘氨酸残基与结合配体形成氢键，以控制配体结合亲和力。代表性的高亲和力和选择性抑制剂在 FXa 和凝血酶的活性部位的对接、评分和结合构象比较表明，关键残基（S214、Y99、W60D）通过直接和远程静电相互作用介导选择性。有趣的是，大多数凝血酶选择性抑制剂在凝血酶中呈 S 形构象，而 FXa 选择性抑制剂在 FXa 中呈 L 形构象。使用野生型和突变 Y99L FXa 与凝血酶选择性抑制剂 2 结合的分子动力学模拟进一步证实了 FXa 和凝血酶第 99 位残基在控制蛋白酶选择性方面的作用。此外，还开发和验证了预测性 CoMFA（FXa q² = 0.814；thrombin q² = 0.667）和 CoMSIA（FXa q² = 0.807；thrombin q² = 0.624）模型，以使用基于对接的构象排列（DBCA）来确定配体结合亲和力的分子决定因素，该排列涉及 141 个（88 个（train）+53 个（test））和 39 个（27 个（train）+11 个（test））非胍类 FXa（0.004≤Ki（nM）≤4700）和凝血酶（0.001≤Ki（nM）≤940）抑制剂，分别。有趣的是，配体基础的见解与基于结构的见解在控制两种蛋白酶选择性的立体、静电和疏水性参数方面非常吻合。本研究提出的新计算见解有望为理解和设计有效的、选择性的抗血栓形成药物提供有价值的信息。
