In silico design and in vitro evaluation of anti-microbialactivity of Melittin analogs

Background and Aim : The antimicrobial activity of melittin (MLT) is now confirmed against awide variety of Gram-negative and Gram-positive bacteria. However, low stability and nonspecifictoxicity are the main limiting factors for the clinical applications of this natural membrane-activepeptide. This study aimed to design and synthesize new analogs of MLT to increase stability,reduce toxicity, and retain their antimicrobial properties against bacterial pathogens.Methods : Initially, peptide analogs were designed computationally through single mutations inthe template peptide and evaluation of their physicochemical properties. The stability of theanalogs was determined by Gromacs software, and the peptides with the highest scores inantimicrobial activity and stability were selected to synthesize and further evaluations. Minimuminhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determinedagainst seven standard bacterial strains. A cell viability assay was used to determine the cellulartoxicity of the analogs.Results : Two peptide analogs, M۱ and M۲, with higher stability and antimicrobial activity wereselected based on Cell PPD data. The M۱ analog was created by replacing alanine with leucine atthe ۱۵th position, and the M۲ analog was designed via substituting alanine with leucine andisoleucine with arginine at the ۱۵th, and ۱۷th positions, respectively. According to the Gromacsresults, the M۱ peptide stabilized at ۵۰ ns, and the M۲ peptide maintained its alpha-helix structurein ۵۰-۲۰۰ ns, indicating more stability of the M۲ analog. RMSD and RMSF results showed noundesirable fluctuations during the ۲۰۰ns MD simulation. The MIC and MBC values for the M۱peptide were calculated in a range of ۸-۱۲۸ ?g/ml, while the M۲ peptide limited the bacterialgrowth at ۳۲-۱۲۸ ?g/mL and killed them at ۶۴-۲۵۶ ?g/ml. Both peptides indicated less toxicitythan natural MLT, based on MTT assay results. Besides, the hemolytic activity of the M۱ and M۲analogs at ۱۶ ?g/mL concentration was ۴۱% and ۱۴.۲۵%, respectively, indicating a notabledecrease in the hemolytic properties of the designed analogs.Conclusion : In this study, we developed two analogs of MLT with low toxicity, low hemolyticactivity, and higher stability, along with retaining antimicrobial properties against gram-negativeand positive bacteria compared to natural MLT.

کلیدواژه ها:

Melittin ، MD simulation ، antibacterial activity ، toxicity ، peptide