RNA lariat debranching enzyme as a novel drug target

描述(申请人提供)：神经退行性疾病肌萎缩侧索硬化症(ALS)和获得性免疫缺陷综合征(AIDS)是具有完全不同的病因和症状的疾病，但它们与人类RNA去分支酶DBR1有着重要的联系。DBR1的主要细胞作用是通过断裂内含子套索RNA分支点的2‘-5’键，帮助内含子RNA在前mRNA剪接后循环。有趣的是，DBR1活性的丧失抑制了人类免疫缺陷病毒(HIV)的复制，HIV是艾滋病的逆转录病毒制剂。令人惊讶的是，最近发现，由于DBR1活性丧失而导致的内含子RNA网的积累，也减轻了ALS相关RNA结合蛋白TDP-43的细胞质聚集所引起的毒性。这些发现的结果是，DBR1抑制剂具有作为抗ALS和抗艾滋病毒/艾滋病药物的潜力。我们研究的长期目标是创新基于DBR1的治疗策略来治疗ALS和HIV感染。这项应用的目标是开发一种高通量的DBR1抑制剂的表型筛选。我们的中心假设是，使用发芽酵母酿酒酵母的表型筛选可以识别人类DBR1抑制剂。利用一种新的酵母套索内含子报告菌株，研究将集中在两个特定的目标上。 目的1是鉴定酵母菌DBR1抑制剂。为此，将进行小分子文库的中试高通量表型筛选，分析我们酵母报告菌株中的内含子RNA套索信号，作为DBR1活性的替代。HIT化合物将通过独立的内含子RNA套索积累和DBR1酶活性的检测来验证。精选的HIT化合物将进一步评估它们对ALS和HIV酵母模型的影响。AIM2将通过测试酵母DBR1抑制剂在人体细胞和人DBR1酶上的作用来识别人DBR1抑制剂。后续研究将检查人类DBR1抑制剂对TDP-43细胞毒性和人类细胞中HIV-1复制的影响。这项工作的意义在于，它将创造一种手段，以确定有可能成为治疗肌萎缩侧索硬化症和艾滋病毒/艾滋病的全新疗法的先导化合物。

DESCRIPTION (provided by applicant): The neurodegenerative disease amyotrophic lateral sclerosis (ALS) and acquired immunodeficiency syndrome (AIDS) are diseases with quite different etiologies and symptoms yet they share an important connection to the human RNA debranching enzyme Dbr1. The main cellular role of Dbr1 is to help recycle intron RNAs following pre-mRNA splicing by cleaving the 2'-5' bond at intron lariat RNA branch points. Interestingly, loss of Dbr1 activity inhibits replication of Human Immunodeficiency Virus (HIV), the retroviral agent of AIDS. Surprisingly, it was recently discovered that accumulation of intron RNA lariats resulting from loss of Dbr1 activity also alleviates toxicity caused by cytoplasmic aggregation of the ALS associated RNA binding protein TDP-43. A consequence of these findings is that Dbr1 inhibitors have potential as anti-ALS and anti-HIV/AIDS drugs. The long-term goal of our research is to innovate novel Dbr1-based therapeutic strategies for treating ALS and HIV infection. The objective of this application is to develop a high-throughput phenotypic screen for Dbr1 inhibitors. Our central hypothesis is that a phenotypic screen using the budding yeast Saccharomyces cerevisiae can identify human Dbr1 inhibitors. Taking advantage of a novel yeast lariat intron reporter strain, research will focus on two specific aims. Aim 1 is to identify yeast Dbr1 inhibitors. For this aim a pilot high-throughput phenotypic screen of small molecule libraries will be performed, assaying for the intron RNA lariat signal in our yeast reporter strain as a proxy for Dbr1 activity. Hit compounds will be validated with independent assays for intron RNA lariat accumulation and Dbr1 enzyme activity. Select hit compounds will be further assessed for their effects on yeast models for ALS and HIV. Aim2 will be to identify human Dbr1 inhibitors by testing yeast Dbr1 inhibitors on human cells and the human Dbr1 enzyme. Follow up studies will examine the effects of human Dbr1 inhibitors on TDP-43 cytotoxicity and HIV-1 replication in human cells. The significance of the work is it will create the means to identify lead compounds that have potential as completely novel treatments for ALS and HIV/AIDS.