新型氮唑类抗真菌先导物结构优化及其与靶酶结合模式研究

Azole antifungals are the first-line drugs in treating invasive fungal infections, and keep being hotpoint in antifungal study. Facing the problems including resistance, antifungal spectrum to be widen, and high motality of invasive fungal infections (about 50%), azole antifungal has been developed slowly in the past decade due to the lack of innovation in optimizing the side chain in their structures, thus it urgently need new drug design method based on true mode of azole antifungal, especially the side chain of it, binding its target. We previously found a novel antifungal compound NT-2, which exhibits higher efficacy and wider antigunngal spectrum than voriconazole and fluconazole. NT-2 was prepared by novel asymmetric synthesis. In this project, we plan to optimize NT-2, and initially prepare and screen 200 derivatives. Based on the CYP51 crystal of Aspergillus funigatus reported in 2015, we will set up a new method for virtue screening with SYBAL software, then run several rounds of “design-virtue screening-synthesis-screening- method optimization and design”. By investigating the relationships of “structure-activity-virtue docking score”, we are expecting to obtain further insight of the binding mode of CYP51 with the side chain in azoles. Next, we plan to introduce photoaffinity group into the side chain of the active compounds to prepare photoaffinity small-molecule probes which can capture their binding sites and peptide sequences in CYP51, thus can validate the virtue docking and optimize our drug design method. Another 100 compounds, designed by new method, will be prepared and screened. This project is aimed to find out 2-3 novel antifungal compounds with more potential than NT-2 against Candida albicans, Crytococcus Neofonmans and Aspergillus funigatus.

氮唑类药物是抗深部真菌感染的主力和研究热点，面临耐药、抗菌谱窄、深部真菌感染致死率高（约50%）等问题，近十年来发展缓慢，关键是其侧链结构创新乏力，迫切需要基于准确的靶酶与药物侧链结合模式的药物设计方法。我们前期运用不对称合成新路线获得优效广谱、结构新颖的手性化合物NT-2。本课题拟采用2015年报道的首个致病真菌CYP51晶体结构，运用SYBAL软件，开展多轮次“设计-对接-合成-测活-优化设计”循环，合成并测活200个NT-2类似物，研究“结构-活性-对接打分”三者关系，获得高可信度的CYP51与药物结合模式。随后，选取高活性化合物，将光亲和基团引入其侧链，设计合成筛选活性小分子探针，捕获其侧链部分与CYP51的结合位点和序列，验证并修正虚拟对接模式，优化药物设计方法，再设计合成100个化合物并测活，目标获得2-3个对白念珠菌、烟曲霉菌和新生隐球菌的药效均优于NT-2的新化合物。

氮唑类药物是抗深部真菌感染的主力和研究热点。近三十年来真菌耐药性问题愈发严重，氮唑类药物研发进展缓慢且受自身毒性、抗菌谱、药物相互作用等问题制约，导致深部真菌感染致死率仍居高不下（约50%）。本课题立足前期优选获得的广谱、结构新颖的手性先导化合物NT-2，针对其侧链结构积极改造创新，利用报道的致病真菌CYP51晶体结构，结合计算机辅助SYBAL软件，开展多轮次的“设计-对接-合成-测活-优化设计”循环，第一轮基于氮唑类药物活性片段，设计合成并测活200个新型氮唑类似物，研究“结构-活性-对接”三者关系，指导后续合理设计；第二轮选取高活性化合物，将光亲和基团引入其侧链，合成筛选活性小分子探针，通过生物实验捕获其侧链部分与CYP51的结合位点和序列，验证并修正虚拟对接模式；第三轮基于上述优化的药物设计方法，再次合成并测活100个新型氮唑类似物，形成具体的构效关系（SAR）。通过多轮次循环，我们建立了一套基于靶点CYP51与氮唑类药物结合模式的药物设计方法，筛选获得了1个对白念珠菌、新生隐球菌和烟曲霉菌均有高活性的化合物NT-a9，小鼠体内药效表明，优选化合物对小鼠的保护作用显著优于对照组，NT-2和NT-a9均转化至企业正在进行临床前研究，NT-2的临床前研究被列为2019国家新药创制科技重大专项的支持品种。在本项目支持下，共发表相关SCI论文6篇，授权专利1项，申请专利3项，培养研究生6名，其研究结果为后续开展解决真菌耐药问题和新药研发提供了新思路、新策略。

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