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:
  • 金额:
    $ 28.28万
  • 依托单位:
  • 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

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(由申请方提供):本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)中药物流入的新方法,解决对抗抗菌药物耐药性药物的关键医疗需求。由于渗透性差的外膜和几种多药外排泵的联合作用,这些物种表现出内在的耐药性。该项目的方法是开发新的连续治疗方法,以增加新的和现有的抗生素的细胞内有效水平。铜绿假单胞菌和A.鲍曼不动杆菌似乎是许多现有抗生素流入的主要途径,并且可能是许多新抗菌剂流入的主要途径,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(=95%)以双结构域、封闭通道形式存在,其跨越外膜和肽聚糖层以稳定细胞结构。与肠杆菌如大肠杆菌相比,低水平的单结构域开放通道构象异构体使外膜渗透性降低一到两个数量级。该建议的策略是鉴定药物样小分子,其将平衡转向开放通道孔蛋白构象异构体,从而通过打开孔蛋白和使细菌细胞结构不稳定来改善抗菌剂流入。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的两个结构域、封闭通道的基于结构的偏倚所致。初步研究确定,铜绿假单胞菌OprF中的几种特定突变使比率向开放通道形式转变,并且仅当OprF孔蛋白处于单结构域开放通道形式时,在残基312处以缺乏其他半胱氨酸的OprF形式产生的半胱氨酸才暴露在细胞表面上。用荧光的、膜不可渗透的染料标记cys-312提供了一种检测和定量开放通道形式的分析。在第一阶段,将完成检测方法的开发和优化,以实现高通量筛选,以鉴定将折叠途径显著转向开放通道构象异构体的小分子。优化的筛选将应用于= 300,000个离散小分子的多样化文库。将在筛选试验中选择并确认命中,然后在二级试验中验证特异性和浓度依赖性效价,包括在存在L-阿拉伯糖的情况下含OprF的蛋白脂质体的渗透溶胀率、OprF蛋白酶敏感性和铜绿假单胞菌中头孢菌素的降低MIC。将根据各种抗菌药物与多种铜绿假单胞菌和A.鲍曼不动杆菌临床分离株以及相关种,如洋葱伯克霍尔德氏菌和嗜麦芽窄食单胞菌。在第二阶段,这些流入促进剂中最有前途的将被优化,以开发先导化合物,用于动物模型的功效和毒性测试。

项目成果

期刊论文数量(0)
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Donald T Moir其他文献

Donald T Moir的其他文献

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{{ truncateString('Donald T Moir', 18)}}的其他基金

Inhibitors of the viral nucleoprotein-polymerase co-factor interaction for human RSV and MPV therapy
用于人类 RSV 和 MPV 治疗的病毒核蛋白-聚合酶辅因子相互作用的抑制剂
  • 批准号:
    9200084
  • 财政年份:
    2016
  • 资助金额:
    $ 28.28万
  • 项目类别:
Inhibitors of isoprenoid synthesis for antibacterial therapy
用于抗菌治疗的类异戊二烯合成抑制剂
  • 批准号:
    8602834
  • 财政年份:
    2013
  • 资助金额:
    $ 28.28万
  • 项目类别:
Inhibitors of isoprenoid synthesis for antibacterial therapy
用于抗菌治疗的类异戊二烯合成抑制剂
  • 批准号:
    8522430
  • 财政年份:
    2013
  • 资助金额:
    $ 28.28万
  • 项目类别:
Inhibitors of S. aureus bNOS for adjunctive therapy
用于辅助治疗的金黄色葡萄球菌 bNOS 抑制剂
  • 批准号:
    8492028
  • 财政年份:
    2012
  • 资助金额:
    $ 28.28万
  • 项目类别:
Validating targets in p. aeruginosa type III secretion using chemical probes
验证第 14 页中的目标。
  • 批准号:
    8283415
  • 财政年份:
    2012
  • 资助金额:
    $ 28.28万
  • 项目类别:
Aminoglycoside potentiators for P. aeruginosa therapy
用于治疗铜绿假单胞菌的氨基糖苷类增效剂
  • 批准号:
    8454071
  • 财政年份:
    2012
  • 资助金额:
    $ 28.28万
  • 项目类别:
Validating targets in p. aeruginosa type III secretion using chemical probes
验证第 14 页中的目标。
  • 批准号:
    8452056
  • 财政年份:
    2012
  • 资助金额:
    $ 28.28万
  • 项目类别:
Inhibitors of S. aureus bNOS for adjunctive therapy
用于辅助治疗的金黄色葡萄球菌 bNOS 抑制剂
  • 批准号:
    8393335
  • 财政年份:
    2012
  • 资助金额:
    $ 28.28万
  • 项目类别:
Aminoglycoside potentiators for P. aeruginosa therapy
用于治疗铜绿假单胞菌的氨基糖苷类增效剂
  • 批准号:
    8585819
  • 财政年份:
    2012
  • 资助金额:
    $ 28.28万
  • 项目类别:
Validating targets in p. aeruginosa type III secretion using chemical probes
验证第 14 页中的目标。
  • 批准号:
    8636993
  • 财政年份:
    2012
  • 资助金额:
    $ 28.28万
  • 项目类别:

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