Small Molecule Inhibition of a Multidrug Efflux Pump of Pseudomonas aeruginosa

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Project summary/Abstract Small Molecule Inhibition of a Multidrug Efflux Pump of Pseudomonas aeruginosa Multidrug resistance (MDR) is a major global threat to the public health and has posed an economic burden worldwide. Each year, more than 2.8 million antibiotic-resistant infections occur in the U.S. alone, causing around 36,000 deaths. Resistant bacteria have evolved with both intrinsic and acquired resistance mechanisms to protect, escape and avoid antibiotics, causing inefficacy of almost all available antibiotics. One of the important pathogens classified as an urgent threat to public health is Pseudomonas aeruginosa (P. aeruginosa). MDR strains of Pseudomonas are resistant to nearly all available antibiotics and thus identified as a serious threat by the Centers for Disease Control and Prevention. The primary cause for multidrug resistance in Gram-negative bacteria including P. aeruginosa is overexpression of resistance-nodulation-cell division (RND) family multidrug efflux pumps by exporting drug molecules out of the cells. Therefore, inhibition of multidrug efflux pumps by efflux pump inhibitors (EPIs), is an attractive and promising approach to potentiate and revive antibacterial activities of existing antibiotics by synergizing or inhibiting efflux pumps of resistant bacteria. However, better understanding of drug efflux and efflux inhibition by EPIs is essential to develop novel and potent EPIs/antibacterials with better permeation and efflux inhibition. So, the goal of the proposed research is to identify potent inhibitors of MexEF-OprN, a multidrug efflux pump of P. aeruginosa and understand the biochemical mechanisms of drug efflux and efflux inhibition. We will use an interdisciplinary approach, including microbiological, biochemical, biophysical, cell-based assays and in vitro functional assays. We will map the drug binding and efflux inhibition site(s) of the efflux pump transporter. Specifically, we will identify the specific amino- acid residues of efflux pump drug transporter essential for drug binding, efflux, and efflux inhibition using a combinatorial unbiased approach of directed evolution and cell-based assays. We will also measure the specificity of inhibitors and/or substrates using both biophysical and in vitro functional assays. We will use a powerful biophysical tool, Surface Plasmon Resonance (SPR) to determine the direct binding, specificity and affinity of substrates and inhibitors for MexF efflux pump transporter. We will measure the kinetics of small- molecule interactions with MexF to determine their affinity using purified MexF efflux pump using a real-time in vitro PLs-based transport assay.