Inhibitors of the twin arginine translocase system in burkholderia pseudomallei

坦率和机会性细菌病原体的关键毒力决定因素是通过双胞胎分泌的 精氨酸转位酶(TAT)分泌系统只由三个基因编码(即talABC)。缺乏TAT 铜绿假单胞菌、假结核耶尔森菌和大肠杆菌O157：O7的突变株显著 在相关感染模型中减弱。我们现在还显示(请参阅此应用程序中的数据) 铜绿假单胞菌atatABC基因的功能替代 突变体，以及泰兰伯克霍尔德氏菌Tat突变体的生长和存活在 巨噬细胞。这些发现支持这样的概念，即TAT是开发的一个有吸引力的目标 新的治疗药物，通过干扰TAT来降低8.假鼻疽的致病潜能 功能。广泛的复合库的可用性增加现在使进行 在更常规的基础上进行高通量筛选。因此，我们进行了这样一次筛选，以识别 TAT功能的抑制剂。一种利用依赖于TAT的细胞外分泌物的分析方法 磷脂酶(PLC)就是为此而设计的。-83,986个化合物的高通量筛选 确定了147个“命中”，这表明检测到的PLC活动量显著减少。次要的 进行检测以区分那些仅抑制PLC活性的化合物和那些 抑制PLC通过TAT的分泌。然后使用额外的表型分析来更直接地 在抑制剂存在的情况下评估TAT的功能。对最初的147个“点击”进行后续筛选，并提供更多内容 对TAT功能的具体分析表明，这些诱导的表型模式中有39种最能反映 那些出现在TAT缺失突变体中的基因。其他方法(如过表达TAT、细胞分级)有 目前正在进行评估，以确定是否有任何这些化合物将被证明是有效的和 TAT的特异性抑制剂以减轻假鼻疽杆菌的致病潜能。我们还在评估 6-聚体合成多肽池可能与一个共识多肽竞争的抑制作用 TAT分泌蛋白的信号序列。每种氨基酸都被其他20种氨基酸取代(包括 鸟氨酸)用于每一轮筛查。我们已经筛选了两个合成多肽和 发现具有最高抑制活性的四个是WF、WN、WNIE和WW 共识序列。我们将最终确定抑制作用最强的6聚体多肽 TAT功能。来自这些实验的小分子化合物和多肽将是 评估它们在真核细胞中抑制假鼻疽杆菌的生长、运输和存活的能力。 这项研究项目符合RMRCE关于细菌治疗的综合研究重点， 并将直接与RP 2.1、2.6交互，并将利用核心D(产品 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).