地衣内生真菌中清除外排泵高表达耐药真菌成分的发现及抗真菌活性研究

In recent years, the tendency of fungal infection in clinic has been more and more intense, and the antifungal resistance has also been more and more serious, which has brought extremely severe challenge for antifungal treatment. At present, the effective antifungal drugs used in clinic are rather limited, so it is of great significance to find new antifungal drugs with high efficiency and low toxicity, especially to solve the problem of fungal resistance. In the preliminary study, we have established the fluorescence labeled strain model and the reversal drug resistance model, and screened 4 strains that selectively eradicate resistant fungi with activated efflux pumps from 208 strains of endolichenic fungi, and obtained a series of active compounds from two strains. Based on the above results, this aims of the present project are listed as follow: i) To utilize the established screening model and two strategies of overcome fungal resistance (single use of compounds to selectively eradicate resistant fungi or the combination of the compounds and antifungal drugs can reverse drug resistance) to separate the other two strains and rapidly obtain the novel bioactive secondary metabolites. ii) to design and prepare the derivatives of the active compounds, analyze the structure-activity relationship and obtain the most potent compounds. iii) to carry out the research on internal and external antifungal effects and mechanism of the potent compounds，and to evaluate their application in fungal infection. The results of this project will make basis for the discovery of new resistant fungi scavengers and resistance reversal agents.

近年来临床真菌感染多发，其耐药性也日趋严重，这给抗真菌治疗带来极为严峻的考验。目前临床上使用的有效抗真菌药物相当有限，因而发现新型高效低毒的抗真菌尤其是解决真菌耐药这一问题的药物具有重要意义。前期研究中，我们建立了荧光标记菌株模型和逆转耐药模型，从208株地衣内生真菌中筛选得到了4株能清除外排泵高表达耐药真菌的活性菌株，并从其中两株中发现一系列活性化合物。本项目拟在前期研究的基础上，综合利用已建立的抗耐药真菌活性筛选模型，基于克服真菌耐药的两种策略（化合物单用选择性清除耐药菌；化合物与抗真菌药物联用可逆转耐药），对筛选出的另外两株内生真菌进行研究，发掘其中具有清除耐药真菌的成分；对活性化合物进行结构修饰，揭示其构效关系，获得活性更强、选择性更高的候选化合物；开展重要代表化合物体内外抗活性和作用机制研究，评价其在抗真菌感染中应用。研究成果将为新型耐药菌清除剂和耐药逆转剂的研发奠定基础。

近年来临床真菌感染多发，其耐药性也日趋严重，这给抗真菌治疗带来极为严峻的考验。目前临床上使用的有效抗真菌药物相当有限，因而发现新型高效低毒抗真菌尤其是解决真菌耐药这一问题的药物具有重要意义。本项目对地衣内生真菌的次生代谢产物展开研究，共发现了60个次生代谢产物，其中有34个为新化合物，包括2个新骨架化合物，17个Perylenequinonoid类化合物，12个heptaketides类化合物和3个其他类型的化合物。开展了新次生代谢产物作为外排泵特异性抑制剂的逆转真菌耐药和抗肿瘤活性的探讨，对作用机制进行的研究，探索化合物的作用靶点、抗真菌和抗肿瘤的作用机制，对上述两类衍生物的构效关系进行了初步探讨。研究发现，Perylenequinonoid类化合物具有高效低毒的特点和显著的逆转耐药活性，可以通过特异性调控MDR1基因表达以抑制外排泵活性，进而实现逆转真菌唑类药物耐药，并通过机制研究证明ROS是化合物SP1介导细胞死亡的因素之一，Cys40是SP1的靶点之一。heptaketides类化合物Biatriosporins D对唑类耐药菌表现出很强的逆转耐药活性，但是在1号位官能团改变后，heptaketides类化合物Ulosporin G表现出显著的抗肿瘤活性，通过DNA损伤引起G0/G1细胞周期阻滞诱导A549细胞凋亡。上述发现为两类化合物的药物研究以及后续的合成修饰提供了很好的先导化合物和思路。