Inhibitors of Pseudomonas aeruginosa efflux pumps to combat multi-drug resistance

Abstract Multi drug resistanceMDRin Gram negative pathogensincluding Pseudomonas aeruginosaposes a significant threat for effective treatment of infections caused by these organismsA major component in the development of the MDR phenotype in Paeruginosa is overexpression of the Resistance Nodulation DevelopmentRNDfamily efflux pumpswhich pump antibacterial agents and biocides out of the celland are involved in virulencebiofilm formationand acquired antibiotic resistanceThe overall goal of this proposal is to identify potent inhibitors of the RND family efflux pumps in Paeruginosa and develop them into innovative therapeuticswhich will be used to increase the potency of existing and new antibiotics and decrease the emergence of MDR bacteriaOur strategy is to use an innovative cellular bioluminescent reporter assay to screen a library of small molecules and natural products for efflux pump inhibitorsEPIsIn preliminary studieswe developed a high throughput screenHTSfor fatty acid synthesis IIFASIIinhibitors using a PfabDluxCDABE reporter strainwhich induces a strong and reproducible luminescent signal in response to FASII inhibitors in an efflux deficientbut not an efflux proficient strainindicating the FASII inhibitors are RND pump substratesThereforethe efflux proficient PfabD luxCDABE reporter strain grown in the presence of a FASII inhibitor provides a sensitivegain of signal reporter assay to screen for inhibitors of RND efflux pumpswhich we verified using a known EPIPA NIn Phase Iwe will optimize the PfabD luxCDABE cell based reporter assay for high throughput screening and we will validate the assay in a small scale pilot screen of known bioactive compoundsThe validated assay will be applied to screen a library ofcompounds at Microbiotix and at the ICCBHarvard Medical SchoolPrimary hit compounds will be confirmedtested against a counter screen to eliminate non specific inhibitorsand prioritized based on their potency and medicinal chemistry propertiesThe high priority compounds will be evaluated in a panel of secondary assays that will assess EPI activityspectrum of activitycytotoxicityselectivitysolubilityand liver microsome stabilityWe will order commercially available analogsas availableof high priority hits to establish preliminary structure activity relationshipsSARWe expect to identifyvalidated hits that meet the stringent criteria described in the milestones of each specific aimThese compounds will be optimized in Phase II to generate Lead compounds for drug developmentTo achieve the goal of this Phase I proposal we will complete the following Specific AimsAimDevelop and optimize a cell based reporter assay for efflux pump inhibitors in PaeruginosaAimScreen a diverse small molecule library for compounds that are potent inhibitors of efflux pumps in PaeruginosaAimValidate confirmed hits using secondary assays and identify hit to lead series