EFFlux pump inhibitors to Overcome antibiotic ResisTance – EFFORT

Molecular biology and genetics approaches will be used to define if the efflux pump inhibitors act on the pumps themselves or otherwise. Antibiotic susceptibility studies will allow for the evaluation of the spectrum of antibiotics that can be boosted in a given bacteria, including checkerboard assays and time kill assays. The use of recombinant efflux pump expression systems will allow for the analysis of the relative susceptibility of different bacterial RND pumps to the developed inhibitors. Structural biology will allow for the definition of the binding pocket of the inhibitors and allow for gaining insight into how to improve the inhibitors. Structural biology of unstudied Gram negative RND pumps will help reveal their structures which will further help inhibitor development. Molecular dynamics simulations will allow for the analysis of inhibitor uptake and interactions. Medicinal chemistry will allow for the generation of a robust structure activity relationship, improve potency and optimise the ADME properties of inhibitors to allow for in vivo proof of concept studies. In vivo studies will help to define the clinical potential of these inhibitor.

Current work has clearly defines that EFFORT is developing a bona vide novel class of RND efflux pump inhibitor that is unique to antibiotic research. These inhibitors have been shown in bacteria and by structural biology to act in a new binding pocket and allow for the complete inhibition of RND efflux pump activity. This concomitant allows for the boosting of the antibiotic activity of all RND efflux pump substrates in Enterobacteriaceae, but also other Gram negative bacteria. Medicinal chemistry has allowed for the development of analogues with 20 fold greater potency, making current inhibitor superior to PABN. Medicinal chemistry has also allowed improved drug like properties, with current molecules approaching in vivo proof of concept studies.

In addition,the development of the technology needed for the structural biology of novel alternative Gram negative RND pumps by cryo-EM and crystallography is advancing well.

To further optimise the inhibitors to be potent on other gram negative bacteria where inhibitor penetration is more challenging. To define in vivo proof of activity in a mouse model where these inhibitors will be given in combination with RND efflux pump substrates. To finalise crystallography, single particle and in situ cryo-TEM for the elucidation of the structures of novel Gram negative efflux pumps involved in antibiotic resistance.

Overall description of tasks and timeline of research is progressing as origionally planned

To date, one patent has been prepared with the help of INSERM transfer for the IP on the efflux pump inhibitors, and is ready for submission. Submission will proceed just prior to publication of the research.

A manuscript has been prepared and ready for submission (early July 2021) describing the discovery and characterisation of these novel class of efflux pump inhibitors.

The discovery of novel classes of antibiotics for Enterobacteriaceae, A. baumannii and P. aeruginosa (the most critically classed pathogens on the WHO priority list), is particularly obstructed by their efficient and promiscuous xenobiotic efflux pumps that prevent the efficacy of new chemical entities. The upregulation of these pumps in clinical strains also results in resistance to current antibiotics. It is therefore clear that developing inhibitors to these efflux systems, as well as enriching our knowledge of the basic biology of these pumps will be a major step forward in breaking the xenobiotic defence system of these bacteria.

Efflux pump inhibitors (EPIs) previously discovered have thus far not been developed into clinical candidates but have greatly helped understand the mechanism by which the pumps work, and proven the druggability of these targets. It is clear that strengthening and expanding this basic knowledge on efflux pumps to bacteria such as A. baumannii is fundamental and synergistic to the development of novel EPIs to help fight Gram-negative infections. Through a fragment-screening we have identified a small water soluble compound able to boost the activity of a broad spectrum of AcrAB-TolC efflux pump substrates in both E. coli and K. pneumoniae. Preliminary data within the EFFORT partnership show that this EPI may bind the “hydrophobic trap” of AcrB, a critical pocket important for pump inhibition. In A. baumannii, this EPI was found to boost the activity of chloramphenicol. By drawing similarities with E. coli, the new EPI may therefore also be a novel inhibitor for efflux in A. baumannii.

The EFFORT consortium will aim to pinpoint the binding pocket of the new EPI in E. coli, K. pneumonia and A. baumannii, and investigate its ability to act as a chemical scaffold for inhibiting other efflux pumps in A. baumannii and P. aeruginosa (such as silent efflux pumps). This whole effort will be supported by structural studies to define the exact binding position of the inhibitors in E. coli AcrB, in K. pneumoniae AcrB and in the to be uncovered A. baumannii efflux pump. The structural biology will be supported by both X-ray crystallography and state-of-the-art Cryo-EM imaging that will maximise the likelihood of enriching out knowledge in efflux pump biology and EPIs. The size and chemical properties of the novel EPI make it very accommodating for easy chemical diversification, rationally guided by structural biology data, molecular dynamics simulations and biological activities, that will aim to improve the potency of the EPI. The physico-chemical and pharmacokinetic properties of the compounds will also be monitored to rapidly identify optimised molecules ready to be tested for in vivo efficacy. Overall EFFORT will both enlighten the basic structural biology of efflux pumps in Gram-negative bacteria by state of the art techniques, and develop a promising EPI.