Structure and Inhibition of the Conjugative DNA Relaxase-Helicase

描述(申请人提供)：耐药细菌感染每年导致美国人死亡的人数超过结肠癌、前列腺癌和卵巢癌的总和。接合DNA转移产生了感染人类的大多数抗药性细菌株。我们最近已经证明，这种DNA转移过程中必不可少的松弛酶可以通过使用各种小分子药物，包括一些骨质疏松药物，以纳摩尔的效果被抑制。松弛酶抑制可防止DNA转移，并选择性地杀死抗药性细菌。接合的F质粒的松弛酶是大的多功能Trai蛋白的一部分，该蛋白还含有高效的解旋酶和可能的蛋白结合C末端结构域。TRAI的松弛酶、解旋酶和C末端都是DNA连接转移所必需的。这项应用的重点是扩大我们的初步结构和化学生物学研究，目标是了解DNA转移的分子基础，并开发能够杀死抗生素耐药性细菌的小分子。本项目将完成四个具体目标：1.阐明一系列松弛酶抑制剂复合体的晶体结构。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-末端结构域在结合转移中的作用。4.研究TRAI结合解旋酶区的结构、功能及其抑制作用。这些研究的结果将为最早发现的DNA操纵系统之一提供详细的机械学见解。此外，由于保守的松弛酶存在于一系列致病微生物中，我们的结果可能提供一种新的方法来针对最危险的感染细菌-那些对抗生素具有耐药性并能够将其耐药性传播到邻近细胞的细菌。公共卫生相关性：结合DNA转移是抗生素耐药性基因在细菌种群中传播的主要途径，由DNA松弛酶和解旋酶启动和驱动。我们最近已经证明，结合松弛酶可以被纳摩尔效应抑制，这种抑制阻止了DNA结合，并选择性地杀死了耐抗生素细菌。该项目将扩大我们的初步结构和化学生物学发现，目标是了解DNA转移的分子基础，并开发有效杀死抗生素耐药性细菌的药物。

DESCRIPTION (provided by applicant): 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 application 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. PUBLIC HEALTH RELEVANCE: 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.