Identification of KRAS mutants (G12C, G12D, and G12V) inhibitors

To identify and validate novel small-molecule inhibitors targeting KRAS G12C, G12D, and G12V mutants through a structure-based drug design and experimental approach. We employed molecular docking, molecular dynamics (MD) simulations, MM-PBSA binding free energy calculations, and principal component analysis (PCA) to screen and evaluate potential inhibitors targeting the Switch-II pocket of KRAS mutants. Top-ranking compounds were experimentally validated using Bio-Layer Interferometry (BLI) for binding affinity and MTT assays to assess anticancer activity in breast and lung cancer cell lines. Compound C797–1505 showed strong binding to KRAS G12V (Dissociation constant (KD) = 141 µM), outperforming the reference Sotorasib (KD = 345 µM). C190–0346 displayed weak affinity toward KRAS G12C. MTT assays revealed that C797–1505 reduced breast cancer cell viability (Half-maximal Inhibitory Concentration (IC50) = 43.51 µM), while both compounds demonstrated significant cytotoxicity against lung cancer cells (IC50 = 18.78 µM and 22.93 µM, respectively). Our integrated computational and experimental strategies successfully identified selective KRAS mutant inhibitors with promising anticancer activity, particularly against G12V and G12C driven tumors. These findings support further development and preclinical evaluation of these compounds as targeted therapeutics.

本研究旨在通过基于结构的药物设计与实验手段，筛选并验证靶向KRAS G12C、G12D及G12V突变体的新型小分子抑制剂。我们采用分子对接、分子动力学（molecular dynamics, MD）模拟、MM-PBSA结合自由能计算以及主成分分析（principal component analysis, PCA）等方法，对靶向KRAS突变体开关II口袋（Switch-II pocket）的潜在抑制剂进行筛选与评价。我们对排名靠前的化合物开展实验验证：通过生物层干涉术（Bio-Layer Interferometry, BLI）检测其结合亲和力，通过MTT实验评估其在乳腺癌与肺癌细胞系中的抗癌活性。化合物C797–1505对KRAS G12V展现出较强结合能力（解离常数（Dissociation constant, KD）=141 µM），其活性优于参比药物索托拉西布（Sotorasib，KD=345 µM）。C190–0346对KRAS G12C的结合亲和力较弱。MTT实验结果显示，C797–1505可降低乳腺癌细胞存活率（半数抑制浓度（Half-maximal Inhibitory Concentration, IC50）=43.51 µM）；两种化合物对肺癌细胞均展现出显著的细胞毒性，对应的IC50分别为18.78 µM与22.93 µM。本研究整合计算与实验策略，成功筛选得到具有良好抗癌活性的选择性KRAS突变体抑制剂，尤其对G12V与G12C驱动的肿瘤效果显著。上述研究结果为这些化合物作为靶向治疗药物的进一步开发与临床前评价提供了坚实支撑。