Data_Sheet_1_A Novel Dual-Targeted α-Helical Peptide With Potent Antifungal Activity Against Fluconazole-Resistant Candida albicans Clinical Isolates.docx

Due to compromised immune system, fungal infection incidences have markedly increased in the last few decades. Pathogenic fungi have developed resistance to the clinically available antifungal agents. Antifungal resistance poses a great challenge to clinical treatment and has stimulated the demand for novel antifungal agents. A promising alternative to the treatment of fungal diseases is the use of antimicrobial peptides (AMPs). However, the antifungal activities of AMPs have not been fully determined. Therefore, this study aimed at designing and screening α-helical peptides with potential antifungal activities. The effects of key physicochemical parameters on antifungal activities were also investigated. A series of lengthened and residue-substituted derivatives of the template peptide KV, a hexapeptide truncated from the α-helical region of porcine myeloid antimicrobial peptide-36, were designed and synthesized. Enhancement of hydrophobicity by introducing aromatic hydrophobic amino acids (tryptophan and phenylalanine) significantly increased the efficacies of the peptides against Candida albicans strains, including fluconazole-resistant isolates. Increased hydrophobicity also elevated the toxic properties of these peptides. RF3 with moderate hydrophobicity exhibited potent anticandidal activities (GM = 6.96 μM) and modest hemolytic activities (HC10 > 64 μM). Additionally, repeated exposure to a subinhibitory concentration of RF3 did not induce resistance development. The antifungal mechanisms of RF3 were due to membrane disruptions and induction of reactive oxygen species production. Such a dual-targeted mechanism was active against drug-resistant fungi. These results show the important role of hydrophobicity and provide new insights into designing and developing antifungal peptides. Meanwhile, the successful design of RF3 highlights the potential utility of AMPs in preventing the spread of drug-resistant fungal infections.近数十年来，机体免疫系统功能受损导致真菌感染的发病率显著攀升。致病性真菌已对临床可用的抗真菌药物产生耐药性，这一问题给临床治疗带来了巨大挑战，同时也催生了新型抗真菌药物的研发需求。抗菌肽（Antimicrobial Peptides, AMPs）是治疗真菌感染性疾病的极具潜力的替代方案，但目前抗菌肽的抗真菌活性尚未被完全阐明。因此，本研究旨在设计并筛选具有潜在抗真菌活性的α-螺旋肽（α-helical peptides），同时探究关键理化参数对抗真菌活性的影响。本研究以模板肽KV为基础——该六肽由猪髓源抗菌肽-36的α-螺旋区域截短得到——设计并合成了一系列经加长修饰与残基替换的衍生物。通过引入芳香族疏水氨基酸色氨酸（tryptophan）与苯丙氨酸（phenylalanine）以提升疏水性，可显著增强肽类对白色念珠菌（Candida albicans）菌株（包括氟康唑（fluconazole）耐药分离株）的抑菌效能，但疏水性的提升也会增强这些肽类的毒性。疏水性适中的RF3展现出强效的抗念珠菌活性（几何平均抑菌浓度GM=6.96 μM），同时溶血活性温和（HC10＞64 μM）。此外，反复暴露于亚抑菌浓度的RF3并不会诱导耐药性的产生。RF3的抗真菌机制主要通过破坏细胞膜以及诱导活性氧（Reactive Oxygen Species, ROS）的产生实现，这种双靶点机制对耐药真菌同样有效。上述研究结果阐明了疏水性的关键作用，为抗真菌肽的设计与开发提供了全新的研究思路。同时，RF3的成功设计也凸显了抗菌肽在遏制耐药真菌感染传播方面的应用潜力。