Discovery of a Membrane-Active, Ring-Modified Histidine Containing Ultrashort Amphiphilic Peptide That Exhibits Potent Inhibition of Cryptococcus neoformans

The new structural classes of ultrashort peptides that exhibit potent microbicidal action have potential as future drugs. Herein, we report that C-2 arylated histidines containing tripeptides His­(2-Ar)-Trp-His­(2-Ar) exhibit potent antifungal activity against Cryptococcus neoformans with high selectivity. The most potent peptide 12f [His­(2-biphenyl)-Trp-His­(2-biphenyl)] displayed high in vitro activity against C. neoformans (IC50 = 0.35 μg/mL, MIC = MFC = 0.63 μg/mL) with a selectivity index of >28 and 2 times higher potency compared to amphotericin B. Peptide 12f exhibited proteolytic stability, with no apparent hemolytic activity. The mechanism of action study of 12f by confocal laser scanning microscopy and electron microscopy indicates nuclear fragmentation and membrane disruption of C. neoformans cells. Combinations of 12f with fluconazole and amphotericin B at subinhibitory concentration were synergistic against C. neoformans. This study suggests that 12f is a new structural class of amphiphilic peptide with rapid fungicidal activity caused by C. neoformans.具有强效杀菌活性的新型超短肽结构类别，具备成为未来候选药物的潜力。本文报道，含有C-2芳基化组氨酸的三肽His(2-Ar)-Trp-His(2-Ar)对新型隐球菌（Cryptococcus neoformans）展现出强效抗真菌活性与高选择性。活性最优的肽类12f [His(2-联苯基)-Trp-His(2-联苯基)]在体外对新型隐球菌表现出优异活性：半数抑制浓度（IC50）为0.35 μg/mL，最低抑菌浓度（MIC）与最低杀菌浓度（MFC）均为0.63 μg/mL，选择性指数大于28，其活性较两性霉素B（amphotericin B）高出2倍。肽12f具备蛋白水解稳定性，且未表现出明显的溶血活性。通过共聚焦激光扫描显微镜（confocal laser scanning microscopy）与电子显微镜（electron microscopy）对12f的作用机制进行研究，结果显示新型隐球菌细胞出现核碎裂与细胞膜破坏现象。在亚抑菌浓度下，12f与氟康唑（fluconazole）、两性霉素B联用对新型隐球菌具有协同抗真菌作用。本研究表明，12f属于一类新型两亲性肽结构，可对新型隐球菌展现出快速杀菌活性。