强心苷探针的设计、合成及其增强KRAS突变型肺癌对化疗药敏感性的靶标研究

KRAS is one of the most frequently mutated genes in lung cancer. Up to now, no targeted therapy has been approved for the treatment of KRAS-mutant lung cancer. Hence, there remains a clinically unmet need to develop effective strategies that can increase the therapeutic response of KRAS mutant lung cancer. During our preliminary study, we found that cardiac glycosides (CGs) derived from the plants of Thevetia peruviana and Antiaris toxicaria significantly suppressed the chemotherapeutic drug camptothecin (CPT)-induced Chk1 phosphorylation in KRAS mutant lung cancer cells. Remarkably, CGs selectively enhanced the growth inhibition effect of chemotherapeutic drugs on KRAS mutant cancer cells compared to KRAS wild type lung cancer cells. Our results indicate a potential strategy to specifically target KRAS mutant lung cancer by combining cardiac glycosides with chemotherapy. However, the precise mechanisms and the underlying target proteins of CGs remain unclear. In this proposal, we will introduce a azido group or a alkynyl group with 3-[3-(but-3-yn-1-yl)-3H-diazirin-3-yl]propanoic acid to the C-6´ of the sugar moiety of cardiac glycoside, synthesizing no tag cardiac glycoside probes. Subsequently, bioimaging coupled with chemoproteomics (pull-down/LC-MS/MS) will be employed to identify cellular proteins that bind cardiac glycoside in KRAS mutant type lung cancer cells. Further, the functions of potential target proteins will be characterized. Our study may discover new targets through which CGs enhance the anticancer effect of chemotherapeutic drugs in KRAS mutant type lung cancer cells. This work will lay groundwork for future application of CGs in the clinic through combined administration with chemotherapeutic agents.

KRAS突变是肺癌中最常见的突变之一。目前缺乏针对KRAS突变型肺癌的靶向治疗药物，因此急需寻找新的治疗手段。前期我们发现来源于植物的强心苷类物质能够有效抑制化疗药引起的肺癌细胞内具有保护作用的DNA损伤应答的激活，且能够选择性地增强KRAS突变型肺癌对化疗药的敏感性。但是，强心苷是如何增强KRAS突变型肺癌对化疗药敏感性，其作用靶标及机制尚不明确。本课题拟对具有较强活性的强心苷化合物进行修饰，引入叠氮基团或连有光亲和基团的炔基来合成化学小分子探针，一方面进行细胞内化合物定位研究；另一方面“钓取”细胞内与强心苷直接相互作用的靶标蛋白。结合LC-MS/MS技术对“钓取”的靶标蛋白进行鉴定，并进一步对鉴定的靶标蛋白进行功能分析，揭示强心苷增强KRAS突变型肺癌对化疗药敏感性的机理。这些工作将阐明强心苷发挥协同抗肿瘤作用的机制，并为强心苷联合化疗药物治疗KRAS突变型肺癌的临床应用提供科学依据。

KRAS突变是肺癌中最常见的突变之一。目前临床上缺乏针对KRAS突变型肺癌的靶向药物，化疗仍然是其重要的治疗手段，但疗效极为有限。我们基于DNA损伤应答通路建立了化疗增敏剂的筛选模型，发现强心苷能够显著抑制化疗药激活的DNA损伤应答通路，并在细胞及动物水平验证了其能够增敏化疗药对KRAS突变型肺癌的抗癌作用。.为了探究强心苷增敏化疗药的机制，我们一方面设计合成了7个强心苷探针（P1-P7）其中包括3个连有光亲和标签的强心苷探针（P5-P7），并评价了其活性，结果显示探针P1-P6较好的维持了强心苷抗肿瘤作用及增敏化疗药的疗效，但只有连有光交联基团的强心苷探针（P5和P6）具有较好的靶标蛋白标记作用，可用于进一步强心苷靶标研究。同时简化了探针合成路线，并将其应用于天然活性糖苷化合物探针的合成；另一方面，我们利用氨基酸稳定同位素标记的方法发现强心苷能显著下调DNA双链断裂（DSB）相关修复蛋白UHRF1。进一步通过多种生化、药理手段研究发现强心苷通过抑制KRAS突变型肺癌中升高的ERK信号，进而选择性抑制KRAS突变型肺癌中UHRF1的表达及其调控的DNA损伤通路的激活，最终靶向KRAS突变型的肺癌细胞，增强其对化疗药物的敏感性。同时，还考察了六个临床使用的强心苷药物增敏化疗药的疗效，发现它们与AT2表现了相似的活性。.以上研究，揭示了强心苷增强化疗药抗肿瘤作用的一种新的机制，有望开发出更强效、毒性更小的强心苷类化合物作为化疗药物增敏剂，用于治疗KRAS突变型肺癌。.

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