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

摘要 铜绿假单胞菌(PA)是重症监护病房(ICU)患者呼吸道分离最多的细菌。 在美国，它是导致气管插管患者肺炎的主要原因。此外，感染PA基因- ALI的预后很差，估计有40%-69%的PA呼吸机相关肺炎(VAP)病例会复发。 死亡率下降，超过30%的幸存者在标准护理抗菌药物治疗后复发 心理治疗。该项目的总体目标是通过发现新的小鼹鼠来满足这一关键的医疗需求。 由POPB和PopD组成的三型分泌系统(T3SS)易位子的CULE抑制剂在主要... 马里细胞膜，并将其开发为治疗PA的新药物。T3SS是主要的 毒力因素有助于PA感染的建立和传播，其存在也是如此- 与感染患者的临床转归差和死亡有关。这个项目的战略是管理 T3SS抑制剂对肺炎支原体肺炎患者强化护理标准功能的辅助作用 通过使吞噬细胞清除持久菌和抗药性细菌来产生抗生素。这样的代理人将 绕过病原菌的内在抗性机制--渗透性差的外膜和多重作用 流量泵。这项建议的方法是确定POPB/PopD转位的小分子抑制剂 在细胞膜上组装。在初步研究中，论证如下：(A)POPB和POPD形式 模型脂质双层中的杂二聚体，(B)POPB辅助PopD插入培养的细胞膜 细胞，以及(C)只有功能性的杂寡体使PopD的N端暴露在宿主胞浆中。简而言之- 通常，PopD N-末端暴露在宿主胞浆中将被用作准确的T3SS的报告 转位基因组装。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。截短的优化超文件夹-gfp(gfp 1-10)缺少16个氨基酸残基β- 链(GFP11)，将在宿主哺乳动物细胞中稳定表达。缺失的GFP 11链已被添加 到PopD的N-末端作为表位标记。功能性转座子的组装将暴露GFP 11 PopD上的表位结合到宿主细胞胞浆中，在那里它将补充GFP1-10，并通过荧光检测。 我们的研究已经证明，当GFP11-PopD加入到一个 PA∆PopD菌株。在第一阶段，我们将建立这种荧光细胞HTS方法来检测T3SS反式- Locon组件。化合物将通过二次分析进行筛选、确认和确认 反屏幕。符合分析漏斗标准的化合物将优先进行化学优化 并在第二阶段证明体内疗效。具体目标是：(1)完成细胞筛选的开发，以识别- T3SS转运子组装的抑制剂(YR 1)；(2)优化T3SS转运子组装筛选 HTS，将其应用于不同化合物的文库，并确认抑制剂(YR 1-2)；(3)验证T3SS转位 组装抑制剂以确定效力、选择性、初步的SAR和预测的ADME特性(年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).