Inhibitors of the twin arginine translocase system in burkholderia pseudomallei

鼻疽伯克霍尔德氏菌双精氨酸易位酶系统的抑制剂

• 批准号: 7675634
• 负责人: Michael L. Vasil
• 金额: $ 29.58万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7675634
• 关键词: Acids; Affinity; Amino Acids; Arginine; Attenuated; Biochemical; Biological Assay; Burkholderia; Burkholderia pseudomallei; Cell Fractionation; Cellular biology; Consensus; Consensus Sequence; Data; Development; Emerging Communicable Diseases; Escherichia coli O157; Eukaryotic Cell; Failure; Genes; Genomics; Growth; Infection; Laboratories; Lead; Libraries; Melioidosis; Methods; Modeling; Modification; Molecular; Molecular Weight; New Agents; Ornithine; Pasteurella pseudotuberculosis; Pattern; Peptide Signal Sequences; Peptides; Pharmaceutical Chemistry; Phospholipase; Positioning Attribute; Proteins; Proteomics; Pseudomonas aeruginosa; Research; Research Project Grants; Resistance; Resources; Screening procedure; Signal Transduction; Specificity; System; Therapeutic; Therapeutic Agents; Twin Multiple Birth; Virulence; antimicrobial; antimicrobial drug; base; biodefense; design; extracellular; follow-up; high throughput screening; inhibitor/antagonist; macrophage; mutant; novel; novel therapeutics; pathogen; product development; research study; synthetic peptide; trafficking; translocase

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).

通过双胞胎，坦率的毒力决定因素和机会性细菌病原体是分泌的 精氨酸易位酶（TAT）分泌系统仅由三个基因（即tatabc）编码。缺陷 铜绿假单胞菌的突变体，Yersinia pseudotuberculcolisos和大肠杆菌O157：O7显着 在相关的感染模型中减弱。现在，我们还显示了（请参阅本应用程序中的数据） burkholderia pseudomallei的tatabc基因在功能上代替了atatabcp。p.铜绿假单胞菌 突变体，泰国人的伯克霍尔德（Burkholderia Tat）突变体在其生长和生存中衰减 巨噬细胞。这些发现支持以下概念，即TAT是开发的目标 新型治疗剂，通过干扰tat来降低8。假单胞菌的致病潜力 功能。现在，广泛的复合库的可用性增加使得进行 高通量屏幕在更常规的基础上。因此，我们执行了这样的屏幕以识别 TAT功能的抑制剂。一种利用细胞外TAT依赖性分泌的测定 为此目的而设计的磷脂酶（PLC）。高通量筛选-83,986种化合物 鉴定出147个“命中”，显示出检测到的PLC活性量的显着降低。次要 进行测定以区分仅抑制PLC活性的化合物和那些化合物 通过TAT抑制了PLC的分泌。然后将其他表型测定用于更直接 在存在抑制剂的情况下评估TAT功能。最初的147个“命中”的后续筛选，更多 TAT功能的特定测定表明，这些引起的表型模式中有39种最反射的 那些在TAT缺失突变体中看到的。其他方法（例如，TAT的表达，细胞分馏）是 目前正在评估以确定这些化合物中的任何一种是否会被证明是有效的，并且 TAT的特异性抑制剂，以减轻假单胞菌的致病潜力。我们也在评估 6-mer合成肽池的抑制作用，可能与共有肽竞争 TAT分泌蛋白的信号序列。每种氨基酸都被其他20个取代（包括 每轮筛查）。我们已经筛选了这些合成肽的两个池， 发现抑制活性最高的四个是WF，WN，WNIE和WW的前两个位置 共识序列。我们最终将在抑制方面确定最有效的6-Mer肽 TAT功能。这些实验的小分子量化合物和肽将是 评估了它们抑制假单胞菌在真核细胞中生长，运输和存活的能力。 该研究项目符合RMRCE综合研究的重点，重点是细菌疗法， 并将直接与RP 2.1、2.6互动，并将利用核心D的资源（产品 开发和制造核心）和核心E（基因组学，蛋白质组学核心）。