Kang Wei, Liu Hanhan, Ma Lingman, Wang Mengxiao, Wei Shanshan, Sun Pengbo, Jiang Meiling, Guo Min, Zhou Changlin, Dou Jie
State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu 210009, PR China.
State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu 210009, PR China.
Eur J Pharm Sci. 2017 Jul 15;105:169-177. doi: 10.1016/j.ejps.2017.05.030. Epub 2017 May 15.
Broad spectrum activities, a unique mode of actions and rare resistant variants make antimicrobial peptide (AMP) a potential alternative to antibiotics. However, AMPs still have limitations in clinical development due to their physiological stability, toxicity and manufacturing costs. Cbf-14, derived from cathelicidin-BF has been proven to be effective against drug-resistant bacteria. Herein, a series of Cbf-14 mutants were designed to overcome these limitations. Design strategies included substitutions of lysine (Lys) or leucine (Leu) with similar residues such as ornithine (Orn) and norleucine (Ile), which are unnatural amino acids, to generate AMPs with enhanced therapeutic potential. Antimicrobial activity, hemolytic activity and cytotoxicity against mouse spleen cells of the peptide mutants were investigated. Among all of the mutants, Cbf-14-2 was regarded as the most potent candidate with the amino acid sequence of RLLR-Orn-FFR-Orn-LKKSV-NH, which exhibited a superior antimicrobial activity with a minimum inhibitory concentration (MIC) of 4-32μg/ml. Meanwhile, Cbf-14-2 displayed low levels of hemolysis in sheep red blood cells (sRBCs) and negligible cytotoxicity against mouse spleen cells, suggesting low toxicity against mammalian cells. A circular dichroism (CD) study indicated that Cbf-14-2 has a higher alpha-helix content than Cbf-14 (68.3% vs 35.1%) in SDS, which may contribute to its superior activity. Time-killing curves showed Cbf-14-2 can eliminate all tested bacteria within 240min, suggesting its rapid bactericidal effect. Transmission electron microscopy (TEM), flow cytometry and calcein release assays revealed its excellent antimicrobial potency by inducing membrane permeation and disruption. In addition, Cbf-14-2 (10mg/kg) could significantly elevate the survival rate of clinical strain infected mice, with a survival rate of 70.0%. Taken together, the data suggest that Cbf-14-2 possesses effective antimicrobial activity against penicillin-resistant bacteria in vitro and in vitro, thus rendering it as a potential anti-infective agent in clinical settings.
广谱活性、独特的作用模式和罕见的耐药变体使抗菌肽（AMP）成为抗生素的潜在替代品。然而，由于其生理稳定性、毒性和制造成本，AMPs在临床开发中仍存在局限性。源自cathelicidin-BF的Cbf-14已被证明对耐药细菌有效。在此，设计了一系列Cbf-14突变体以克服这些局限性。设计策略包括用鸟氨酸（Orn）和正亮氨酸（Ile）等类似残基取代赖氨酸（Lys）或亮氨酸（Leu），这些都是非天然氨基酸，以产生具有增强治疗潜力的AMPs。研究了肽突变体对小鼠脾细胞的抗菌活性、溶血活性和细胞毒性。在所有突变体中，Cbf-14-2被认为是最有效的候选物，其氨基酸序列为RLLR-Orn-FFR-Orn-LKKSV-NH₂，其最小抑菌浓度（MIC）为4-32μg/ml，表现出优异的抗菌活性。同时，Cbf-14-2在绵羊红细胞（sRBCs）中溶血水平较低，对小鼠脾细胞的细胞毒性可忽略不计，表明对哺乳动物细胞毒性较低。圆二色性（CD）研究表明，在SDS中，Cbf-14-2的α-螺旋含量高于Cbf-14（68.3%对35.1%），这可能有助于其优异的活性。时间杀灭曲线表明Cbf-14-2可在240分钟内消除所有测试细菌，表明其快速杀菌作用。透射电子显微镜（TEM）、流式细胞术和钙黄绿素释放试验通过诱导膜渗透和破坏揭示了其优异的抗菌效力。此外，Cbf-14-2（10mg/kg）可显著提高临床菌株感染小鼠的存活率，存活率为70.0%。综上所述，数据表明Cbf-14-2在体外和体内对耐青霉素细菌均具有有效的抗菌活性，因此使其成为临床环境中潜在的抗感染剂。
