Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Connective Tissue Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Probiotics Antimicrob Proteins. 2024 Apr;16(2):490-500. doi: 10.1007/s12602-023-10066-6. Epub 2023 Mar 29.
The low stability and nonspecific toxicity are the main limiting factors for the clinical applications of melittin (MLT). This study aimed to design and synthesize new analogs of MLT to increase stability, reduce toxicity, and retain their antimicrobial properties against bacterial pathogens. At first, peptide analogs were designed computationally by inducing single mutations in MLT peptides and evaluating their physicochemical properties. The stability of the analogs with the highest scores was determined by Gromacs software. In vitro assays were performed to examine the antimicrobial activity and toxicity of the selected analogs. Two peptide analogs, M1 and M2, were selected based on the SVM score in cell PPD. The M1 analog was created by replacing alanine with leucine on the 15th. The M2 analog was designed by substituting alanine with leucine and isoleucine with arginine at the 15th and 17th positions. According to the Gromacs results, the M2 peptide indicated more stability. RMSD and RMSF results showed no undesirable fluctuations during the 200 ns MD simulation. The MIC and MBC values for the M1 peptide were calculated in a range of 8-128 μg/ml, while the M2 peptide limited the bacterial growth to 32-128 μg/mL. Both peptides indicated less toxicity than natural MLT, based on MTT assay results. The hemolytic activity of the M1 analog was more than M2 at 16 μg/mL concentration. M1 peptide displayed the highest selectivity index against S. aureus and A. baumannii, which were approximately 5.27-fold improvements compared to MLT. In conclusion, we introduced two analogs of MLT with low toxicity, low hemolytic activity, and higher stability, along with retaining antimicrobial properties against gram-negative and positive bacteria.
蜂毒素（MLT）的低稳定性和非特异性毒性是其临床应用的主要限制因素。本研究旨在设计和合成 MLT 的新类似物，以提高稳定性、降低毒性，并保留其对细菌病原体的抗菌特性。首先，通过在 MLT 肽中诱导单点突变并评估其理化性质，通过计算设计肽类似物。通过 Gromacs 软件确定得分最高的类似物的稳定性。进行体外测定以检查所选类似物的抗菌活性和毒性。根据细胞 PPD 中的 SVM 评分，选择了两种肽类似物，M1 和 M2。M1 类似物是通过在第 15 位用亮氨酸替换丙氨酸而产生的。M2 类似物是通过在第 15 位和第 17 位用亮氨酸和精氨酸取代丙氨酸设计的。根据 Gromacs 的结果，M2 肽显示出更高的稳定性。RMSD 和 RMSF 结果表明在 200 ns MD 模拟过程中没有出现不良波动。M1 肽的 MIC 和 MBC 值在 8-128μg/ml 范围内，而 M2 肽将细菌生长限制在 32-128μg/ml。根据 MTT 测定结果，两种肽都比天然 MLT 的毒性小。在 16μg/ml 浓度下，M1 类似物的溶血活性高于 M2。M1 肽对金黄色葡萄球菌和鲍曼不动杆菌的选择性指数最高，与 MLT 相比提高了约 5.27 倍。总之，我们引入了两种 MLT 类似物，它们具有低毒性、低溶血活性和更高的稳定性，同时保留了对革兰氏阴性和阳性细菌的抗菌特性。
