Small Molecule Inhibition of a Multidrug Efflux Pump of Pseudomonas aeruginosa

铜绿假单胞菌多药外排泵的小分子抑制

• 批准号: 10435576
• 负责人: HELEN I ZGURSKAYA
• 金额: $ 7.06万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-21 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10435576
• 关键词: Affect; Affinity; Amino Acids; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Bacteria; Binding; Biochemical; Biological Assay; Biophysics; Cell division; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Complement; Cystic Fibrosis; Development; Directed Molecular Evolution; Drug Efflux; Economic Burden; Essential Drugs; Family; Goals; Gram-Negative Bacteria; Hospitals; Immune system; In Vitro; Individual; Infection; Kinetics; Maps; Measures; Mediating; Microbial Biofilms; Microbiology; Modeling; Multi-Drug Resistance; Nodule; Pathogenesis; Patients; Pharmaceutical Preparations; Plasmids; Proteins; Pseudomonas; Pseudomonas aeruginosa; Public Health; Research; Resistance; Resources; Risk Factors; Site; Small Molecule Chemical Library; Specificity; Structure-Activity Relationship; Substrate Specificity; Surface Plasmon Resonance; Time; Training; Virulence; Work; antibiotic resistant infections; bacterial resistance; base; biophysical tools; combinatorial; efflux pump; graduate student; inhibitor; innovation; interdisciplinary approach; multidrug-resistant Pseudomonas aeruginosa; mutant; next generation; novel; overexpression; pathogen; proteoliposomes; reconstitution; resistance mechanism; resistant strain; response; small molecule; small molecule inhibitor; undergraduate student

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.

项目概要/摘要 铜绿假单胞菌多药外排泵的小分子抑制 多重耐药性（MDR）是对全球公共卫生的主要威胁，并造成了经济负担 全世界。每年，仅在美国就发生超过 280 万例抗生素耐药性感染，造成约 36,000 人死亡。耐药细菌已经进化出内在的和获得性的耐药机制 保护、逃避和避免抗生素，导致几乎所有可用的抗生素无效。其中重要的一项 被列为对公众健康构成紧急威胁的病原体是铜绿假单胞菌（P. aeruginosa）。多重耐药 假单胞菌菌株对几乎所有可用的抗生素都具有抗药性，因此被认定为严重威胁 疾病控制和预防中心。革兰氏阴性菌多重耐药的主要原因 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（EPI）是一种有吸引力且有前途的增强和恢复抗菌作用的方法 通过协同或抑制耐药细菌的外排泵来抑制现有抗生素的活性。不过，更好的是 了解 EPI 的药物流出和流出抑制对于开发新颖有效的药物至关重要 EPI/抗菌剂具有更好的渗透和外排抑制作用。因此，拟议研究的目标是确定 MexEF-OprN（铜绿假单胞菌多药外排泵）的有效抑制剂，并了解其生化机制 药物外排和外排抑制机制。我们将采用跨学科的方法，包括 微生物学、生物化学、生物物理、细胞分析和体外功能分析。我们将绘制药物图 外排泵转运蛋白的结合和外排抑制位点。具体来说，我们将确定特定的氨基 外排泵药物转运蛋白的酸残基对于药物结合、外排和外排抑制至关重要 定向进化和基于细胞的测定的组合无偏方法。我们还将测量 使用生物物理和体外功能测定来确定抑制剂和/或底物的特异性。我们将使用一个 强大的生物物理工具，表面等离子共振 (SPR)，可确定直接结合、特异性和 MexF 外排泵转运蛋白的底物和抑制剂的亲和力。我们将测量小分子的动力学 使用纯化的 MexF 外排泵使用实时分析仪与 MexF 进行分子相互作用，以确定其亲和力 基于 PLs 的体外转运测定。