Small Molecule Inhibition of a Multidrug Efflux Pump of Pseudomonas aeruginosa

项目摘要/摘要 铜绿假单胞菌多药外排泵的小分子抑制作用 多药耐药(MDR)是对公共健康的重大全球威胁，并已造成经济负担 全世界。每年，仅在美国就发生了超过280万例耐药感染，导致约 36,000人死亡。抗药性细菌进化出了内在和获得性的抗药性机制 保护、逃避和避免抗生素，导致几乎所有可用的抗生素无效。其中一个重要的 被列为公共卫生紧急威胁的病原体是铜绿假单胞菌(P.铜绿假单胞菌)。MDR 假单胞菌菌株对几乎所有可用的抗生素都有抗药性，因此被认为是一种严重的威胁 疾病控制和预防中心。革兰氏阴性菌多药耐药的主要原因 包括铜绿假单胞菌在内的细菌过度表达耐药结节细胞分裂(RND)家族多药 通过将药物分子输出到细胞外来进行外排泵。因此，通过抑制多药外排泵 外排泵抑制剂(Epis)是一种诱人的、有前途的增强和恢复抗菌药物的方法 通过协同或抑制耐药细菌的外排泵来发挥现有抗生素的活性。然而，更好的是 了解Epis对药物外排和外排的抑制对于开发新的有效的药物外排是必不可少的 EPIS/抗菌药物具有更好的渗透性和外排抑制作用。因此，这项拟议研究的目标是确定 铜绿假单胞菌多药外排泵MexEF-OprN的有效抑制剂和了解生化 药物外排和抑制外排的机制。我们将使用跨学科的方法，包括 微生物学、生化、生物物理学、细胞分析和体外功能分析。我们会绘制出药物地图 外排泵转运体的结合和外排抑制部位(S)。具体地说，我们将确定特定的氨基- 外排泵药物转运体的酸残留物对于药物结合、外排和外排抑制至关重要 定向进化和基于细胞的分析的组合无偏方法。我们还将测量 使用生物物理和体外功能分析的抑制剂和/或底物的特异性。我们将使用 强大的生物物理工具，表面等离子体共振(SPR)，以确定直接结合，特异性和 MexF外排泵转运体的底物和抑制剂的亲和力。我们将测量小行星的动力学。 用纯化的MexF外排泵实时测定分子与MexF的相互作用 基于PLS的体外转运试验。

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.