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

 描述(由申请人提供)：这个项目的总体目标是通过一种新的方法来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)的药物流入，以满足药物对抗抗菌素耐药性的关键医学需求。由于渗透性差的外膜和几个多药外排泵的共同作用，这些物种显示出内在的耐药性。这个项目的方法是开发新的辅助疗法，以增加新的和现有的抗生素在细胞内的有效水平。在铜绿假单胞菌和鲍曼不动杆菌中，OprF/OmpA孔蛋白家族似乎是许多现有抗生素流入的主要途径，也可能是许多新的抗菌药物的主要途径，因为这些孔蛋白是药物大小的分子通过外膜非特异性扩散的主要途径。然而，这些孔蛋白主要(=95%)以两个结构域、闭合通道的形式存在，该形式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、开放通道构象使外膜通透性降低一到两个数量级。这一建议的策略是识别类药物小分子，这些小分子将平衡转移到开放通道的孔蛋白构象，从而通过打开孔蛋白和破坏细菌细胞结构的稳定来改善抗菌药物的流入。这两种构象并不是快速平衡的，但似乎是由于新生蛋白质折叠途径中的两个结构域、闭合通道的基于结构的偏向造成的。初步研究证实，铜绿假单胞菌OprF中的几个特定突变使比率向开放通道形式移动，并且只有当OprF孔蛋白处于单结构域、开放通道形式时，在残基312处以OprF形式生成的半胱氨酸才会暴露在细胞表面。用一种不透膜的荧光染料标记Cys-312提供了一种检测和量化开放通道形式的方法。在第一阶段，将完成分析的开发和优化，以实现高通量筛选，以识别显著将折叠途径转向开放通道构象的小分子。优化的屏幕将应用于=300,000个离散小分子的多样化文库。HITS将在筛选试验中进行选择和确认，然后在二次试验中验证其特异性和浓度依赖性效力，包括含有OprF的蛋白脂质体在L-阿拉伯糖存在下的渗透肿胀率、OprF蛋白酶敏感性和头孢菌素在铜绿假单胞菌中的降低MIC值。由此产生的无细胞毒性、有效的HITS将优先考虑各种抗菌药对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窄食单胞菌)的最低抑菌浓度的增强程度。在第二阶段，这些流入促进剂中最有希望的将被优化，以开发用于动物模型有效性和毒性测试的先导化合物。

 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.