RNA lariat debranching enzyme as a novel drug target

描述（由申请人提供）：神经退行性疾病肌萎缩性侧索硬化症（ALS）和获得性免疫缺陷综合征（AIDS）是具有完全不同病因和症状的疾病，但它们与人RNA脱支酶Dbr 1有重要联系。Dbr 1的主要细胞作用是在前mRNA剪接后通过切割内含子IRNA分支点的2 '-5'键来帮助回收内含子RNA。有趣的是，Dbr 1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。令人惊讶的是，最近发现，由Dbr 1活性丧失引起的内含子RNA突变体的积累也加剧了由ALS相关RNA结合蛋白TDP-43的细胞质聚集引起的毒性。这些发现的结果是Dbr 1抑制剂具有作为抗ALS和抗HIV/AIDS药物的潜力。我们研究的长期目标是创新基于Dbr 1的治疗ALS和HIV感染的新治疗策略。本申请的目的是开发Dbr 1抑制剂的高通量表型筛选。我们的中心假设是，使用芽殖酵母酿酒酵母的表型筛选可以识别人类Dbr 1抑制剂。利用一种新的酵母lactinron报告菌株，研究将集中在两个特定的目标。 目的1是鉴定酵母Dbr 1抑制剂。为此目的，将进行小分子文库的试验性高通量表型筛选，测定我们的酵母报告菌株中的内含子RNA干扰信号作为Dbr 1活性的代表。命中化合物将用内含子RNA酶积累和Dbr 1酶活性的独立测定法进行验证。将进一步评估选定的命中化合物对ALS和HIV酵母模型的影响。目标2将通过检测人细胞和人Dbr 1酶上的酵母Dbr 1抑制剂来鉴定人Dbr 1抑制剂。后续研究将检查人Dbr 1抑制剂对TDP-43细胞毒性和人细胞中HIV-1复制的影响。这项工作的意义在于它将创造一种方法来识别有潜力作为ALS和HIV/AIDS全新治疗方法的先导化合物。

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.