Abstract：
The main proteinase (Mpro) of SARS-CoV-2 plays a critical role in cleaving viral polyproteins into functional proteins required for viral replication and assembly, making it a prime drug target for COVID-19. It is well known that noncompetitive inhibition offers potential therapeutic options for treating COVID-19, which can effectively reduce the likelihood of cross-reactivity with other proteins and increase the selectivity of the drug. Therefore, the discovery of allosteric sites of Mpro has both scientific and practical significance. In this study, we explored the binding characteristics and inhibiting process of Mpro activity by two recently reported allosteric inhibitors, pelitinib and AT7519 which were obtained by the X-ray screening experiments, to probe the allosteric mechanism via molecular dynamic (MD) simulations. We found that pelitinib and AT7519 can stably bind to Mpro far from the active site. The binding affinity is estimated to be -24.37 ± 4.14 and - 26.96 ± 4.05 kcal/mol for pelitinib and AT7519, respectively, which is considerably stable compared with orthosteric drugs. Furthermore, the strong binding caused clear changes in the catalytic site of Mpro, thus decreasing the substrate accessibility. The community network analysis also validated that pelitinib and AT7519 strengthened intra- and inter-domain communication of Mpro dimer, resulting in a rigid Mpro, which could negatively impact substrate binding. In summary, our findings provide the detailed working mechanism for the two experimentally observed allosteric sites of Mpro. These allosteric sites greatly enhance the \'druggability\' of Mpro and represent attractive targets for the development of new Mpro inhibitors.
摘要：
SARS-CoV-2的主要蛋白酶（Mpro）在将病毒多蛋白裂解为病毒复制和组装所需的功能蛋白中起着关键作用。使其成为COVID-19的主要药物靶标。众所周知，非竞争性抑制为治疗COVID-19提供了潜在的治疗选择，可以有效降低与其他蛋白质交叉反应的可能性，并增加药物的选择性。因此,发现Mpro的变构位点具有科学和现实意义。在这项研究中,我们探索了两种最近报道的变构抑制剂对Mpro活性的结合特性和抑制过程，pelitinib和AT7519通过X射线筛选实验获得，通过分子动力学(MD)模拟来探索变构机制。我们发现pelitinib和AT7519可以稳定地结合远离活性位点的Mpro。pelitinib和AT7519的结合亲和力估计分别为-24.37±4.14和-26.96±4.05kcal/mol，与正构药物相比相当稳定。此外，强结合导致Mpro催化位点发生明显变化，从而降低基材的可接近性。社区网络分析还验证了pelitinib和AT7519增强了Mpro二聚体的域内和域间交流，导致了刚性的Mpro，这可能会对底物结合产生负面影响。总之,我们的发现为Mpro的两个实验观察到的变构位点提供了详细的工作机制。这些变构位点极大地增强了Mpro的“可药用性”，并代表了开发新的Mpro抑制剂的有吸引力的目标。
