Antimicrobial activity, membrane interaction and stability of the D-amino acid substituted analogs of antimicrobial peptide W3R6.

Antimicrobial peptide W3R6 was derived from chensinin-1b and showed potential as a novel antibiotics. However, W3R6 was susceptible to protease cleavage, which limited its therapeutic application. To improve the proteolytic resistance of W3R6, D-amino acids were incorporated into its sequence by specific amino acid substitution or whole sequence substitution according to the specificity of the cleavage site. In this study, partially substituted analog W3R6 and completely substituted D-enantiomer D-W3R6 were synthesized. The resistance of W3R6 and D-W3R6 to cleavage by the tested protease increased, particularly of D-W3R6. The antimicrobial activity of W3R6 was almost the same as that of the parent peptide W3R6, but the antimicrobial activity of D-W3R6 was slightly decreased. The hemolytic activity of both W3R6 and D-W3R6 was negligible. The CD spectrum of D-W3R6 exhibited symmetry with that of W3R6 in a membrane-mimetic environment. The membrane interaction between the D-amino acid substituted analogs and a real/mimic bacterial cell membrane was examined. The outer membrane depolarization, inner membrane permeability and dye leakage in three types of liposomes treated with W3R6 and D-W3R6 were not obviously different from W3R6, which could be due to the similar physical and chemical properties. In addition, these three peptides showed the binding ability with LPS micelles detected by ITC, and their ability to disrupt the LPS micelles was examined by DLS experiment and even neutralize the surface negative charge of E. coli cells. These results suggest that W3R6 is a promising antibiotic molecule.