Structure and Inhibition of the Conjugative DNA Relaxase-Helicase

项目摘要 每年死于耐抗生素细菌感染的美国人比死于结肠癌、前列腺癌和卵巢癌的美国人还多 癌症综合接合DNA转移产生大多数抗生素抗性细菌菌株， 感染人类我们最近已经证明，这种DNA转移过程所必需的松弛酶可以 使用各种小分子，包括一些骨质疏松症药物，以纳摩尔的功效抑制。 松弛酶抑制剂阻止DNA转移并选择性地杀死抗生素耐药细菌。的松弛酶 接合F质粒是大的多功能TraI蛋白的一部分，也含有高效解旋酶 和推定的蛋白质结合C末端结构域。TraI的松弛酶、解旋酶和C末端区域都是 是接合DNA转移所必需的。该提案的重点是扩展我们的初步结构和 化学生物学研究的目的是了解DNA转移的分子基础和发展 能够杀死耐抗生素细菌的小分子。该项目将实现四个具体目标： 1.阐明一系列松弛酶抑制剂复合物的晶体结构。 2.发现和合成新的松弛酶抑制剂，并测试它们对接合和细菌存活的影响。 3.揭示TraI C端结构域在接合转移中的作用。 4.检查TraI接合解旋酶区域的结构、功能和抑制。 这些研究的结果将提供详细的机制见解之一的第一个DNA 操纵系统被发现此外，由于保守的松弛酶存在于一系列的细胞中， 病原微生物，我们的研究结果可能提供一种新的方法，以针对最危险的感染性细菌  那些具有抗生素抗性并能够将其抗性传播到邻近细胞的细胞。项目叙述 接合DNA转移，抗生素耐药基因传播的主要途径 细菌种群的DNA松弛酶和解旋酶启动和驱动。我们最近的研究表明 接合松弛酶可以以纳摩尔效力被抑制，并且这种抑制防止DNA 结合并选择性地杀死抗生素抗性细菌。该项目将扩大我们的初步结构 和化学生物学发现，目的是了解DNA转移的分子基础， 开发出能有效杀死耐抗生素细菌的药物。

PROJECT SUMMARY Antibiotic resistant bacterial infections kill more Americans each year than colon, prostate and ovarian cancer combined. Conjugative DNA transfer generates most of the antibiotic resistant strains of bacteria that infect humans. We have recently shown that the relaxase enzyme essential to this DNA transfer process can be inhibited with nanomolar efficacy using a variety of small molecules, including some osteoporosis drugs. Relaxase inhibition prevents DNA transfer and selectively kills antibiotic resistant bacteria. The relaxase of the conjugative F plasmid is part of the large multifunctional TraI protein that also contains highly efficient helicase and putative protein-binding C-terminal domains. The relaxase, helicase and C-terminal regions of TraI are all essential for conjugative DNA transfer. This proposal focuses on extending our preliminary structural and chemical biology studies with the goals of understanding the molecular basis of DNA transfer and developing small molecules capable of killing antibiotic resistant bacteria. This project will accomplish four specific aims: 1. Elucidate crystal structures of a range of relaxase-inhibitor complexes. 2. Discover and synthesize new relaxase inhibitors and test their impact on conjugation and bacterial survival. 3. Unravel the role the TraI C-terminal domain plays in conjugative transfer. 4. Examine the structure, function and inhibition of the TraI conjugative helicase region. Results from these studies will provide detailed mechanistic insights into one of the first DNA manipulation systems discovered. In addition, because conserved relaxases are present in a range of pathogenic microbes, our results may provide a novel method to target the most dangerous infectious bacteria  those that are antibiotic resistant and are capable of spreading their resistance to neighboring cells. PROJECT NARRATIVE Conjugative DNA transfer, the primary route by which antibiotic resistance genes spread through bacterial populations, is initiated and driven by DNA relaxase and helicase enzymes. We have recently shown that conjugative relaxases can be inhibited with nanomolar efficacy, and that this inhibition prevents DNA conjugation and selectively kills antibiotic resistant bacteria. This project will extend our preliminary structural and chemical biology discoveries with the goal of understanding the molecular basis of DNA transfer and developing drugs that potently kill antibiotic resistant bacteria.