糖酵解新型抑制剂的合成优化及其对肺癌肿瘤干细胞杀灭作用的研究

Lung cancer is a common malignant disease that severely threatens the lives and health of human beings. Currently, there is a lack of effective drugs that can improve the long-term survival of lung cancer patients. The persistence of cancer stem cells, which have high potential to form tumor with drug-resistant property, may be an important reason for why lung cancer are so difficult to treat and cure. Our preliminary study has shown that the side population (SP) of lung cancer cells with stem-cell properties exhibit significantly high glycolytic activity and are very sensitive to glycolytic inhibitors 3-brompyruvate (3-BrPA) and propyl 3-bromopyruvate (3-BrOP), suggesting that inhibition of glycolysis may be a new strategy to effectively kill lung cancer stem cells and improve therapeutic effect. However, the two glycolytic inhibitors 3-BrPA and 3-BrOP are chemically unstable, have high covalent binding to serum proteins, and are not highly specific against the glycolytic enzyme GAPDH. These shortcomings limit the clinical utility of these compounds as therapeutic drugs. We have modified these two compounds, and have obtained some novel glycolytic inhibitors with better properties. In this research project, we propose to (1) develop novel glycolytic inhibitors as anticancer drug candidates with better chemical stability and higher target specificity by further chemical modifications and optimization of the structures of 3-BrPA and 3-BrOP; (2) test the ability of the new compounds to eliminate lung cancer stem cells in vitro and in vivo, and elucidate the underlying mechanisms; and (3) develop novel strategy to combine new glycolytic inhibitors with traditional chemotherapeutic drugs to completely kill lung tumor cells. Since currently there is no effective drugs against lung cancer stem cells, this research project will lay a foundation for the development of effective drugs that eliminate cancer stem cells, and thus has high scientific significance with potential clinical applications.

肺癌是一种常见的恶性肿瘤，目前缺乏有效提高病人长期生存的药物。肿瘤干细胞具有很强的成癌潜能和抗药性，是肺癌难以治疗和根除的重要原因。至今尚未有针对肺癌干细胞的特效药物。我们前期研究发现，具有肿瘤干性的肺癌侧群细胞糖酵解活性显著升高，糖酵解抑制剂3-BrPA及衍生物3-BrOP能高效杀灭该类细胞。提示，抑制糖酵解是有效清除肺癌干细胞和提高肺癌疗效的新策略。但3-BrPA等因稳定性差和对糖酵解关键酶GAPDH的选择性不高等原因，限制其用于临床。通过优化合成其类似物，我们发现了更好的糖酵解新型抑制剂。本项目拟：1，进一步优化3-BrPA等化合物，获得理化性质更好和选择性更强的糖酵解新型抑制剂候选药；2，验证其在体内外杀灭肺肿瘤干细胞的活性，并阐明其作用机制；3，探讨糖酵解新型抑制剂与传统化疗药物联用治疗肺癌的新策略。本项目为开发靶向肺肿瘤干细胞的新型药物提供实验依据，具有重要科学价值和临床意义。

肺癌是一种常见的恶性肿瘤，目前缺乏有效提高病人长期生存的药物。肿瘤干细胞具有很强的成癌潜能和抗药性，是肺癌难以治疗和根除的重要原因。至今尚未有针对肺癌干细胞的特效药物。我们前期研究发现，具有肿瘤干性的肺癌侧群细胞糖酵解活性显著升高，糖酵解抑制剂3-BrPA及衍生物3-BrOP能高效杀灭该类细胞。提示，抑制糖酵解是有效清除肺癌干细胞和提高肺癌疗效的新策略。但3-BrPA等因稳定性差和对糖酵解关键酶-3-磷酸甘油醛脱氢酶（GAPDH）的选择性不高等原因，限制其用于临床。本项目在实施过程中，1）针对3-BrPA和3-BrOP等化合物进行了结构优化，发现了抗肿瘤活性最好、干预肺癌细胞能量分子ATP最好的新颖糖酵解抑制剂LM340，该化合物容易制备；2）进一步实验证实LM340对肺癌、结直肠癌、肝癌、白血病等多种肿瘤细胞都具有比3-BrPA更好的抗肿瘤活性；3）通过含有牛血清培养基预孵育后的凋亡实验和巯基丙醇处理的核磁实验证实了LM340比3-BrPA更稳定，能够抵抗血清和巯基化合物的孵育；4）通过等温滴定量热和酶动力实验阐明LM340强有力结合GAPDH，与底物3-磷酸甘油醛处在不同位置，而辅酶NAD+可以增强LM340和GAPDH的结合能力；5）阐明LM340和抗风湿临床药物金诺芬能够抑制肺癌细胞ATP的生成，海马代谢仪实验或葡萄糖摄取等实验证实这两化合物都能干预糖酵解活动，进而杀伤具有肿瘤干性类似性质的SP细胞，下调ABCG2和SOX4等肿瘤干性标记物表达，诱导肺癌细胞凋亡；6）验证了LM340在体内能够明显抑制肺癌细胞A549和HCC827移植瘤的生长，金诺芬和临床化疗药物阿霉素可以协同抑制A549移植瘤生长。本项目为开发靶向肺肿瘤干细胞的新型药物提供实验依据，具有重要科学价值和临床意义。