构效优化的新型抗菌肽AaeAP1a抗真菌作用与机制研究

It is very significant to search for new anti-fungal substances and study their anti-fungal mechanism for development of more effective new drugs because of the resistance of clinical antifungal compounds.AaeAP1a is a kind of derivative polypeptide obtained by the applicant through optimizing the discovered natural scorpion venom antibacterial peptide AaeAP1, which has antifungal function and weak toxicity. However, its antifungal mechanism of action is still unclear. In preliminary studies, it was discovered that when FITC-AaeAP1a was put to interact with Candida albicans, AaeAP1a could aggregate on cell membrane and go inside cells, indicating that AaeAP1a can go through cell membrane to work with intracellular substances and generate antifungal activity through not only one mechanism. It was meanwhile detected that the expression level of cAMP-PKA pathway negative regulation factor PDE2 increased, indicating that AaeAP1 can complete transition to Candida albicans state through hindering normal operation of cAMP-PKA pathway. On the basis of clarifying that derivative polypeptide AaeAP1a outperforms natural scorpion venom antibacterial polypeptide AaeAP1in antifungal activity and stability and has a weak cell toxicity, this project verified the target organ of AaeAP1a exerting its antifungal effect on Candida albicans. In addition, it also explored the regulating role of cAMP-PKA pathway in AaeAP1a during antifungal effect, defined AaeAP1a’s antifungal mechanism of action through detecting the established animal pathogenic fungus infection model indicators, and explained AaeAP1a’ work mechanism in terms of cell, molecular level and animal overall level, hence laying the theoretical and experimental foundation for the development of new-type antifungal drugs.

针对病原真菌耐药性问题，寻找新型抗真菌物质、研究其作用机制对开发疗效更高的新药具有重要科学意义。AaeAP1a是申请者对自身发现的天然蝎毒抗菌肽AaeAP1构效优化得到的衍生多肽，但其抗真菌作用机制尚不清楚。在前期研究中，申请者发现FITC-AaeAP1a与白色念珠菌作用后能够在细胞膜上聚集且可到达胞内，提示可能通过不止一种机制产生抗真菌活性；同步检测到cAMP-PKA通路负向调控因子PDE2表达水平增加，提示AaeAP1a可能通过抑制该通路的正常运转发挥抗真菌效应。本项目在明确AaeAP1a抗真菌活性和稳定性优于AaeAP1且细胞毒性弱的基础上，确认AaeAP1a发挥抗真菌效应的靶器官，探讨cAMP-PKA途径在其发挥抗真菌效应中的调控作用，并建立动物病原真菌感染模型指标检测确认，从细胞、分子和动物整体水平阐明AaeAP1a抗真菌作用机制，为开发新型抗真菌药物提供理论基础和实验依据。

AaeAP1a是从天然蝎毒抗菌肽AaeAP1构效优化得到的衍生多肽，申请者发现FITC-AaeAP1a与白色念珠菌作用后能够在细胞膜上聚集且可到达胞内，提示可能通过不止一种机制产生抗真菌活性；同步检测到cAMP-PKA通路负向调控因子PDE2表达水平增加，提示AaeAP1a可能通过抑制该通路的正常运转发挥抗真菌效应。本项目在明确AaeAP1a抗真菌活性和稳定性优于AaeAP1且细胞毒性弱的基础上，确认AaeAP1a发挥抗真菌效应的靶器官，探讨cAMP-PKA途径在其发挥抗真菌效应中的调控作用，并建立动物病原真菌感染模型指标检测确认，从细胞、分子和动物整体水平阐明AaeAP1a抗真菌作用机制，为开发新型抗真菌药物提供理论基础和实验依据。课题组发现并优化改造新型抗菌肽肽kassinatuerin-3，并通过实验证明其对革兰氏阳性菌具有抗菌活性,在金黄色葡萄球菌和耐甲氧西林金黄色葡萄球菌上的生物膜清除以及在所选癌症细胞系的抗增殖方面具有活性，这表明对kassinatuerin-3有很高的治疗指数也为抗菌衍生物的设计提供了一个新的视角。此外,课题组发现DM-PC衍生的短阳离子两亲性α-螺旋肽在预防和治疗慢性和急性感染方面有很大的潜力，也说明设计较短的皮肤抑制素衍生物有助于化学合成，并有可能降低生产成本，这将为开发新的抗生素替代品带来新的见解。我们的研究揭示了抗菌肽的电荷和疏水性的结构-活性关系。疏水性的增加，特别是疏水表面的疏水性的增加，与膜的通透性增强有关，而在疏水范围较低时增加电荷有利于膜的选择性。因此，我们的研究针对病原真菌耐药性问题，寻找新型抗真菌物质、研究其作用机制对开发疗效更高的新药具有重要科学意义。