Antibiotic potentiators maximizing the formation of open- channel OprF-type outer membrane porins

抗生素增效剂可最大限度地形成开放通道 OprF 型外膜孔蛋白

基本信息

批准号：
8980003
负责人：
Donald T Moir
金额：
$ 28.28万
依托单位：
MICROBIOTIX, INC
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-06-15 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8980003
关键词：
Acinetobacter baumannii Acute Address Animal Model Anti-Bacterial Agents Antibiotic Resistance Antibiotics Arabinose Bacteremia Bacterial Drug Resistance Bacterial Infections Biological Assay Burkholderia cepacia Burn injury Cell Membrane Permeability Cell surface Cells Cellular Structures Cephalosporins Chemicals Chemosensitization Clinical Critical Illness Cysteine Detection Development Diffusion Drug Efflux Drug resistance Dyes Equilibrium Escherichia coli Exhibits Family Fluoroquinolones Goals Gram-Negative Bacteria HIV Human Immune In Vitro Infection Label Lead Libraries Malignant Neoplasms Mammalian Cell Measures Mechanical ventilation Mediating Medical Membrane Molecular Conformation Multi-Drug Resistance Mutation Pathway interactions Patients Penicillins Peptide Hydrolases Peptidoglycan Pharmaceutical Preparations Phase Pneumonia Prevalence Proteins Pseudomonas aeruginosa Pump Research Resistance Route Series Specificity Stenotrophomonas maltophilia Structure Swelling Therapeutic Toxicity Tests Treatment Failure Virulence Virulent assay development base combat conformer cystic fibrosis patients cytotoxicity efficacy testing efflux pump high throughput screening improved inhibitor/antagonist mortality mutant novel novel strategies pathogen porin proteoliposomes public health relevance resistance frequency resistant strain screening small molecule

项目摘要

DESCRIPTION (provided by applicant): The overall goal of this project is to address the critical medical need for agents to combat antibacterial drug resistance by a novel approach of potentiating drug influx in Gram-negative non-fermenters such as Pseudomonas aeruginosa and Acinetobacter baumannii. These species exhibit intrinsic drug resistance due to the combined effects of a poorly permeable outer membrane and several multi-drug efflux pumps. The approach of this project is to develop novel adjunctive therapeutics to increase the intracellular effective levels of new and existing antibiotics. The OprF/OmpA family of porins in P. aeruginosa and A. baumannii appears to be the major route for influx of many existing antibiotics and likely for many new antibacterial since these porins are the major route for non-specific diffusion of drug-sized molecules across the outer membrane. However, these porins exist predominately (=95%) in a two-domain, closed-channel form, which spans the outer membrane and peptidoglycan layer to stabilize the cell structure. The low levels of one-domain, open- channel conformers reduce the outer membrane permeability by one to two orders of magnitude as compared to that of enterobacteriaciae such as Escherichia coli. The strategy of this proposal is to identify drug-like small molecules that shift the balance towards the open-channel porin conformer, thus improving antibacterial influx by opening the porin and destabilizing the bacterial cell structure. The two conformations are not in rapid equilibrium but appear to result from a structure-based bias for the two-domain, closed-channel from in the folding pathway of nascent proteins. Preliminary studies established that several specific mutations in P. aeruginosa OprF shift the ratio toward the open-channel form, and that a cysteine created at residue 312 in a form of OprF devoid of other cysteines is exposed on the cell surface only when OprF porins are in the single- domain, open-channel form. Labeling cys-312 with a fluorescent, membrane-impermeable dye provides an assay to detect and quantify open-channel forms. In Phase I, development and optimization of the assay will be completed to enable high throughput screening to identify small molecules that significantly shift the folding pathway toward open-channel conformers. The optimized screen will be applied to a diverse library of =300,000 discrete small molecules. Hits will be selected and confirmed in the screening assay and then validated for specificity and concentration-dependent potency in secondary assays including osmotic swelling rates of proteoliposomes containing OprF in the presence of L-arabinose, OprF protease sensitivity, and reduced MICs for cephalosporins in P. aeruginosa. Resulting non-cytotoxic, validated hits will be prioritized by their extent of potentiation of the MICs of a variety of antibacterials vs. multiple P. aeruginosa and A. baumannii clinical isolates as well as related species such as Burkholderia cepacia and Stenotrophomonas maltophilia. In Phase II, the most promising of these influx facilitators will be optimized to develop lead compounds for efficacy and toxicity testing in animal models.

描述（由申请人提供）：该项目的总体目标是通过一种增强革兰氏阴性非发酵菌（如铜绿假单胞菌和鲍曼不动杆菌）药物流入的新方法来解决对抗抗菌药物耐药性的药物的关键医疗需求。由于渗透性差的外膜和多种药物外排泵的综合作用，这些物种表现出内在的耐药性。该项目的目的是开发新型辅助疗法，以提高新的和现有抗生素的细胞内有效水平。铜绿假单胞菌和鲍曼不动杆菌中的孔蛋白 OprF/OmpA 家族似乎是许多现有抗生素流入的主要途径，并且可能是许多抗生素流入的主要途径。新型抗菌剂，因为这些孔蛋白是药物大小的分子穿过外膜非特异性扩散的主要途径。然而，这些孔蛋白主要存在。 (=95%) 呈双域、封闭通道形式，跨越外膜和肽聚糖层以稳定细胞结构。低水平的单域、开放通道构象异构体可将外膜渗透性降低一到一倍。与大肠杆菌等肠杆菌相比，该研究的策略是识别药物样小分子，将平衡转向开放通道孔蛋白构象异构体，从而改善。通过打开孔蛋白并破坏细菌细胞结构的稳定性来实现抗菌流入。这两种构象并不处于快速平衡状态，但似乎是由于新生蛋白质折叠途径中基于结构的两域、封闭通道的偏差所致。研究表明，铜绿假单胞菌 OprF 中的几个特定突变将比例转向开放通道形式，并且在 OprF 形式的残基 312 处产生的半胱氨酸缺乏仅当 OprF 孔蛋白处于单结构域、开放通道形式时，其他半胱氨酸才会暴露在细胞表面。用荧光、膜不可渗透的染料测定法标记 cys-312 提供了一种检测和定量开放通道形式的测定法。在第一阶段，将完成测定分析的开发和优化，以实现高通量筛选，以识别显着将折叠途径转向开放通道构象异构体的小分子。优化的筛选将应用于多种库。 = 300,000 个离散小分子将在筛选测定中被选择和确认，然后在二次测定中验证其特异性和浓度依赖性效力，包括在 L-阿拉伯糖存在下含有 OprF 的蛋白脂质体的渗透膨胀率、OprF 蛋白酶敏感性和铜绿假单胞菌中头孢菌素的 MIC 降低将根据其作用范围优先考虑产生的非细胞毒性、经过验证的命中。各种抗菌药物相对于多种铜绿假单胞菌和鲍曼不动杆菌临床分离株以及相关物种（如洋葱伯克霍尔德菌和嗜麦芽寡养单胞菌）的 MIC 增强在第二阶段，将优化开发这些最有前途的流入促进剂。用于动物模型功效和毒性测试的先导化合物。