抗菌肽RIKL的分子设计及生物学活性分析

Molecular design and biological activity analysis of antimicrobial peptide RIKL

摘要:

天然抗菌肽具有较强的杀菌能力，但高生物相容性抗菌肽的构建一直阻碍着该领域的发展。为了提高抗菌肽的选择特异性，通过分子动力学分析探讨了抗菌肽的结构特性，并检测其生物学活性。首先以(RXKY)₂(YRY)₂ (X代表Ile，Y代表Leu) 为模板设计新型抗菌肽分子RIKL。通过圆二色谱(circular dichroism, CD) 检测RIKL的二级结构，并通过分子动力学分析模拟了RIKL在水溶液和POPC/POPG膜环境下的结构，同时通过检测抑菌活性、溶血活性、细胞毒性及盐离子稳定性等指标进一步研究其生物学活性。CD结果表明，RIKL在细菌膜模拟环境下呈现α-螺旋结构，分子动力学模拟预测了RIKL可以在水溶液和POPG环境下保留部分二级结构，而在POPC环境下分子的二级结构含量有所降低。抑菌试验表明RIKL具有较高的抑菌活性，其最小抑菌浓度(minimum inhibitory concentration, MIC) 的几何平均值为3.1 μmol/L; 溶血和细胞毒性试验表明，RIKL在检测范围内无溶血活性、细胞毒性较低; 稳定性试验发现，RIKL在不同pH值、不同浓度血清和盐离子存在的环境下仍保持抑菌活性。综合以上结果，RIKL具有较高的细胞选择性，有成为高效抗菌药物的发展潜力。

关键词:抗菌肽;分子动力学;抑菌活性;毒性;稳定性

Abstract:

Natural antimicrobial peptides have strong bactericidal activities. An obstacle of the development of antimicrobial peptides resides in the difficulty of developing peptides with high biocompatibility. In this study, molecular dynamics analysis was employed to assess the structural characteristics and biological activities of peptides. A (RXKY)₂(YRY)₂ structure was used as a template to design an antimicrobial peptide RIKL of high-efficiency and low-toxicity, where X represents Ile and Y represents Leu. The secondary structure of the antimicrobial peptide was detected by circular dichroism (CD), and the structures of RIKL in water and in POPC/POPG membrane environment were measured using molecular dynamics. The biological activity of RIKL was further studied by assessing its antimicrobial activity, hemolytic activity, eukaryotic cytotoxicity, and salt ion stability. CD results showed that RIKL presented an α-helical structure in a simulated bacterial membrane environment. Molecular dynamics simulation predicted that the secondary structure of RIKL could be partly retained in water and POPG environment, while this secondary structure was weakened in the POPC environment. Antimicrobial test suggested that RIKL had high antimicrobial activities, and the geometric mean of the Minimum Inhibitory Concentration (MIC) was 3.1 μmol/L. The hemolysis indicated that RIKL had no hemolytic activity within the detection range, and cytotoxicity test suggested the cytotoxicity of RIKL was low. Stability test showed that RIKL maintained antimicrobial activities under different pH, serum concentrations and salt environments. Based on the above results, RIKL has high cell selectivity and has the potential as a highly effective antibacterial drug.

Key words:antimicrobial peptides;molecular dynamics;antimicrobial activity;toxicity;stability