Development of Efflux Pump Inhibitors for overcoming innate and acquired AntiMicrobial Resistance in Enterobacterale infections – EPI-4-AMR

This project involves medicinal chemistry to identify optimized lead efflux pump inhibitors (EPIs), evaluate their in vitro efficacy on both drug-sensitive and resistant strains, and assess the in vivo efficacy of EPI/antibiotic combinations.

Pyridylpiperazine EPIs will be optimised by rational design and by synthesizing a chemical library of triazoles, using a click-chemistry reaction. The physicochemical properties, in vitro stabilities and toxicity will then be assessed. Best compounds will be resynthesized in larger scale to perform multi-organ PK studies in mice.

New EPIs will be tested on E. coli and K. pneumoniae wild-type strain in the presence of a subactive concentration of pyridomycin, the AcrAB-TolC substrate antibiotic used historically for this screening. Most potent compounds will then be tested in combination with a panel of antibiotics on E. coli and K. pneumoniae wild-type strain, and on K. pneumoniae resistant strains bearing clinically relevant mutations. Finally, EPIS will be tested on resistant clinical isolates to select the most interesting combinations of clinical strain/antibiotic/PyrPip.

The best PyrPip/antibiotic combination will be used in a murine model of K. pneumoniae lung infection with sensitive and resistant strain.

A chemical library of more than 600 triazoles was synthesised using a copper-catalysed cycloaddition reaction and more than 100 compounds were obtained by rational design.

All the compounds were tested on E. coli WT in combination with a subactive dose of pyridomycin. This led to the identification of around ten efflux pump inhibitors that were ten times more potent than the reference compound. The physicochemical properties, microsomal stability and cytotoxicity of these compounds were measured. This led to the selection of 3 compounds from different chemical families, which were used to perform multi-organ pharmacokinetic studies in mice.

These 3 compounds were tested on E. coli and K. pneumoniae WT in combination with a panel of antibiotics from different families and were able to potentiate the activity of all antibiotics substrates of the AcrAB/TolC pump.

2 compounds were tested in combination with antibiotics on resistant strains bearing clinically relevant mutations. Both EPI restore antibiotic susceptibility to the clinical breakpoint for different antibiotic classes on K. pneumoniae resistant strains.

These two EPIs were also tested on a panel of clinical isolates of multidrug-resistant K. pneumoniae and were able to overcome resistance by boosting the activity of several antibiotic families.

In vivo testing in a mouse model of lung infection is currently ongoing.

The next objectives are to

1) Continue to optimise this family of efflux pump inhibitors for Enterobacterales (Escherichia coli and Klebsiella pneumoniae), in order to obtain a compound with the best profile for pre-clinical development.

2) Validate the compound in vitro on multidrug-resistant clinical isolates.

3) Carry out the first proof of concept in a mouse model of pulmonary infection by a resistant strain of Klebsiella pneumoniae.

Drug resistant Enterobacterale are a major threat to global healthcare, and it has become clear in the last years that new tools are urgently needed to combat these bacteria. In the endeavour to develop new antibiotics against these Gram-negative bacteria, a major hurdle is the bacterial expression of efficient and promiscuous xenobiotic efflux pumps that limit the efficacy of known antibiotics and new chemical entities under development. Potent inhibitors of these efflux systems would be truly game-changers in breaking the xenobiotic defence system of these bacteria.
Previously discovered efflux pump inhibitors (EPIs) have not yet reached clinical phases but have proven the druggability of these targets. We have recently discovered a novel class of efflux pump inhibitors able to boost the activity of a large panel of antibiotics in both Escherichia coli and Klebsiella pneumoniae Gram-negative bacteria.
The EPI-4-AMR consortium will aim to further optimise this chemical series to identify more potent, specific and safe EPIs which will boost the antibiotic efficacy in drug sensitive and resistant clinical Enterobacterales strains and in relevant mouse infection models. This class of EPIs, in combination with efflux pump substrate antibiotics, will offer a new therapeutic alternative to treat multidrug-resistant infections by Enterobacterales.