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: 10286575
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10286575
• 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/antagonist; 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 多药耐药（Multidrug resistance，MDR）是全球性的公共卫生威胁，并已造成严重的经济负担 国际吧每年，仅在美国就有超过280万例耐药性感染，造成约 36,000人死亡耐药细菌已经进化出内在和获得性耐药机制， 保护，逃避和避免抗生素，导致几乎所有可用的抗生素无效。其中一个重要 被归类为对公众健康的紧急威胁的病原体是铜绿假单胞菌（P. aeruginosa）。MDR 假单胞菌菌株对几乎所有可用的抗生素都有抗性，因此被鉴定为严重威胁， 疾病控制和预防中心。革兰阴性杆菌多药耐药的主要原因 包括铜绿假单胞菌在内的细菌是耐药-增殖-细胞分裂（RND）家族多药耐药基因的过度表达。 外排泵通过将药物分子输出到细胞外。因此，多药外排泵的抑制 外排泵抑制剂（EPIs）是一种有吸引力和有前途的增强和恢复抗菌药物活性的方法， 通过协同或抑制耐药细菌的外排泵来增强现有抗生素的活性。然而，更好地 了解药物外排和外排抑制的EPI是必不可少的开发新的和有效的 具有更好的渗透和外排抑制作用的EPI/抗菌药物。因此，拟议研究的目标是确定 MexEF-OprN（铜绿假单胞菌的多药外排泵）的有效抑制剂，并了解其生物化学特性 药物外排和外排抑制的机制。我们将采用跨学科的方法，包括 微生物、生物化学、生物物理、基于细胞的测定和体外功能测定。我们将绘制药物 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- 使用药物结合、外排和外排抑制所必需的外排泵药物转运蛋白的酸性残基， 定向进化的组合无偏方法和基于细胞的测定。我们还将测量 使用生物物理学和体外功能测定来测定抑制剂和/或底物的特异性。我们将使用一个 强大的生物物理工具，表面等离子体共振（SPR），以确定直接结合，特异性和 底物和抑制剂对MexF外排泵转运蛋白的亲和力。我们将测量小分子- 分子与MexF的相互作用，以使用纯化的MexF外排泵，使用实时荧光定量PCR测定其亲和力。 基于PL的体外转运测定。