Type 3 secretion system inhibitors to treat Pseudomonas aeruginosa keratitis

隐形眼镜相关的细菌性角膜炎是一种严重的眼部感染，如果治疗可能导致失明 是无效的。机会性革兰氏阴性病原菌铜绿假单胞菌是最常见的 与接触透镜使用相关的病原体和多药耐药菌株的流行正在增加。P. 铜绿假单胞菌在角膜上皮上形成生物膜，导致对抗生素的耐药性增加， 中性粒细胞，进一步使这些感染的治疗复杂化。这些感染的大多数临床分离株 是表达ExoU的细胞毒性菌株，ExoU是一种通过3型分泌物注入宿主细胞的强效细胞毒素 系统（T3SS），这与增加的毒力，抗生素耐药性和更高的发病率。 显然，需要新的方法来治疗这些感染。铜绿假单胞菌的T3SS是一种毒力 该因子是该生物体在眼睛中建立感染所必需的。本提案的总体目标是 开发一种新的局部疗法，用于治疗接触镜相关性角膜炎，靶向P的T3SS。 铜绿。在Microbiotix（MBX）正在进行的一项计划中，我们发现并化学优化了一种新的 苯氧基乙酰胺（PhA）化学系列的铜绿假单胞菌的T3SS的有效抑制剂，导致 两性离子（ZW）类似物亚系列的PhA，具有优异的体外效力和ADM E性质。几个ZW 类似物在具有铜绿假单胞菌感染的鼠非血小板减少性肺炎模型中是有效的， 其在体外试验和感染的鼠模型中的功效。我们的发展战略 铜绿假单胞菌角膜炎的治疗是使用体外试验评估这些ZW PhA T3SS抑制剂的选择集， 以及设计用于鉴定适合于进一步开发为局部治疗剂的先导候选物的体内测定 抗假单胞菌抗生素治疗铜绿假单胞菌角膜炎。在第一阶段的提案中，我们将优先考虑 ZW类似物库的成员使用一组基于体外细胞的T3SS抑制活性和细胞毒性， 测定和角膜炎临床分离物与人角膜上皮细胞系。根据这些结果， 研究中，我们将鉴定多达6种类似物，用于评价在鼠铜绿假单胞菌中的耐受性和功效。 角膜炎模型作为眼用抗生素的连续治疗，目的是选择主要候选药物 这适合于在后续第二阶段提案中进一步开发。第11阶段研究将包括 用于眼科适应症的先导优化、眼科抗生素组合的评价、配方，以及 向FDA提交IND申请所需的临床前研究。 我们将通过完成以下具体目标来实现第一阶段的目标：目标1。优先选择PhA 使用与眼部感染相关的体外测定的T3SS抑制剂。目标二。基于以下内容确定潜在客户 在鼠铜绿假单胞菌角膜炎模型中的体内功效。

Contact lens-associated bacterial keratitis is a serious infection of the eye that can result in blindness if treatment is not effective. The opportunistic Gram-negative pathogen Pseudomonas aeruginosa is the most common pathogen associated with contact lens use, and multi-drug resistant strains are increasing in prevalence. P. aeruginosa forms biofilms on the corneal epithelium, resulting in increased resistance to antibiotics and killing by neutrophils, further complicating the treatment of these infections. Most clinical isolates from these infections are cytotoxic strains that express ExoU, a potent cytotoxin that is injected into host cells by the type 3 secretion system (T3SS), which is associated with increased virulence, antibiotic resistance, and greater morbidity. Clearly, novel approaches to treating these infections are needed. The T3SS of P. aeruginosa is a virulence factor that is essential for this organism to establish infection in the eye. The overall goal of this proposal is to develop a novel topical therapy for the treatment of contact lens-associated keratitis that targets the T3SS of P. aeruginosa. In an ongoing program at Microbiotix (MBX), we discovered and chemically optimized a novel phenoxyacetamide (PhA) chemical series of potent inhibitors of the T3SS of P. aeruginosa, resulting in the zwitterionic (ZW) analog sub-series of PhAs with excellent in vitro potency and ADM E properties. Several ZW analogs were efficacious in a murine non-neutropenic pneumonia model with P. aeruginosa infection consistent with their efficacy in in vitro assays and in murine models of infection. Our strategy for developing novel treatments for P. aeruginosa keratitis is to evaluate a select set of these ZW PhA T3SS inhibitors using in vitro and in vivo assays designed to identify a lead candidate suitable for further development as a topical adjunctive therapy to anti-pseudomonal antibiotics for P. aeruginosa keratitis. In this Phase I proposal, we will prioritize members of a library of ZW analogs for T3SS inhibitory activity and cytotoxicity using a panel of in vitro cellbased assays and keratitis clinical isolates with human corneal epithelial cell lines. Based on the results of these studies, we will identify up to 6 analogs for evaluation of tolerability and efficacy in a murine P. aeruginosa keratitis model as an adjunctive therapy with an ophthalmic antibiotic, with the goal of selecting a lead candidate that is suitable for further development in a follow-on Phase II proposal. The Phase 11 research will comprise lead optimization for an ophthalmic indication, evaluation of ophthalmic antibiotic combinations, formulation, and pre-clinical studies that are required for an IND application to the FDA. We will accomplish the goals of Phase I by completing the following Specific Aims: Aim 1. Prioritize select PhA T3SS inhibitors using in vitro assays relevant to ophthalmic infection. Aim 2. Identify a lead candidate based on in vivo efficacy in a murine P. aeruginosa keratitis model.