上海交通大学学报（医学版）

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2025 , Vol. 45 >Issue 6: 705 - 716

DOI: https://doi.org/10.3969/j.issn.1674-8115.2025.06.005

论著 · 基础研究

KRAS R68G继发突变引发KRASG12D靶向抑制剂MRTX1133耐药的机制研究

  • 王高明 ,
  • 崔然 ,
  • 黎彦璟 ,
  • 刘颖斌
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  • 1.上海交通大学医学院附属仁济医院胆胰外科，上海 200127
    2.上海交通大学医学院附属仁济医院肿瘤系统医学全国重点实验室，上海市肿瘤研究所，上海 200127
    3.上海交通大学医学院附属仁济医院上海市肿瘤系统调控与转化重点实验室，上海 200127
    4.同济大学附属东方医院肝胆胰外科，上海 200120
王高明（1999—），男，博士生；电子信箱：wgm657158702@sjtu.edu.cn。
第一联系人：王高明和黎彦璟设计该研究，王高明和崔然进行分子动力学模拟和统计分析，王高明完成论文写作，王高明、黎彦璟和刘颖斌就KRASG12D对MRTX1133可能的耐药机制进行广泛的文献检索和讨论，黎彦璟和刘颖斌修改稿件。所有作者阅读并同意最终稿件的提交。
黎彦璟，研究员，博士；电子信箱：liyanjing@sjtu.edu.cn
刘颖斌，主任医师，博士；电子信箱：laoniulyb@163.com。

收稿日期: 2024-12-18

  录用日期: 2025-03-07

  网络出版日期: 2025-06-23

基金资助

肿瘤系统医学全国重点实验室自主课题(ZZ-RCPY-23-25);国家自然科学基金(32471528)

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Study on the mechanism of KRAS R68G secondary mutation-induced resistance to KRASG12D-targeted inhibitor MRTX1133

  • WANG Gaoming ,
  • CUI Ran ,
  • LI Yanjing ,
  • LIU Yingbin
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  • 1.Department of Biliary-Pancreatic Surgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
    2.State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
    3.Shanghai Key Laboratory for Cancer Systems Regulation and Clinical Translation (CSRCT-SHANGHAI), Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
    4.Department of Hepatopancreatobiliary Surgery, East Hospital Affiliated Tongji University, Shanghai 200120, China
First author contact:WANG Gaoming and LI Yanjing designed the study. WANG Gaoming and CUI Ran performed molecular dynamics simulations and statistical analyses. WANG Gaoming wrote the manuscript. WANG Gaoming, LI Yanjing, and LIU Yingbin conducted an extensive literature review and discussed potential resistance mechanisms of KRASG12D to MRTX1133. LI Yanjing and LIU Yingbin revised the manuscript. All authors have read the final version of the paper and consented to its submission.
LI Yanjing, E-mail: liyanjing@sjtu.edu.cn
LIU Yingbin, E-mail: laoniulyb@163.com.

Received date: 2024-12-18

  Accepted date: 2025-03-07

  Online published: 2025-06-23

Supported by

State Key Laboratory of Systems Medicine for Cancer(ZZ-RCPY-23-25);National Natural Science Foundation of China(32471528)

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摘要

目的·从原子层面探索KRASG12D/R68G突变诱发肿瘤细胞对MRTX1133耐药的机制。方法·从RCSB蛋白质数据库（Protein Data Bank，PDB）获取KRASG12D与MRTX1133相互作用复合物的晶体结构数据。使用PyMOL软件将KRAS第68位的精氨酸突变为甘氨酸（R68G），构建KRASG12D和KRASG12D/R68G分别与MRTX1133相互作用体系的初始构象。使用LEaP程序构建带有周期性边界的模拟体系，应用ff19SB力场计算KRAS中标准氨基酸的力场参数，应用GAFF（general AMBER force field）力场计算MRTX1133的力场参数，应用TIP3P（intermolecular potential three point）力场计算水分子的力场参数。使用Amber软件对体系进行能量最小化，体系升温至300 K后，进行等温等容平衡和等温等压运动的计算。使用cpptraj轨迹分析软件计算每个体系的均方根偏差（root mean square deviation，RMSD）、体系中每个氨基酸的均方根波动（root mean square fluctuation，RMSF），对轨迹进行主成分分析（principal component analysis，PCA），计算MRTX1133和GDP的溶剂可及表面积（solvent-accessible surface area，SASA）。测量区域之间氢键形成的数量，并计算氨基酸之间的动态交叉相关矩阵（dynamic cross-correlation matrix，DCCM）。结果·RMSD分析显示KRASG12D/R68G体系中KRAS的变化幅度大于KRASG12D体系。RMSF分析显示KRASG12D/R68G体系中KRAS的Switch Ⅰ和Switch Ⅱ区域的波动幅度明显大于KRASG12D体系。PCA分析提示KRASG12D/R68G体系中KRAS的Switch Ⅰ和Switch Ⅱ区域更多地处于向外打开的状态。两体系中Switch Ⅰ与P-loop之间距离以及Switch Ⅱ与P-loop之间距离的比较显示了KRASG12D/R68G体系中的GDP和MRTX1133的结合口袋与KRASG12D体系相比均显著扩大。SASA分析显示KRASG12D/R68G体系中的GDP和MRTX1133的溶剂暴露面积与KRASG12D体系相比均明显增加。DCCM分析显示KRASG12D/R68G体系中Switch Ⅰ、Switch Ⅱ和P-loop区域之间存在更多的分离运动。结论·KRASG12D/R68G突变破坏了Switch Ⅰ和Switch Ⅱ区域之间的相互作用，导致了Switch Ⅰ和Switch Ⅱ的分离，继而导致MRTX1133的结合口袋开放，增加了MRTX1133的溶剂暴露面积，从而加速了MRTX1133的解离，最终导致KRASG12D/R68G对MRTX1133耐药。

关键词： MRTX1133; KRAS继发突变; 分子动力学模拟; 耐药机制

本文引用格式

王高明 , 崔然 , 黎彦璟 , 刘颖斌 . KRAS R68G继发突变引发KRASG12D靶向抑制剂MRTX1133耐药的机制研究[J]. 上海交通大学学报（医学版）, 2025 , 45(6) : 705 -716 . DOI: 10.3969/j.issn.1674-8115.2025.06.005

Abstract

Objective ·To explore the mechanism at the atomic level by which the KRASG12D/R68G mutation induces tumor cell resistance to MRTX1133. Methods ·The crystal structure data of the KRASG12D-MRTX1133 complex were obtained from the RCSB Protein Data Bank (PDB). PyMOL software was used to mutate arginine at position 68 of KRAS to glycine (R68G), constructing the initial conformations of the KRASG12D-MRTX1133 and KRASG12D/R68G-MRTX1133 complexes. The LEaP module was used to build simulation systems under periodic boundary conditions. The ff19SB force field was applied to standard amino acids in KRAS, GAFF (general AMBER force field) to MRTX1133, and TIP3P (intermolecular potential three point) to water molecules. Energy minimization was performed using the Amber software suite. The systems were then heated to 300 K, followed by NVT (constant volume and temperature) equilibration and NPT (constant pressure and temperature) production. Root mean square deviation (RMSD), root mean square fluctuation (RMSF), principal component analysis (PCA) and solvent-accessible surface area (SASA) of MRTX1133 and GDP were analyzed using cpptraj. The number of hydrogen bonds between regions and the dynamic cross-correlation matrix (DCCM) of amino acid movements were also calculated. Results ·RMSD analysis showed greater structural variation in KRAS in the KRASG12D/R68G system compared to the KRASG12D system. RMSF analysis revealed significantly higher fluctuations in the Switch Ⅰ and Switch Ⅱ regions of the KRASG12D/R68G system. PCA indicated that Switch Ⅰ and Switch Ⅱ in the KRASG12D/R68G system were more frequently in an open conformation. The distances between Switch Ⅰ and the P-loop, and between Switch Ⅱ and the P-loop, were larger in the KRASG12D/R68G system, indicating an expanded binding pocket for GDP and MRTX1133 compared to the KRASG12D system. SASA analysis indicated that both GDP and MRTX1133 had increased solvent exposure in the KRASG12D/R68G system. DCCM analysis revealed more decoupled movements among the Switch Ⅰ, Switch Ⅱ and P-loop regions in the KRASG12D/R68G system. Conclusion ·The KRASG12D/R68G mutation disrupts the interactions between the Switch Ⅰ and Switch Ⅱ regions, leading to their separation and the opening of the MRTX1133 binding pocket. This increases the solvent exposure of MRTX1133, accelerates its dissociation, and ultimately results in KRASG12D/R68G resistance to MRTX1133.

Key words： MRTX1133; KRAS secondary mutation; molecular dynamics simulation; resistance mechanism

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