A Unique Target for Discovery of Novel Anti-infectives

发现新型抗感染药物的独特目标

• 批准号: 6730793
• 负责人: A. WALI KARZAI
• 金额: $ 41.49万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6730793
• 关键词: DNA replication; Francisella tularensis; Yersinia pestis; Yersinia pestis disease; antibacterial agents; antibiotics; antiinfective agents; bioinformatics; bioterrorism /chemical warfare; drug discovery /isolation; drug resistance; gene deletion mutation; gene expression; gene expression profiling; laboratory mouse; microarray technology; microorganism disease chemotherapy; polymerase chain reaction; proteomics; ribosomes; tissue /cell culture; tularemia; yeast two hybrid system

The objective of this research program is to use a combination of molecular genetics, protein biochemistry, global gene expression profiling, proteomics and bioinformatics approaches to elucidate the molecular mechanism of the SmpB.SsrA ribosome rescue system and its role in survival, virulence, and pathogenesis of two category A pathogens, Yersinia pestis and Francisella tularensis. The menace of bioterrorism and the emergence of antibiotic resistance in an increasing number of pathogenic bacterial highlight the need for the development of novel classes of anti-infective agents. Existing antimicrobial drugs target known cellular mechanisms associated with protein synthesis, DNA replication, cell wall and membrane synthesis, cell division, or nutrient uptake and transport. New antibiotic development needs to focus on novel targets and will require a basic molecular understanding of their cellular function. Promisingly, bacteria possess a unique target -the SmpB.SsrA mediated ribosome rescue, protein tagging and directed degradation system- that is thought to participate in the adaptation and survival of pathogenic bacteria in the hostile micro-niches of their hosts. This unique system, also known as trans-translation, is orchestrated by a remarkable RNA (SsrA RNA) that functions both as a tRNA and an mRNA to facilitate the addition of a short degradation tag to the C-terminus of nascent polypeptides. All known activities of SsrA require SmpB, a small protein that binds SsrA specifically and with high affinity to promote its association with stalled ribosomes. The molecular basis for the formation of the SmpB.SsrA complex, its recognition of impaired ribosomes, and the subsequent addition of the degradation tag are not well understood. To assist us in these studies, we will develop an in vitro trans-translation system that should enable us to scrutinize the mechanistic details of the SmpB-SsrA mediated ribosome rescue process. This in vitro system will also be utilized as a screening assay for selection of small molecules that specifically inhibit this process. Furthermore, we wish to identify and characterize additional cellular factors that participate in this process for use as novel targets. Because the trans-translation system exists exclusively in prokaryotes and involves novel RNA and protein factors that are essential for the survival of most (if not all) pathogenic bacteria, a better understanding of this unique process should permit the discovery and/or design of highly specific novel anti-bacterial agents.

该研究计划的目标是结合分子遗传学、蛋白质生物化学、全局基因表达谱、蛋白质组学和生物信息学方法来阐明 SmpB.SsrA 核糖体拯救系统的分子机制及其在生存、毒力和发病机制中的作用两种 A 类病原体：鼠疫耶尔森菌和土拉弗朗西斯菌。生物恐怖主义的威胁和越来越多的病原细菌中抗生素耐药性的出现凸显了开发新型抗感染药物的必要性。现有的抗菌药物针对已知的与蛋白质合成、DNA 复制、细胞壁和膜合成、细胞 分裂，或养分的吸收和运输。新抗生素的开发需要关注新的靶点，并需要对其细胞功能有基本的分子了解。令人鼓舞的是，细菌拥有一个独特的靶点——SmpB.SsrA介导的核糖体拯救、蛋白质标记和定向降解系统——被认为参与病原菌在宿主敌对微环境中的适应和生存。这种独特的系统也称为反式翻译，由一种非凡的 RNA (SsrA RNA) 精心策划，该 RNA 既充当 tRNA，又充当 mRNA，以促进在 C 末端添加短降解标签 新生多肽。 SsrA 的所有已知活性都需要 SmpB，这是一种小蛋白，能够特异性地结合 SsrA，并具有高亲和力，以促进其与停滞核糖体的结合。 SmpB.SsrA 复合物形成的分子基础、其对受损核糖体的识别以及随后添加的降解标签尚不清楚。为了协助我们进行这些研究，我们将开发一种体外转译系统，使我们能够仔细检查 SmpB-SsrA 介导的核糖体救援过程的机制细节。该体外系统还将用作筛选试验，以选择特异性抑制该过程的小分子。此外，我们希望识别和表征参与该过程的其他细胞因子，以用作新靶标。由于反式翻译系统仅存在于原核生物中，并且涉及对大多数（如果不是全部）病原菌的生存至关重要的新型RNA和蛋白质因子，因此更好地理解这种独特的过程应该允许发现和/或设计高度特异性新型抗菌剂。