Peptidoglycan (PG) is an attractive and validated target for antibacterial drug development for two main reasons. First, it is an essential and unique bacterial cell wall polymer with no counterpart in human cells, minimizing the risk of drug toxicity. Second, the essential PG synthases are exposed at the outer surface of the cytoplasmic membrane, making them highly accessible for antibiotic inhibition.
Formation of the PG network requires glycosyltransferases for glycan chain elongation and transpeptidases for peptide cross-linking. Transpeptidation involves two stem peptides that act as acyl donor and acceptor substrates, respectively. The acyl donor site is targeted by the ß-lactams, which form covalent adducts, and this interaction is well characterized. In contrast, nothing is known on the interaction of the transpeptidases with the acceptor substrate. To combat the erosion of the activity of ß-lactams, we propose to identify additional drugable sites in the transpeptidases, including the acceptor binding site, and develop lead antibacterial agents acting on these sites.
Our first objective is to characterize the mode of recognition of the acyl acceptor by transpeptidases and identify compounds blocking the binding of this substrate. We will use NMR spectroscopy to map the acceptor site and develop specific inhibitors based on modeling and virtual screening. Our second objective is to identify the partners of transpeptidases that regulate the coordinated elongation of glycan chains and cross-linking of stem peptides. This will allow us to select additional drugable sites in transpeptidases and associated proteins within the PG polymerization complexes.
We will map key interactions by FRET analyses in live bacteria producing fluorescent proteins and by in vitro transpeptidase/glycosyltransferase assays in complexes obtained by tandem-affinity purification. Microfluidic cultures and time-lapse microscopy will assess the impact of inhibitors on cell division and viability. The interaction of lead compounds with their targets will be characterized by X-ray crystallography. These complementary approaches will enable the consortium to develop novel strategies for transpeptidase inhibition and obtain leads active against ß-lactam-resistant bacteria.
- Michel Arthur, INSERM, France (Coordinator)
- Waldemar Vollmer, Newcastle University, UK
- Tanneke den Blaauwen, University of Amsterdam, The Netherlands
- Jean-Pierre Simorre, CNRS, France
- John Mc Kinney, Swiss Federal Institute of Technology Lausanne (EPFL), Switzerland
- Natalie Strynadka, University of British Columbia, Canada
Antibiotics gradually lose their efficacy due to the emergence and dissemination of resistance in all bacterial pathogens. At the same time, most pharmaceutical companies have disengaged from the development of new drugs since it is not profit-making. In this context, efforts for early steps of drug discovery have partly moved from industry to academic laboratories.
Bacterial resistance to antibiotics is by no means a new issue. It has been mainly fought by modifications of molecules discovered more than 50 years ago. Consequently, antibiotics available in the clinics belong to a very limited number of chemical classes. It is arguable that modification of these drugs to escape resistance has reached, or will soon reach, an unbreakable limit since resistance mechanisms are able to promptly evolve to be effective on drugs with similar structures and the same mode of action.
The JPIAMR-funded project has evaluated new ways to inhibit “old” drug targets. We focused on the targets of penicillin, which have been validated by a long history of successful antibiotherapy. Our consortium brought together research teams with complementary expertise in structural biology to identify new drug binding sites, in chemistry to synthesize molecules specific of these binding sites, and in biochemistry to evaluate target inhibition. By this approach, we have identified new binding sites that are distinct from that of penicillin and are essential for the activity of the targets.
Research spotlight: One-pager on NACPLI (pdf 0,2 MB)
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