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

Abstract

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.

Antimicrobial and Cell-Penetrating Peptides: Understanding Penetration for the Design of Novel Conjugate Antibiotics

“…The conjugate can enter the periplasm, where it can bind to protein 11S, completing a functional luciferase enzyme. The luminescence detected is proportional to the amount of pep86 molecules entering the periplasm [ 36 ]. The advantage of this assay is that it measures compound accumulation in the periplasm, without the confounding addition of a fluorescent group.…”

“…When studying the penetrability of a specific membrane active peptide, the membrane models under which this is conducted should be as close as possible to the actual environment at which the peptide will be applied. If the application is translational, such as their use in novel antimicrobial modalities [ 36 , 37 ], clinical isolates of the strains targeted [ 36 ] or membrane vesicles mimicking bacterial outer membranes [ 81 ] are ideal models.…”

“…Research has also been conducted on the use of membrane active peptides as part of novel classes of antibiotics [ 19 , 20 , 21 , 36 , 37 , 87 ]. In recent years, several groups have attempted to use membrane active peptides as an entry mechanism to transport larger compounds into the bacterial cell.…”

“…Most membrane active peptides have a disordered secondary structure in solution but can assume a more rigid conformation upon membrane interaction [ 7 , 29 , 30 , 31 , 32 ]. Many CPPs have shown antibacterial activity and the majority of AMPs studied are cell penetrating, [ 6 , 33 ] with some showing selectivity for only prokaryotic membranes and some no selectivity between eukaryotic and prokaryotic membranes [ 30 , 34 , 35 , 36 , 37 , 38 ]. As mentioned above, some AMPs have additional immunomodulatory activity which is a feature of their indirect antimicrobial action, [ 6 , 8 ] and thought to be conducted by direct or indirect immune cell membrane receptor interactions [ 8 , 39 ].…”

“…Research around membrane active peptides has intensified [ 6 , 7 ] not only as an invaluable basic research tool in delivering different compounds intracellularly but also as translational research, as they could be used as “vectors” for anti-microbial [ 7 , 21 , 36 , 37 ] or anti-cancer [ 7 , 48 ] drugs or other cargoes. To date, thousands of membrane active peptides have been investigated from all kingdoms of life [ 7 , 9 , 13 , 35 , 49 , 50 , 51 , 52 ].…”