具有P-gp抑制活性的秋水仙碱结合部位抑制剂的发现及优化

P-glycoprotein-mediated multidrug resistance (MDR) was a major obstacle limiting the successful chemotherapy of tumor. To overcome the MDR, increasing the sensitivity of tumor cells to chemotherapeutic drugs by inhibiting P-gp activity is an effective way. Colchicine binding site inhibitors (CBSI) attracted much attention in recent years, due chiefly to their dual proliferation-inhibiting/vascular-disrupting activities. However, some classic CBSI are the substrates of P-gp efflux pump and able to significantly increase in P-gp expression. In our previous work, it was found that a series of imidazole derivatives as CBSI displayed potent antiproliferative activities in drug-resistant tumor cells with significantly anti-tubulin polymerization and reduced P-gp liability. Base on the above results, our objective is to address the P-gp-inhibition liability of CBSI with significantly maintaining their antiproliferative activity, and understand the structural basis and structure-activity relationship of P-gp/tubulin dual inhibitory activities. With our constructed pharmacophores, CBSI can be screened in silico to predict such potential P-gp-inhibition activity without affecting its antiproliferative activity. A set of CBSI with P-gp-inhibition activity are developed via computer aided drug design strategy. Then these target compounds will be synthesized, purified and characterized. The potential druggability of the candidate compounds, including dual activities, mechanism of action, metabolic stability and preliminary toxicity, will be evaluated. Our studies will be helpful to develop new antitumor drugs for the treatment of patients with drug resistance mediated by P-gp.

P-gp高表达导致肿瘤细胞产生多药耐药性（MDR）是当前影响肿瘤化疗疗效的主要障碍。通过抑制P-gp的活性、提高肿瘤细胞对药物的敏感性是目前解决MDR的有效途径。具有增殖抑制/血管破坏双重活性的微管蛋白秋水仙碱结合部位抑制剂（CBSI）是近年来抗肿瘤创新药物研究的热点领域。但某些经典的CBSI不仅是P-gp的外排底物，还能导致P-gp表达增加。在前期研究中，申请人发现了一类咪唑类CBSI，不仅能干扰微管蛋白聚合还能抑制P-gp的活性，对耐药肿瘤疗效显著。在此基础上，本项目拟建立“具有P-gp抑制活性CBSI”的计算机虚拟筛选模型。采用计算机辅助的药物设计方法，完成目标化合物的合成、纯化与表征，并考察其双重活性、代谢稳定性、初步毒性等成药特性；阐明“P-gp抑制活性”与“微管蛋白抑制活性”的构效关系规律与联系，揭示CBSI具有P-gp抑制活性的结构基础。为开发治疗耐药肿瘤的创新药物奠定基础。

P-gp介导的MDR是导致肿瘤化疗失败的主要原因之一。CBSI虽然抗肿瘤活性突出，但易成为P-gp的外排底物导致活性丧失。本项目通过详细研究GQ131028的抗MDR机制，发现其可通过抑制耐紫杉醇A549/Taxol细胞中微管蛋白聚合而导致有丝分裂相阻滞。作为一个CBSI，GQ131028不仅不是P-gp的不良底物，还能通过降低P-gp在蛋白和mRNA水平上的表达来抑制P-gp的功能。GQ131028还能诱导的ROS产生并引起A549/Taxol细胞的DNA损伤和GSH降低。研究发现，GQ131028能够诱导A549/Taxol细胞发生了保护性自噬。这表明抑制自噬可以进一步提示GQ131028的抗MDR功效。在明确作用机理后，进一步拓展了GQ131028的构效关系研究。基于已有GQ131028的类似物研究，利用分子对接、分子动力学模拟以及密度泛函计算等方法并建立一个虚拟筛选模型，并揭示了形成“弯曲”构型对保持此类化合物的活性至关重要。在此基础上，利用计算机辅助药物设计方法成功发展了3类新颖的具有P-gp抑制活性CBSI并发展了一种绿色的苄位氧化方法。本项不仅为研发治疗耐药肿瘤的创新药物奠定了基础，还为药物化学合成的绿色化提供了新方法。