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

doi:10.3969/j.issn.1674-8115.2025.06.005

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (6): 705-716.doi: 10.3969/j.issn.1674-8115.2025.06.005

KRAS R68G继发突变引发KRAS^G12D靶向抑制剂MRTX1133耐药的机制研究

王高明¹^,²^,³, 崔然⁴, 黎彦璟²^,³(), 刘颖斌¹^,²^,³()

^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

收稿日期:2024-12-18 接受日期:2025-03-07 出版日期:2025-06-23 发布日期:2025-06-23
通讯作者: 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。
作者简介:王高明（1999—），男，博士生；电子信箱：wgm657158702@sjtu.edu.cn。
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)

Study on the mechanism of KRAS R68G secondary mutation-induced resistance to KRAS^G12D-targeted inhibitor MRTX1133

WANG Gaoming¹^,²^,³, CUI Ran⁴, LI Yanjing²^,³(), LIU Yingbin¹^,²^,³()

^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

Received:2024-12-18 Accepted:2025-03-07 Online:2025-06-23 Published:2025-06-23
Contact: LI Yanjing, E-mail: liyanjing@sjtu.edu.cn
LIU Yingbin, E-mail: laoniulyb@163.com.
Supported by:
State Key Laboratory of Systems Medicine for Cancer(ZZ-RCPY-23-25);National Natural Science Foundation of China(32471528)

摘要/Abstract

摘要：

目的·从原子层面探索KRAS^G12D/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KRAS^G12D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KRAS^G12D和KRAS^G12D/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KRAS^G12D/R68G体系中KRAS的变化幅度大于KRAS^G12D体系。RMSF分析显示KRAS^G12D/R68G体系中KRAS的Switch Ⅰ和Switch Ⅱ区域的波动幅度明显大于KRAS^G12D体系。PCA分析提示KRAS^G12D/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KRAS^G12D/R68G体系中的GDP和MRTX1133的结合口袋与KRAS^G12D体系相比均显著扩大。SASA分析显示KRAS^G12D/R68G体系中的GDP和MRTX1133的溶剂暴露面积与KRAS^G12D体系相比均明显增加。DCCM分析显示KRAS^G12D/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·KRAS^G12D/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KRAS^G12D/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

Abstract:

Objective ·To explore the mechanism at the atomic level by which the KRAS^G12D/R68G mutation induces tumor cell resistance to MRTX1133. Methods ·The crystal structure data of the KRAS^G12D-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 KRAS^G12D-MRTX1133 and KRAS^G12D/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 KRAS^G12D/R68G system compared to the KRAS^G12D system. RMSF analysis revealed significantly higher fluctuations in the Switch Ⅰ and Switch Ⅱ regions of the KRAS^G12D/R68G system. PCA indicated that Switch Ⅰ and Switch Ⅱ in the KRAS^G12D/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 KRAS^G12D/R68G system, indicating an expanded binding pocket for GDP and MRTX1133 compared to the KRAS^G12D system. SASA analysis indicated that both GDP and MRTX1133 had increased solvent exposure in the KRAS^G12D/R68G system. DCCM analysis revealed more decoupled movements among the Switch Ⅰ, Switch Ⅱ and P-loop regions in the KRAS^G12D/R68G system. Conclusion ·The KRAS^G12D/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 KRAS^G12D/R68G resistance to MRTX1133.

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

中图分类号:

王高明, 崔然, 黎彦璟, 刘颖斌. KRAS R68G继发突变引发KRAS^G12D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.

WANG Gaoming, CUI Ran, LI Yanjing, LIU Yingbin. Study on the mechanism of KRAS R68G secondary mutation-induced resistance to KRAS^G12D-targeted inhibitor MRTX1133[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(6): 705-716.

图/表 6

图1 MRTX1133和KRAS中MRTX1133的结合口袋及相关的耐药突变Note： A. Chemical structure of MRTX1133. B. The MRTX1133 binding pocket (PDB ID: 7RPZ). C. Six secondary mutations, including V9E, V9W, V9Q, T58Y, Y96W and Q99L, cause significant steric hindrance or block the formation of interactions between MRTX1133 and KRAS. Red areas stand for steric clashes.

Fig 1 MRTX1133 and its binding pocket in KRAS, and associated resistance mutations

图2 KRASG12D 和KRASG12D/R68G 的构象动力学Note： A. The initial structures of the KRASG12D and KRASG12D/R68G systems. B. RMSDs of Cα atoms in KRASG12D and KRASG12D/R68G systems. C. RMSFs of each residue in KRASG12D and KRASG12D/R68G systems. Yellow region stands for the Switch Ⅰ region, while pink region represents the Switch Ⅱ region. D. The distribution of delta RMSF values of the KRASG12D/R68G system relative to the KRASG12D system. Red regions represent higher RMSF values, whereas blue regions stand for lower RMSF values.

Fig 2 Conformational dynamics of KRASG12D and KRASG12D/R68G

图3 KRAS蛋白的全局构象转变Note： A. Projections of the first and second principal components (PC1 & PC2) from molecular dynamics simulations of KRASG12D and KRASG12D/R68G systems. B. Conformational changes along PC1.

Fig 3 Global conformational changes of the KRAS protein

图4 KRAS蛋白的构象景观和代表性构象Note： A. Characteristic vectors indicating distances of Cα atoms between D12 and P34 (dD12-P34, red dotted line), between D12 and G60 (dD12-G60, blue dotted line), and between P34 and G60 (dP34-G60, yellow dotted line). B. Conformational landscapes generated using the dD12-P34 and dD12-G60 in KRASG12D (blue) and KRASG12D/R68G (red)systems. C. Surface representations of the representative structures in conformational clusters C1 to C4.

Fig 4 Conformational landscape and representative conformations of the KRAS protein

图5 MRTX1133和GDP的SASANote： A. Diagram of the SASA of MRTX1133. B. Density plots of SASA of MRTX1133 in KRASG12D and KRASG12D/R68G systems. C. Diagram of the SASA of GDP. D. Density plots of SASA of GDP in KRASG12D and KRASG12D/R68G systems.

Fig 5 SASAs of MRTX1133 and GDP

图6 Switch Ⅰ和Switch Ⅱ区域在运动中的关系Note： A. DCCM plot of the KRASG12D system. B. Delta DCCM plot of the KRASG12D/R68G system relative to the KRASG12D system. Region a represents the decoupled motion between Switch Ⅰ and P-loop; region b indicates the decoupled motion between Switch Ⅱ and P-loop; region c suggests the decoupled motion between Switch Ⅰ and Switch Ⅱ. C. Distance distribution of Cα atoms between P34 and G60 (dP34-G60). D. Average hydrogen bonds formed between Switch Ⅰ and Switch Ⅱ during the simulations. E. Representative structures of KRASG12D (magenta) and KRASG12D/R68G (cyan) systems show the distance (yellow dotted line) and polar contacts (green dotted line) between Switch Ⅰ and Switch Ⅱ.

Fig 6 Dynamic relationship between Switch Ⅰ and Switch Ⅱ regions

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KRAS R68G继发突变引发KRAS^G12D靶向抑制剂MRTX1133耐药的机制研究

Study on the mechanism of KRAS R68G secondary mutation-induced resistance to KRAS^G12D-targeted inhibitor MRTX1133

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