Chimeric Peptidomimetic Antibiotics Against Gram-Negative Bacteria
Novel antibiotics against Gram-negative bacteria are urgently needed, particularly because resistance against the last resort antibiotic colistin is on a global rise. A new family of antibiotics targeting the Gram-negative ESKAPE pathogens has now been reported.
The rapid emergence of antimicrobial resistance is now a matter of growing public awareness. Of particular concern according to the WHO are carbapenem and third generation cephalosporin-resistant Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae, which belong to the Gram-negative so-called ESKAPE pathogens. Novel antibiotics against Gram-negative bacteria are urgently needed, particularly because resistance against the last resort antibiotic colistin is on a global rise.
One of the difficulties in trying to discover and develop new antibiotics against Gram-negative bacteria has been their unique double membrane structure. The outer membrane (OM) is an asymmetric bilayer composed of complex glycolipids, called lipopolysaccharides (LPS), located exclusively in the outer leaflet, with glycerophospholipids in the inner leaflet. The long oligosaccharide chains of the tightly packed LPS molecules are directed outwards into the medium and often forms an almost impenetrable barrier for any new antibiotic that tries to gain entry to the periplasm or cytoplasm. And yet the cellular machinery used by the bacteria to build the OM can also become an achilles heel, as we show in our publication. Perhaps surprisingly, none of the current clinically used antibiotics, most of which were discovered during the 1950's-1980's, target the bacterial proteins involved in OM biogenesis. This is also a testament to the difficulties experienced, even for big pharma companies, in efforts to discover new antibiotics targeting OM biogenesis. Fortunately, major advances in understanding of the complex processes involved in OM biogenesis have been made by microbiologists world-wide over the past few years.
The path to the discovery of the new antibiotics had its origins in our earlier work, in which we identified the first antibiotic targeting LptD, an essential ß-barrel OM protein specifically in Pseudomonas spp. (Science 2010, 327, 1010-1013. https://science.sciencemag.org/content/327/5968/1010). LptD is part of the lipopolysaccharide transport (Lpt) protein complex (LptA-LptG) spanning the inner membrane (IM), the OM and the intervening aqueous periplasm, which is responsible for transporting LPS molecules from their site of assembly at the IM to their final location in the OM. This first-in-class antibiotic, called murepavadin, belongs to a new molecule class comprising macrocyclic peptides folded into well-defined ß-hairpin conformations. We could show that such molecules can target bacterial ß-barrel OM proteins. We built upon this observation in the current work, but critically, extended the molecular structure by attaching a second macrocycle that targets LPS and is found in the polymyxin/colistin family of natural products. After extensive optimization of a lead molecule, the resulting chimeric antibiotics showed potent and rather broad action against not only the ESKAPE pathogens, but also many clinically derived multi-drug resistant strains, and even strains that have become resistant to the last-resort antibiotic colistin. We further show that the optimized leads show potent levels of antimicrobial activity in-vivo, in several animal infection models, and appropriate levels of safety for potential clinical applications.
How do the new antibiotics function? Through a variety of biochemical and biophysical experiments we could indeed show that the antibiotics bind to another essential ß-barrel OM protein, called BamA. This OM protein is the central part of the BAM complex, which is required to catalyze folding and insertion into the OM of all ß-barrel OM proteins in Gram-negative bacteria. We hypothesize that the antibiotics inhibit the function of the BAM complex by binding to BamA, thereby cause the build-up of incorrectly folded OM proteins also in the IM, which is highly toxic for the cells.
The lead antibiotic is now advancing into preclinical toxicology studies and may address in the future a major growing unmet medical need.
Reference: Chimeric peptidomimetic antibiotics against Gram-negative bacteria. A. Luther, et al. Nature, 2019. https://www.nature.com/articles/s41586-019-1665-6
Figure Legend: Escherichia coli cells treated with a novel chimeric peptidomimetic antibiotic by STED fluorescence microscopy. Cells in blue are alive while green cells are already killed by the peptidomimetic. As the antibiotic destroys the integrity of the bacterial membranes, we observed “explosive cell lysis” (cells indicated by arrows), which leads to the release of DNA (diffuse green)