Inhibitors of the twin arginine translocase system in burkholderia pseudomallei

坦率的和机会性细菌病原体的关键毒力决定因素是通过孪生分泌的 精氨酸移位酶（达特）分泌系统仅由三个基因（即tatABC）编码。TAT缺陷 铜绿假单胞菌、假结核耶尔森菌和大肠杆菌的突变株。大肠杆菌O 157：O 7对大肠杆菌O 157：O 7 在相关感染模型中减弱。我们现在还表明（见本申请中的数据）， 类鼻疽伯克霍尔德氏菌的tatABC基因在功能上替代AtatABC铜绿假单胞菌的那些基因 突变体，且泰国伯克霍尔德氏菌达特突变体在它们生长和存活中被减弱， 巨噬细胞这些发现支持达特是开发具有吸引力的目标的概念。 新的治疗剂，以减少8.干扰达特治疗类鼻疽 功能广泛的化合物库的可用性增加，现在可以进行 高通量筛选在更常规的基础上。因此，我们进行了这样的筛选，以确定 达特功能抑制剂。一种利用细胞外的TAT依赖性分泌的测定， 磷脂酶（PLC）是为此目的而设计的。约83，986种化合物的高通量筛选 鉴定了147个“命中”，这表明检测到的PLC活性的量显著降低。二次 进行测定以区分仅抑制PLC活性的那些化合物和仅抑制PLC活性的那些化合物。 通过达特抑制PLC的分泌。然后采用额外的表型测定来更直接地 在抑制剂存在的情况下评估达特功能。对最初的147个“命中”进行后续筛查， 达特功能的特异性测定显示，这些诱导的表型模式中有39种最能反映 那些在达特缺失突变体中看到的。其他方法（例如达特的过表达、细胞分级分离）是 目前正在进行评估，以确定这些化合物中是否有任何一种被证明是有效的， 达特的特异性抑制剂以减轻B的致病潜力。假鼻疽我们也在评估 6-mer合成肽库的抑制作用，所述6-mer合成肽库可以与在所述细胞中的共有肽竞争。 达特分泌蛋白信号序列。每个氨基酸被20个其他氨基酸取代（包括 鸟氨酸）。我们已经筛选了这些合成肽的两个库， 发现具有最高抑制活性的四个是WF，WN，WNIe和WW，位于前两个位置。 共有序列我们将最终确定最有效的6聚体肽在抑制 达特函数。来自这些实验的小分子量化合物和肽将被 评价它们抑制B生长、运输和存活的能力。真核细胞中的类鼻疽。 该研究项目符合RMRCE细菌治疗综合研究重点， 并将直接与RP 2.1、2.6交互，并将利用核心D（产品）的资源 开发和制造核心）和核心E（基因组学，蛋白质组学核心）。

Key virulence determinants of frank and opportunistic bacterial pathogens are secreted via the Twin Arginine Translocase (TAT) secretion system encoded by only three genes (i.e. tatABC). TAT-deficient mutants of Pseudomonas aeruginosa, Yersinia pseudotuberculosis and E. coli O157:O7 are significantly attenuated in relevant models of infection. We have now also shown (see data in this application) that the tatABC genes of Burkholderia pseudomallei functionally substitute for those of a AtatABC P. aeruginosa mutant, and that Burkholderia thailandensis TAT mutants are attenuated in their growth and survival in macrophage. These findings support the concept that TAT is an appealing target for the development of novel therapeutic agents, to diminish the pathogenic potential of 8. pseudomallei by interfering with TAT function. The increased availability of extensive compound libraries now makes it possible to conduct highthroughput screens on a more routine basis. Accordingly, we performed such a screen to identify inhibitors of TAT function. An assay, which exploits the TAT-dependent secretion of extracellular phospholipases (PLC), was designed for this purpose. High-throughput screening of -83,986 compounds identified 147 "hits", which showed a significant reduction in the amount of PLC activity detected. Secondary assays were performed to distinguish between those compounds that only inhibited PLC activity and those that inhibited secretion of PLC via TAT. Additional phenotypic assays were then employed to more directly assess TAT function in the presence of the inhibitor. Follow-up screening of the initial 147 "hits" with more specific assays of TAT function revealed that 39 of these induced phenotypic patterns most reflective of those seen in a TAT deletion mutant. Other approaches (e.g. over expression of TAT, cell fractionation) are currently being evaluated to determine whether any of these compounds will prove to be efficient and specific inhibitors of TAT to mitigate the pathogenic potential of B. pseudomallei. We are also evaluating the inhibitory effects of pools of 6-mer synthetic peptides that may compete with a consensus peptide in the signal sequence of TAT secreted proteins. Each amino acid is being substituted with 20 others (including ornithine) for each round of screening. We have already screened two pools of these synthetic peptides and found that the four with the highest inhibitory activity are WF, WN, WNIe, and WW in the first two positions of the consensus sequence. We will ultimately identify the most potent 6-mer peptides in terms of inhibiting TAT function. The small molecular weight compounds and the peptides from these experiments will be evaluated for their ability to inhibit the growth, trafficking and survival of B. pseudomallei in eukaryotic cells. This research project fits within the RMRCE Integrated Research Focus on Bacterial Therapeutics, and will interact directly with RP 2.1, 2.6 and will utilize the resources of the Core D (Product Development and Manufacturing Core) and Core E (Genomics, Proteomics Core).