Discovery of Small Molecules that Block Supt4h1-Supt5h Dimerization for Potential C9FTD/ALS Therapeutics

项目总结 C9FTD/ALS是一种快速进展和衰弱的神经系统疾病，由 基因C9ORF72中的简单串联重复序列。C9FTD/ALS是遗传致病的头号原因 额颞性痴呆(FTD)和肌萎缩侧索硬化症(ALS)。目前还没有有效的治疗方法 对于c9FTD/ALS，以及其他神经重复扩张性疾病 在c9FTD/ALS和大多数其他重复扩张性疾病中，重复 扩展被转录为扩展的串联重复序列RNA，或xtrRNA，它介导 疾病的分子机制。尽管导致C9FTD/ALS的分子链 病理仍不清楚，人们普遍认为减少或阻止xtrRNA的产生将 导致有效的治疗方法，可以阻止疾病的进展。关键在于找出 选择性抑制大重复扩增转录而不影响正常的治疗 基因表达。 一种名为Supt4h1的蛋白质(酵母中的Spt4)作为核心RNA聚合酶II的加工因子 在重复的、结构复杂的或大范围的基因组区域中提高转录的酶。 先前的研究表明，Supt4h1在酵母中基本上是不必要的，它在酵母中的敲除 C9FTD/ALS模型生物和患者来源的细胞显著减轻疾病病理。 Supt4h1通过与核心转录因子Sup5h的二聚化作用与RNA聚合酶II相互作用。 这种二聚体的晶体结构显示了精确的分子接触。因此，我们将开发一款Supt4h1- Supt5h二聚检测，可快速报告二聚状态，并适用于高通量 药物筛查。该方法比色快速，可进行高通量的化学文库筛选 交通便利，性价比高。我们将寻找抑制二聚化的小分子。在柜台后- 筛选和滴定最好的抑制剂，我们将进一步表征顶端分子的能力 阻断C9ORF72 xtrRNA转录和下游疾病病理分子标志物- 派生的细胞。 该项目将潜在地提供铅小分子作为候选药物的进一步开发 C9FTD/ALS。该项目还可能有助于填补一些疾病的治疗方面的紧迫空白。 包括一整类神经性疾病。建立了Supt4h1-Supt5h二聚化实验 这将有助于筛选其他小分子或活性生物制品的文库。

PROJECT SUMMARY C9FTD/ALS is a rapidly progressive and debilitating neurological disease caused by expansion of a simple tandem repeat sequence in the gene C9ORF72. C9FTD/ALS is the number one inherited cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). There are no effective treatments for C9FTD/ALS, nor the other neurological repeat expansion disorders In C9FTD/ALS and most other repeat expansion disorders, of which over two dozen exist, the repeat expansion is transcribed into expanded tandem repeat-containing RNA, or xtrRNA, which mediates the molecular mechanisms of disease. Although the molecular chain of events that lead to C9FTD/ALS pathology are still unclear, it is widely accepted that reducing or blocking production of the xtrRNA will lead to effective therapeutic treatments that can halt disease progression. The key will lie in identifying treatments that can selectively inhibit transcription of large repeat expansions without affecting normal gene expression. A protein called Supt4h1 (Spt4 in yeast) acts as a processivity factor to the core RNA polymerase II enzyme to improve transcription across repetitive, structurally complex, or large regions of the genome. Previous studies have demonstrated that Supt4h1 is largely dispensable in yeast and its knock-down in C9FTD/ALS model organisms and patient-derived cells significantly mitigates disease pathology. Supt4h1 interacts with RNA polymerase II through dimerization with Sup5h, a core transcription factor. A crystal structure of this dimer reveals precise molecular contacts. Thus, we will develop an Supt4h1- Supt5h dimerization assay that can rapidly report dimerization status and is amenable to high throughput drug screening. The assay is colorimetric and quick, making high throughput chemical library screening accessible and cost-effective. We will search for small molecules that inhibit dimerization. After counter- screening and titration of the best inhibitors, we will further characterize the ability of top molecules to block C9ORF72 xtrRNA transcription and downstream molecular markers of disease pathology in patient- derived cells. This project will potentially deliver lead small molecules for further development as drug candidates for C9FTD/ALS. This project may also help fill an urgent gap in therapeutics for a number of diseases comprising an entire class of neurological disorders. The Supt4h1-Supt5h dimerization assay developed here will be useful for screening other libraries of small molecules or active biologics.