DESENHO DE UM NOVO PEPTÍDEO ANTIMICROBIANO ULTRACURTO GUIADO POR FERRAMENTAS COMPUTACIONAIS

Abstract

Antimicrobial peptides have different sites of action than conventional antibiotics. Recent research has been looking for peptides capable of controlling multidrug-resistant bacteria, without presenting side effects against healthy cells. In this work we describe the antimicrobial properties of RK8, an ultra-short peptide, which has 8 amino acid residues. The physicochemical characteristics of RK8 were previously projected through computational tools. The secondary structure of RK8 was determined in the presence of water and conditions that mimic biological membranes by circular dichroism (CD). The peptide was tested against Gram-negative and Gram-positive strains of multidrug-resistant bacteria. The synergistic effect of RK8 and ciprofloxacin was evaluated against E. coli KPC+ and multidrug-resistant A. baumannii. The ability to eradicate multidrug-resistant mature biofilms of A. baumannii was also evaluated. In vitro and in vivo cytotoxicity assays were performed. The stability of RK8 against peptidases was investigated in 25% fetal bovine serum solution and its mechanism of action on the microbial membrane was evaluated through the uptake of the DNA intercalator Sytox green. CD analyzes showed that RK8 assumed an unordered structure in water and SDS, suggesting that even though it was originally designed to assume an alpha-helix secondary structure, due to its reduced size, the peptide acts on membranes with an extended structure. . The peptide showed a minimal inhibitory concentration (MIC) against MRSA ATCC33591 and MRSA ATCC43300 of 8 and 16 µM, respectively. For E. coli KPC+ and A. baumannii, RK8 showed a MIC of 16 and 64 µM, respectively. The combination of RK8 and ciprofloxacin showed a synergistic effect against E. coli KPC+, reducing the concentration of the antibiotic and the peptide by 32-fold and 8-fold, respectively, to exert antimicrobial effects. RK8 was able to eradicate 38% of the mature biofilm of A. baumannii, when used in the MIC or half of it, affecting the biofilm structure and bacterial viability. RK8 showed no cytotoxic effects against murine macrophages (up to 64 µM) or against erythrocytes (up to 100 µM) or against Galleria mellanella larvae (up to 960 µM). In the stability assay against peptidases, RK8 was shown to be stable, maintaining about 60% of the intact molecule after 120 min of incubation. In this same assay time, the Sytox Green reagent achieved 100% membrane permeation of Gram-positive and Gram-negative bacteria. In summary, RK8 represents a promising strategy for the development of a new antimicrobial and antibiofilm agent against multidrug-resistant bacteria.