Understanding the membrane penetration of antimicrobial

Abstract

The studies on β-lactam antibiotics have found great interest since the use of antimicrobial therapy became widespread and as a result, bacteria started to develop resistance against β-lactam antibiotics. The most effective resistance mechanism is the production of β-lactamases. The need of successful inhibitors against β-lactamase led to the studies that revealed the structure of β-lactamase inhibitory protein (BLIP) and its interactions with β-lactamase. The structure of TEM-1-BLIP complex indicated that a type II β turn (residues 46-51) of BLIP made critical interactions with the active site of the TEM-1 β-lactamase, suggesting that a peptide including this loop would be a good lead toward the development of a peptide based inhibitor. In peptide based drug design, one of the most common challenges is penetration through the membrane. It was aimed to investigate the translocation mechanism of the cell-penetrating peptide pVEC (LLIILRRRIRKQAHAHSK) and to analyze it on a residue basis in order to identify mechanism by which bacterial uptake occurs. The wild-type pVEC, eight mutants, retro- and scramble pVEC were chosen to be studied using molecular dynamics (MD) and steered molecular dynamics (SMD) simulations. The peptide transport was observed to occur in three main stages and it was distinguished that for peptides with increased uptake potential, the work performed was higher. Residues L1, R6, R7, R8, R10, K11 and K18 was shown to contribute to the interaction of pVEC with the lipid bilayer. In the experimental part of the study, effect of Peptide 1 (5-Fluorescein-NH-HA AGDYYAY- CONH2), Peptide 2 (5/6-Fluorescein-NH-RRGHYY-COOH) and Peptide 3 (5/6-Fluorescein-NH-LLIILHAAGDYYAY- CONH2) on growth of antibiotic resistant E. coli K12 pUC18 cells expressing β-lactamase was examined. Peptide 1 did not have an inhibitory effect on the cell growth. On the other hand, Peptide 3 was taken into the cells, as revealed by microscopy and incubation with this peptide led to cell death. Our studies showed that inclusion of the five hyrophobic residues at the N-terminus enhanced the uptake potential of the inhibitory peptide.