Screen for efflux Pump Inhibitors and Chemical Engineering to upraise antibiotic efficiency – SpiceUp

Our project focuses on tripartite efflux pumps from the RND family (member of the Resistance, Nodulation, cell Division family). The transporter is an energy-dependent, proton-fueled pump located in the inner membrane that works in conjunction with a membrane fusion periplasmic protein (MFP), and an outer membrane channel (OMF). The cytoplasmic inner membrane transporter is a key target for developing efflux pump inhibitors (EPIs). Such EPIs act as helper- compounds since they do not possess antibiotic activity by themselves but work by blocking efflux pump and therefore improve or restore the activity of existing antibiotics. Research on EPI offers a new route to developing new drugs or to restoring efficiency to known compounds. The recommended combination therapy (an antibiotic of the beta-lactam class together with an aminoglycoside or a fluoroquinolone), now often lead to therapeutic failures because of the poly- specificity of efflux-pumps that can efficiently extrude all of these drugs from the bacteria, and when overproduced, render bacteria multi-resistant. Then, last-resort compounds like polymyxin have to be prescribed. The implementation path of microbial EPIs may be hindered by the fact that manipulation of efflux systems can cause unexpected toxicity due to the multitude of physiological roles transporters play in human cells. Thus, efforts directed at specifically inhibiting efflux pumps operating only in prokaryotes, such as RND pumps, may offer a greater chance of therapeutic success. The development of a clinically applicable universal or species-specific EPI for Gram- negative pathogens remains a challenge.

The novelty of our project is to apply an original and unique methodology that allows to specifically monitor the activity of in vitro reconstituted efflux pumps from Gram negative bacteria. The methodology is already up and working and is available to screen for inhibitors. Our focus will be to characterize EPIs active on efflux pumps MexAB-OprM from Pseudomonas aeruginosa and AcrAB-TolC from Escherichia coli.
- The expression, purification and reconstitution protocol is routinely performed with good yields. Medium throughput liposome permeabilization tests have been developed.

- Three libraries of molecules, including one from a first pharmacomodulation campaign, have been tested on bacteria. Candidate molecules were identified.

- A pool of 37 most interesting molecules were submitted to the in vitro permeabilization test. A minority of molecules having an intrinsic effect on the integrity of the membrane were excluded.

The consortium works very well. The communication between the different teams allows us to communicate our respective progress and to quickly resolve any problems encountered. We are perfectly on time with respect to our schedule.

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The need to overcome antibiotic resistance in common Gram-negative pathogens remains unmet and represents a challenging aspect of the research in the field of antimicrobials. Among the various resistance mechansims developped by bacteria, efflux pumps are on the front line. They consist of membrane proteins that expel noxious compounds outside of the bacteria, across its two-membranes environment. Efflux pumps constitute a promising therapeutic target, as their blockage could restore the actual arsenal utility. Our project is to identify efflux pump inhibitors (EPIc) that will consit of adjuvant molecules without intrinsic antibiotic activity but working by blocking efflux pump and therefore restoring the activity of antibiotics. Usually, the activity of the suspected EPIs is deduced from measurements where the susceptibility of bacteria to different antibiotics is measured in the absence or in the presence of the molecules tested.  The novelty of our project is to apply an original and unique methodology that allows to specifically monitor the activity of in vitro reconstituted efflux pumps from Gram negative bacteria. This test relies on the reconstitution of the efflux pumps in proteoliposomes mimicking the two-membranes architecture of the envelope of Gram-negative bacteria, and on the monitoring of the transfer of substrates through the reconstituted pump using fluorescent reporters.
In the meantime, libraries will be tested against Gram-negative strains from relevant species (P. aeruginosa, E. coli, E. aerogenes) in primary antibiotic enhancement assays: functional and phenotypic assays employing strains that lack or overexpress efflux systems . Hence, the combination of both expertize will make it possible for us to explore new chemical spaces and will be the basis for pharmacomodulation improvement campaigns.
Finally, the methodology will be adapted, miniaturized and automatized on a 96-wells microplate reader, hence making it possible to automatically test large-scale chemical libraries.
Our focus will be to characterize EPIs active on efflux pumps MexAB-OprM from Pseudomonas aeruginosa and AcrAB-TolC from Escherichia coli.