作为许多蜂窝信令网络的重要枢纽,KRAS(Kirsten大鼠肉瘤病毒癌基因同源物)已被鉴定为肿瘤生物标志物。它是人类癌症中经常突变的癌基因,和突变引起的KRAS蛋白激活,比如G12D,已在许多人类肿瘤组织中发现。虽然,KRAS蛋白上有两个特定的变构位点(AS1和AS2),可以用作抑制剂开发的靶标,两个单独的变构位点之间的调节机制的差异仍未报道。这里,采用分子动力学模拟结合分子力学广义表面积(MM/GBSA)分析,我们发现两种抑制剂,位于AS1和AS2,能够降低野生型之间的结合自由能,突变型KRAS(G12/D/V/S/C)和GTP显著,然而,抑制剂对野生型之间的结合自由能的影响,突变KRAS和GDP是有限的。此外,AS2抑制剂引起的KRAS和GTP之间结合自由能的降低程度明显大于AS1抑制剂引起的。进一步分析表明,AS1和AS2的两种抑制剂都能够调节开关Ⅰ和开关Ⅱ的波动,以扩大正构位点(GTP结合位点)的口袋,从而减少KRAS与GTP的结合。值得注意的是,两种抑制剂对开关Ⅰ和开关Ⅱ的调控偏好存在显着差异。作为AS2的抑制剂主要调节开关Ⅱ影响正构位点的口袋,而AS1的抑制剂主要通过调节开关Ⅰ的波动来扩大正构位点的口袋。我们的研究比较了两种抑制剂在调节KRAS蛋白活性方面的差异,揭示了AS2作为小分子药物靶点的优势,旨在为新型KRAS蛋白抑制剂的研究提供理论指导。
As the important hub of many cellular signaling networks, KRAS (Kirsten rat sarcoma viral oncogene homologue) has been identified as a tumor biomarker. It is the frequently mutated oncogene in human cancers, and KRAS protein activation caused by mutations, such as G12D, has been found in many human tumors tissues. Although, there are two specific allosteric sites (AS1 and AS2) on the KRAS protein that can be used as the targets for inhibitor development, the difference of regulatory mechanisms between two individual allosteric sites still not be reported. Here, using molecular dynamics simulations combined with molecular mechanics generalized born surface area (MM/GBSA) analysis, we found that both of the inhibitors, located at AS1 and AS2, were able to reduce the binding free energy between wild type, mutant KRAS (G12/D/V/S/C) and GTP remarkably, however the effect of inhibitors on the binding free energy between wild type, mutant KRAS and GDP was limited. In addition, the degree of decrease of binding free energy between KRAS and GTP caused by inhibitors at AS2 was significantly greater than that caused by inhibitors at AS1. Further analysis revealed that both inhibitors at AS1 and AS2 were able to regulate the fluctuation of Switch Ⅰ and Switch Ⅱ to expand the pocket of the orthosteric site (GTP binding site), thereby reducing the binding of KRAS to GTP. Noteworthy there was significant differences in the regulatory preferences on Switch Ⅰ and Switch Ⅱ between two type inhibitor. The inhibitor at AS2 mainly regulated Switch Ⅱ to affect the pocket of the orthosteric site, while the inhibitor at AS1 mainly expand the pocket of the orthosteric site by regulating the fluctuation of Switch Ⅰ. Our study compared the differences between two type inhibitors in regulating the KRAS protein activity and revealed the advantages of the AS2 as the small molecule drug target, aiming to provide theoretical guidance for the research of novel KRAS protein inhibitors.