Investigating Penetration and Antimicrobial Activity of Vector-Bicycle Conjugates.

Growing antibiotic resistance is rapidly threatening the efficacy of treatments for Gram-negative infections. Bicycle molecules, constrained bicyclic peptides from diverse libraries generated by bacteriophage display that bind with high affinity to a chosen target are a potential new class of antibiotics. The generally impermeable bacterial outer membrane currently limits the access of peptides to bacteria. The conjugation of membrane active peptides offers an avenue for outer membrane penetration. Here, we investigate which physicochemical properties of a specific membrane active peptide (MAP), derived from ixosin-B, could be tweaked to enhance the penetration of conjugates by generating multiple MAP-Bicycle conjugate variants. We demonstrate that charge and hydrophobicity are important factors, which enhance penetration and, therefore, antimicrobial potency. Interestingly, we show that induction of secondary structure, but not a change in amphipathicity, is vital for effective penetration of the Gram-negative outer membrane. These results offer insights into the ways vectors could be designed to deliver Bicycle molecules (and other cargos) through biological membranes.

An Assay for Periplasm Entry Advances the Development of Chimeric Peptide Antibiotics.

The treatment of infection by Gram-negative bacteria is increasingly challenging as resistance to existing antibiotics spreads. Constrained peptides, selected for high target specificity and affinity via library display technologies, are an emerging therapeutic modality in many disease areas and may be a fertile source of new antibiotics. Currently, the utility of constrained peptides and other large molecules as antibiotics is limited by the outer membrane (OM) barrier of Gram-negative bacteria. However, the addition of certain moieties to large molecules can confer the ability to cross the OM; these moieties function as intramolecular trans-OM "vectors". Here, we present a method to systematically assess the carrying capacity of candidate trans-OM vectors using a real-time luminescence assay ("SLALOM", Split Luciferase Assay for Live monitoring of Outer Membrane transit), reporting on periplasmic entry. We demonstrate the usefulness of our tools by constructing a 3800 Da chimeric compound composed of a constrained bicyclic peptide (Bicycle) with a periplasmic target, linked to an intramolecular peptide vector; the resulting chimera is a broad-spectrum inhibitor of pathogenic Gram-negative bacterial growth.