Inhibitors of T3SS translocon assembly to combat multi-drug resistant P. aeruginosa

摘要 铜绿假单胞菌（PA）是最常从ICU患者呼吸道分离的细菌， 在美国，这是导致插管患者肺炎的主要原因。此外，PA基因的感染- 预后不良，估计有40-69%的PA呼吸机相关性肺炎（VAP）病例复发， 导致死亡，超过30%的幸存者在使用标准护理抗菌剂后复发。 疗法该项目的总体目标是通过发现新的小鼹鼠来解决这一关键的医疗需求， 3型分泌系统（T3 SS）易位子的Cule抑制剂，主要由PopB和PopD组成， 并将其开发成新的抗PA治疗药物。T3 SS是主要的 毒力因子有助于PA感染的建立和传播，并且其存在与PA感染阿索。 与感染患者的不良临床结果和死亡有关。该项目的战略是管理 T3 SS抑制剂对PA肺炎患者作为预防性药物，以增强标准治疗的功能 通过使吞噬细胞能够消除持久性和抗药性细菌来使用抗生素。这些代理人将 绕过病原体内在的抗性机制--渗透性差的外膜和多重效应-- 流量泵该建议的方法是鉴定PopB/PopD易位子的小分子抑制剂 组装在细胞膜上。在初步研究中，证明了以下内容：（a）PopB和PopD形式 模型脂质双层中的异源二聚体，（B）PopD B协助PopD插入培养的细胞膜中。 细胞，和（c）只有功能性异源寡聚体暴露N-末端的PopD的宿主胞质溶胶。因此， 随后，PopD N-末端暴露于宿主胞质溶胶将被用作精确的T3 SS的报告基因 转位子组装分裂的绿色荧光蛋白（GFP）的自我互补将用于检测 正确插入PopD。缺失16个氨基酸残基的截短的优化的超折叠体-GFP（GFP 1 -10） 链（GFP 11），将在宿主哺乳动物细胞中稳定表达。缺失的GFP 11链已添加 作为表位标记。功能性translocon的组装将暴露GFP 11 将PopD上的表位转移到宿主细胞胞质溶胶，在那里它将与GFP 1 -10互补并通过荧光检测。 我们的研究已经证明，当将GFP 11-PopD加入到一种新的载体中时， PA β popD菌株。在第一阶段，我们将开发这种荧光细胞HTS检测T3 SS反式- locon组件。将筛选化合物，并通过二级试验确认和验证命中， 反屏幕。符合分析漏斗标准的化合物将优先进行化学优化 和II期体内验证功效。具体目标是：（1）完成细胞筛选的发展，以确定- （2）优化T3 SS易位子组装筛选， HTS，将其应用于不同化合物的文库，并确认抑制剂（Yr 1-2）;（3）β-T3 SS易位子 组装抑制剂，以确定效力、选择性、初步SAR和预测的ADME性质（Yr 2）。

Abstract Pseudomonas aeruginosa (PA) is the bacterium most frequently isolated from the respiratory tract of ICU pa- tients in the US and is a major cause of pneumonia in intubated patients. Furthermore, infection with PA gener- ally has a poor prognosis, with an estimated 40-69% of PA ventilator-associated pneumonia (VAP) cases re- sulting in mortality, and more than 30% of survivors suffer recurrence following standard-of-care antimicrobial therapy. The overall goal of this project is to address this critical medical need by discovering novel small mole- cule inhibitors of the type-three secretion system (T3SS) translocon comprised of PopB & PopD in the mam- malian cell membrane and developing them into new therapeutic agents against PA. The T3SS is the major virulence factor contributing to the establishment and dissemination of PA infections, and its presence is asso- ciated with poor clinical outcomes and death in infected patients. The strategy of this project is to administer T3SS inhibitors to PA pneumonia patients as adjunctive agents to enhance the function of standard-of-care antibiotics by enabling phagocytic cells to eliminate persisters and antibiotic-resistant bacteria. Such agents will by-pass the pathogen intrinsic resistance mechanisms -- a poorly permeable outer membrane and multiple ef- flux pumps. The approach of this proposal is to identify small molecule inhibitors of the PopB/PopD translocon assembly in cell membranes. In preliminary studies, the following was demonstrated: (a) PopB and PopD form a hetero-dimer in model lipid bilayers, (b) PopB assists the insertion of PopD into the membrane of cultured cells, and (c) only functional hetero-oligomers expose the N-terminus of PopD to the host cytosol. Conse- quently, exposure of the PopD N-terminus to the host cytosol will be used as a reporter of accurate T3SS translocon assembly. Self-complementation of a split green fluorescent protein (GFP) will be used to detect properly inserted PopD. A truncated optimized superfolder-GFP (GFP1-10) missing a 16 amino acid residue β- strand (GFP11), will be stably expressed in host mammalian cells. The missing GFP11 strand has been added to the N-terminus of PopD as an epitope label. Assembly of functional translocons will expose the GFP11 epitope on PopD to the host cell cytosol where it will complement GFP1-10 and be detected by fluorescence. Our studies have already demonstrated that GFP11-PopD is active for effector translocation when added to a PA∆popD strain. In Phase I, we will develop this fluorescent cellular HTS assay for inhibitors of T3SS trans- locon assembly. Compounds will be screened and hits confirmed and validated with secondary assays and counter-screens. Compounds that meet the assay funnel criteria will be prioritized for chemical optimization and in vivo proof efficacy in Phase II. Specific Aims are: (1) Complete development of cellular screens to iden- tify inhibitors of the T3SS translocon assembly (Yr 1); (2) Optimize the T3SS translocon assembly screen for HTS, apply it to libraries of diverse compounds, and confirm inhibitors (Yr 1-2); (3) Validate T3SS translocon assembly inhibitors to determine potency, selectivity, preliminary SAR, and predicted ADME properties (Yr 2).