Department of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russian Federation.
Org Biomol Chem. 2020 Apr 29;18(16):3069-3081. doi: 10.1039/d0ob00071j.
The use of selective covalent inhibitors with low binding affinity and high reactivity with the target enzyme is a promising way to solve a long-standing problem of the "undruggable" RAS-like proteins. Specifically, compounds of the ARS family that prevent the activation of the GDP-bound G12C mutant of Kirsten RAS (KRAS) are in the focus of recent experimental research. We report the first computational characterization of the entire reaction mechanism of the covalent binding of ARS-853 to the KRASG12C·GDP complex. The application of molecular dynamics, molecular docking and quantum mechanics/molecular mechanics approaches allowed us to model the inhibitor binding to the protein and the chemical reaction of ARS-853 with Cys12 in the enzyme binding site. We estimated a full set of kinetic constants and carried out numerical kinetic analysis of the process. Thus, we were able to compare directly the physicochemical parameters of the reaction obtained in silico and the macroscopic parameters observed in experimental studies. From our computational results, we explain the observed unusual dependence of the rate constant of covalent complex formation, kobs, on the ARS concentration. The latter depends both on the non-covalent binding step with the equilibrium constant, Ki, and on the rate constant of covalent adduct formation, kinact. The calculated ratio kinact/Ki = 213 M-1 s-1 reproduces the corresponding experimental value of 250 ± 30 M-1 s-1 for the interaction of ARS-853 with KRASG12C. Electron density analysis in the reactive region demonstrates that covalent bond formation occurs efficiently according to the Michael addition mechanism, which assumes the activation of the C[double bond, length as m-dash]C bond of ARS-853 by a water molecule and Lys16 in the binding site of KRASG12C. We also refine the kinact and Ki constants of the ARS-107 compound, which shares common features with ARS-853, and show that the decrease in the kinact/Ki ratio in the case of ARS-107 is explained by changes in both Ki and kinact constants.
利用与靶标酶具有低结合亲和力和高反应性的选择性共价抑制剂是解决长期存在的“不可成药”RAS 样蛋白问题的一种有前途的方法。具体来说，防止 Kirsten RAS (KRAS) 的 GDP 结合 G12C 突变体激活的 ARS 家族化合物是最近实验研究的重点。我们报告了 ARS-853 与 KRASG12C·GDP 复合物的共价结合的整个反应机制的首次计算特征。分子动力学、分子对接和量子力学/分子力学方法的应用使我们能够模拟抑制剂与蛋白质的结合以及 ARS-853 在酶结合位点与 Cys12 的化学反应。我们估计了一组完整的动力学常数，并对该过程进行了数值动力学分析。因此，我们能够直接比较在计算机上获得的反应的物理化学参数和在实验研究中观察到的宏观参数。从我们的计算结果中，我们解释了观察到的共价复合物形成速率常数 kobs 对 ARS 浓度的异常依赖性。后者不仅取决于与平衡常数 Ki 的非共价结合步骤，还取决于共价加合物形成的速率常数 kinact。计算得到的 kinact/Ki = 213 M-1 s-1 与 ARS-853 与 KRASG12C 相互作用的相应实验值 250 ± 30 M-1 s-1 相吻合。反应区域的电子密度分析表明，根据 Michael 加成机制有效地形成了共价键，该机制假定 ARS-853 的 C[双键，长度为 m-dash]C 键被水分子和 KRASG12C 结合位点中的 Lys16 激活。我们还改进了 ARS-107 化合物的 kinact 和 Ki 常数，该化合物与 ARS-853 具有共同特征，并表明 ARS-107 情况下的 kinact/Ki 比值降低是由 Ki 和 kinact 常数的变化引起的。
