Genome wide assessment of essential genes in Trypanosoma brucei

描述（由申请人提供）：布鲁氏锥虫是世卫组织一类病原体（新出现且不受控制），引起非洲人类锥虫病（HAT，又名非洲昏睡病），除非治疗，否则总是致命的。由于目前的治疗受到毒性、给药困难和耐药性扩散的限制，迫切需要新的药物。在过去的20-30年里，通过各种假设驱动的研究项目，对布鲁氏体的分子和细胞生物学积累了丰富的知识。虽然从这些研究中衍生出了一些经过验证的药物靶点，但目前还没有系统的综合努力来利用基因组计划的成果和布鲁氏菌遗传操作的最新进展来具体识别和评估药物靶点的潜力。此外，在建立药物发现项目方面投入了大量资源，包括在世界各地的几个学术中心建立高通量筛选（HTS）设施。然而，尽管做出了这些努力，但经过验证的既重要又可能“可药物化”的药物靶点清单很少，因此没有足够的高价值靶点来证明HTS或专门的药物发现工作的巨大努力和资源是合理的。该提案是我们总部设在美国的团队（Stuart and Phillips）和我们在英国的合作者(dr。M. Fergusson和J. Mottram)，在一项全基因组调查中进行RNAi（干扰RNA）分析，以寻找布鲁氏杆菌的基本靶点和“可药物”靶点。世卫组织/TDR正在编制一个全基因组数据库，其中列出了药物发现过程中评估靶点所需的关键数据，并将成为选择靶点的关键资源。虽然本研究的重点是HAT，但布鲁氏T.的基因验证靶点可能转化为克氏T.和利什曼原虫，因此这些研究将为整个着丝体群落提供资源。项目产生的所有数据将提供给tdrtargets.org的管理员，以便在该网站上公开分发。这项为期三年的研究的目标是：1)在评估潜在“药物性”的基础上，从布鲁氏体基因组中选择200 - 300个基因进行RNAi必要性分析；2)通过RNAi分析确定所选基因是否为血型布氏体寄生虫体外和体内生长所必需的基因；3)开发高产的异源蛋白表达系统，并对必要的基因进行合适的酶分析；4)整理资料和试剂，向布鲁氏体研究界提供分发。

DESCRIPTION (provided by applicant): Trypanosoma brucei, a WHO category one pathogen (emerging and uncontrolled), causes human African trypanosomiasis (HAT, aka African Sleeping Sickness), which is invariably fatal unless treated. New drugs are badly needed since current therapy is limited by toxicity, difficulty of administration, and spreading drug resistance. A wealth of knowledge has accumulated over the last 20-30 years on the molecular and cellular biology of T. brucei through a variety of hypothesis driven research programs. While several validated drug targets have arisen as a spin-off of these studies, there has been no systematic comprehensive effort to exploit the output of the genome projects and the recent advances in genetic manipulation of T. brucei to specifically identify and assess potential of drug targets. In addition, significant resources have been invested in setting up drug discovery programs including the building of facilities for high throughput screening (HTS) in several academic centers around the world. However, despite these efforts the list of validated drug targets that are both essential and likely to be "druggable" is small, and thus there are not enough high value targets that justify the considerable effort and resource of a HTS, or a dedicated drug discovery effort. This proposal is part of a coordinated effort by our US-based group (Stuart and Phillips) and our collaborators in the UK (Drs. M. Fergusson and J. Mottram), to undertake RNAi (interfering RNA) analysis in a genome-wide survey for essential, and "druggable" targets in T. brucei. A genome-wide data-base that lists key data required for target assessment in drug discovery is being compiled by the WHO/TDR and will be a key resource for target selection. While this proposal focuses on HAT, genetically validated targets in T. brucei may translate to T. cruzi and Leishmania species, and thus these studies will provide a resource for the entire kinetoplastid community. All data generated from the project will be provided to the tdrtargets.org curators for public distribution on that web site. The goals of this three year study are to 1) Select 200 - 300 genes in the T. brucei genome for RNAi essentiality analysis based on an assessment of potential "druggability"; 2) to determine if the selected genes are essential for the growth of blood form T. brucei parasites in vitro and in vivo by RNAi analysis; 3) to develop high-yield heterologous protein expression systems and suitable enzyme assays for genes that are shown to be essential; and 4) to organize data and reagents to provide distribution to the T. brucei research community. PUBLIC HEALTH RELEVANCE: The goal of the National Institute of Allergy and Infectious Diseases (NIAID) is to support basic and applied research leading to a better understanding of infectious, immunologic, and allergic diseases with the goal of generating new treatments or prevention programs for these diseases. The direct goal of this proposal is to identify new drug targets for the development of novel treatment programs against several priority trypanosomal pathogens that have been of long standing interest in the NIAID portfolio.