Yeast Based Assays for Chemical Screens Against SARS-CoV Targets

描述（由申请人提供）：摘要人类冠状病毒是一组由四种抗原性不同的病毒组成的多样化组，它们是引起上呼吸道和下呼吸道感染的重要人类病原体。在幼儿和老年人中，下呼吸道感染可能很严重。最近出现的人类冠状病毒，SARS-CoV，导致严重的肺炎和死亡。它是一个强大的经验模型，用于识别生物功能的药理学探针，可以加强我们对疾病建立中涉及的蛋白质和途径的理解。此外，SARS-CoV是鉴定用于治疗冠状病毒引起的危及生命的疾病的广谱抗病毒药物的理想选择。在这项提案中，我们开发和验证新的检测方法，并计划在第2年由分子库生产中心网络（MLPCN）进行高通量筛选工作。基于酵母的测定将用于鉴定对SARS-CoV靶标Nsp 1和PLP具有活性的化合物。当在酵母中表达时，这两种靶点都会产生明显的表型，从而形成了抑制表型的化学筛选的基础。重要的二次筛选将揭示具有抗SARS-CoV活性的命中，以及对人类冠状病毒具有广谱活性的命中。将使用2000个成员的化合物多样性集文库手动进行中试验证筛选。冠状病毒模型是稳健的，因为存在分子克隆，已经鉴定了对病毒复制至关重要的靶基因，两种靶标的结构信息都存在，并且稳健的细胞培养和严重终末期呼吸道疾病的鼠模型允许在体外和体内对命中进行生物学评价。在目标1中，我们将优化Nsp 1和PLP蛋白在酵母中的表达和生长抑制表型，表征它们在96孔板中的行为，并进行中试筛选。在目标2中，我们将评估来自针对重组SARS-CoV和编码报告基因的其他人类冠状病毒的筛选的命中的抗病毒活性。该提案的最终目标是开发一种合理的、高通量的基于酵母的筛选，以鉴定靶向新型病毒基因的小分子抑制剂。其影响很大，因为我们的研究将开发一种新的高通量模式，以鉴定将用作冠状病毒功能分子探针的新型药物。同样重要的是，还将确定针对任何微生物病原体内编码的靶基因的候选广谱抗病毒药物。弗吉尼亚大学和查佩尔山的北卡罗来纳州大学的经验丰富的研究人员建议开发新的方法来发现化学化合物（“药物”），可用于研究严重急性呼吸道综合征（SARS）冠状病毒（称为SARS-CoV）的生物学。芽殖酵母酿酒酵母将被用作活试管，以测量特定SARS-CoV蛋白对生长的影响。然后，将使用单个化合物来确定哪些化合物可以逆转这些影响。这里的想法是，可以逆转酵母中SARS-CoV蛋白的作用的化合物也将能够逆转SARS感染期间它们在人体中的作用。我们假设，我们可以通过识别哪些化合物可以抑制SARS-CoV来了解SARS-CoV的生物学，然后研究它们是如何工作的。

DESCRIPTION (provided by applicant): Abstract Human coronaviruses are a diverse group of four antigenically distinct viruses that are important human pathogens that cause upper and lower respiratory tract infections. In young children and the elderly, lower respiratory tract infections can be severe. The recently emerged human coronavirus, SARS-CoV, causes severe pneumonia and death. It is a robust empirical model for identifying pharmacological probes of biological function that can strengthen our understanding of the proteins and pathways involved in establishment of disease. In addition, SARS-CoV is an ideal choice for the identification of broad spectrum antivirals for the treatment of coronavirus induced life threatening disease. In this proposal, we develop and validate novel assays, with plans for high throughput screening efforts during year 2 by the Molecular Libraries Production Centers Network (MLPCN). A yeast based assay will be used for the identification of compounds with activity against the SARS-CoV targets Nsp1 and PLP. Both targets give pronounced phenotypes when expressed in yeast, forming the basis for chemical screens to suppress the phenotypes. Important secondary screens will reveal hits that have anti-SARS-CoV activity, as well as those with broad spectrum activity against human coronaviruses in general. A pilot validation screen will be performed manually with the 2000 member Diversity Set library of compounds. The coronavirus model is robust because molecular clones exist, target genes have been identified that are essential for virus replication, structural information exists for both targets, and robust cell culture and murine models of severe end stage respiratory disease allow for biological evaluation of hits both in vitro and in vivo. In aim 1, we will optimize the expression and growth-inhibitory phenotypes of the Nsp1 and PLP proteins in yeast, characterize their behavior in 96-well plates, and perform the pilot screen. In aim 2, we will evaluate the antiviral activity of hits from the screen against recombinant SARS-CoV and other human coronaviruses encoding reporter genes. The ultimate goal of this proposal is to develop a rational, high throughput yeast-based screen that identifies small molecule inhibitors that target novel viral genes. The impact is high because our studies will develop a new high throughput paradigm to identify novel pharmaceuticals that will be used as molecular probes of coronavirus function. Equally important is that candidate broad spectrum antivirals against the target genes encoded within any microbial pathogen will also be identified. Project Narrative Experienced investigators at the University of Virginia and the University of North Carolina at Chapel Hill propose to develop new ways to discover chemical compounds ("drugs") that can be used to study the biology of the Severe Acute Respiratory Syndrome (SARS) coronavirus, called SARS-CoV. The budding yeast Saccharomyces cerevisiae will be used as a living test tube to measure the effects of specific SARS-CoV proteins on growth. Then, individual chemical compounds will be used to determine which compounds can reverse these effects. The idea here is that compounds that can reverse the effects of SARS-CoV proteins in yeast will also be able to reverse their effects in the human body during a SARS infection. We hypothesize that we can learn about the biology of SARS-CoV by identifying which chemical compounds can inhibit it, and then study how they work.