F1Fo-ATP合酶β亚基作为抗白念珠菌新靶点的确证研究

Candida albicans is an emerging multidrug-resistant fungal pathogen representing an important source of invasive disease in humans, and the scarcity of antifungal agents and their limited efficacy contribute to the unacceptably high mortality rates associated with these infections. Both the limited spectrum of antifungal drugs currently in clinical use and the emergence of resistances make the development of new effective antifungal drug necessary for anti-Candida albicans, however, such researches are limited by the small number of specific target sites identified to date. Our previous studies have demonstrated that the absence of the β subunit in Candida albicans fails to lead to a fatal infection in mice disseminated models, with the growth conditions, time-series, cell viability being significantly reduced，suggesting that the β subunit is a potential drug target for anti-Candida albicans. Our project intends to perform structure-based virtual screening (SBVS) based on the homology modeling approximation, molecular docking, and molecular dynamics simulation to identify novel candidate compounds as potential β subunit inhibitors. Then, protein-small molecule interaction, the co-crystal structure analysis together with site-directed mutagenesis are determined to clarify the active sites of the small molecules. Last, in vitro and in vivo activity evaluation will be measured to judge whether β subunit has the potential value as a drug target. The present project will provide solid evidence for confirming whether the F1Fo-ATP synthase β subunit of Candida albicans is a new antifungal drug target. Also, it is the first attempt to combined SBVS approach with drug target identification for antifungal drug development, which is proved to be a cost efficient and simple strategy.

侵袭性白念珠菌感染致死率高，耐药现象日趋严重，新药研发进展缓慢，候选分子结构难有新突破，新靶点确证困难但甚为必要。我们前期发现，白念珠菌F1Fo-ATP合酶β亚基缺失后，无法对小鼠造成致死性损害，且生长繁殖能力、时序寿命、细胞活力均显著降低，提示其可能是抗白念珠菌药物的潜在靶点。因此，本项目拟通过同源模建、分子对接以及分子动力学技术进行基于结构的虚拟筛选（SBVS）以期发现β亚基抑制剂的候选化合物；进而通过靶蛋白-小分子相互作用、共晶体结构解析结合定点突变验证小分子作用位点，通过体内外模型评价其抑制活性，从而综合评价其作为药物靶点的潜在价值。项目成果将为确认F1Fo-ATP合酶β亚基是否为抗白念珠菌药物新靶点提供扎实而系统的证据，同时也首次尝试将SBVS结合靶点验证为核心的这一高效、简便的方案用于抗真菌药物研发。

白念珠菌侵袭性感染致死率高，耐药现象日趋严重，新药研发进展缓慢，候选分子结构难有新突破，新靶点确证困难但甚为必要。.本项目主要研究内容：①F1Fo-ATP合酶β亚基是否是抗白念珠菌的潜在药物靶点。②F1Fo-ATP合酶β亚基是否是抗白念珠菌的新靶点。.研究发现：（1）白念珠菌F1Fo-ATP合酶β亚基在致病过程中起关键作用：①β亚基缺失后无法对小鼠造成致死性损害；②β亚基缺失后白念珠菌毒力明显降低；③β亚基缺失后菌株与宿主互作明显减弱。（2）获得白念珠菌β亚基三维结构，以及小分子结合活性位点。（3）基于β亚基结构虚拟筛选出毒性低、活性好的小分子S496。（4）对S496进行初步优化，其中S496-2表现出更为强效、广谱、低毒的抗真菌活性。（5）β亚基与S496-2直接结合：①ITC结果显示β亚基与S496-2特异性结合；②S496-2未能进一步降低β亚基缺失株的活性和F1Fo-ATP合酶活性；③β亚基点突变后与与S496-2非特异性结合；④S496-2未能进一步降低β亚基点突变株的活性和F1Fo-ATP合酶活性。（6）S496-2体内外抗菌活性良好。.研究结果揭示，F1Fo-ATP合酶β亚基是抗白念珠菌的新靶点，基于其结构筛选的S496-2为抗真菌新抑制剂。.如期完成了合同书内容。发表论文SCI论文3篇。正在投稿1篇。