Inhibitors for Bacterial Efflux Pumps – IBEF

Our project links biophysical, biochemical, chemical and microbiological aspects with multidisciplinary and complementary expertise from the partners in order to decipher the various parameters of drug transport. During this project, prominent points have been studied such as the biological activity of the E. aerogenes AcrB pump and its selectivity towards various substrates, the modeling and dynamics simulation of the pump, the docking and location of some potential substrates inside AcrB pump, the identification/design of pharmacophoric groups in the substrate, and rational synthesis of original compounds that inhibit antibiotic efflux. This generated model has been used to study the specific binding site of the different molecules inside the E. aerogenes AcrB pump and to identify key interactive regions. The rational synthesis has been carried out using the various chemical pathways previously used and the activity of these compounds determined using the collection of strains previously characterized.

The main result corresponds to the identification of pharmacophores in some inhibitor molecules, a key information for improving the design of drugs.

The pharmacomodulation of specific chemical groups (in the putative inhibitor) exhibiting a strong affinity for internal pump sites and causing a defect in efflux properties is the attractive application for potentiating antibiotic concentrations in MDR pathogens.

All the publications are listed at the end of this document
5 mono-partner publications, 2 multi-partners in preparation; 3 reviews and book chapter, one rmulti-partner review scheduled for 2017. 5 lectures, 7 oral communications, and 3 posters presented from the beginning of IBEF.

In Gram-negative bacteria, the over-expression of efflux pumps (EP) is involved in the Multi-Drug Resistance phenotype (MDR). EPs recognize a large panel of substrates including all antibiotic and biocide families. These EPs favour the acquisition of additional resistance mechanisms such as target mutation or drug modification. AcrAB-TolC pump belonging to RND super family of drug transporters, is active in the majority of MDR enterobacterial clinical isolates. A functional, pharmaco-chemical and structural analysis of pump-substrate interactions will be engaged to decipher the molecular bases of interaction of the pump with specific ligands allowing us to develop the rational synthesis of efflux inhibitors.
Our proposal links biophysical, biochemical, chemical and microbiological aspects with multidisciplinary and complementary expertises in order to decipher the molecular basis of drug transport. During this project, prominent points must be studied: the biological activity of the E. aerogenes AcrB pump and its selectivity towards various substrates, the modelling and dynamic simulation of the pump, the 3D-resolution of substrate affinity domains in the pump, the identification/design of pharmacophoric groups, and rational synthesis of original compounds that inhibit antibiotic efflux. With these objectives, we will use four experimental tasks : - biological and functional analyses of EP: this part includes a bacteriological determination of activity of the EP and its contribution in E. aerogenes clinical isolates, the study of pump selectivity (quinolones, ß-lactams, etc), and the characterization and activity of new pump inhibitors. - molecular modelling and dynamic simulation : we will study the pump by using various molecular modelling methods and dynamic simulations (such as docking, free and targeted MD). This new generated model will be used to study the specific binding of the different molecules inside the E. aerogenes AcrB pump and to identify key interactive regions. - structural analyses of EP : the determination of the 3D structure of E. aerogenes AcrB will be carried out (alone or with substrates) in order to precise the location of affinity sites. This part will be used to determine the involved residues in the affinity pockets and to improve the model. - QSAR and rational synthesis: using the results from structure and modelling parts we will design and then synthesize molecules exhibiting improved affinity for EP sites. The rational synthesis will be carried out using the various chemical pathways previously used. The activity of these compounds will be determined using the collection of strains characterized during the second task. The main result will focus on the efflux dynamics and mechanism: the identification of pharmacophores in efflux-substrate molecules will be a key information for the design of drugs, e.g. the modification of specific chemical groups exhibiting a strong affinity for internal pump sites (inhibitor) or a decrease affinity causing a defect in efflux properties (bad substrate) thereby potentiating antibiotic concentrations. Additional data regarding membrane transporters is a fruitful gain for the biologist, the physico-chemist and the pharmacologist taking into account the very restricted information available on bacterial membrane proteins (only two RND pumps have been crystallized).