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

项目摘要 C9 FTD/ALS是一种快速进行性和使人衰弱的神经系统疾病， 基因C9 ORF 72中的简单串联重复序列。C9 FTD/ALS是导致糖尿病的头号遗传原因。 额颞叶痴呆（FTD）和肌萎缩侧索硬化（ALS）。没有有效的治疗方法 对于C9 FTD/ALS，也没有其他神经重复扩张疾病 在C9 FTD/ALS和大多数其他重复扩增障碍中，其中存在超过20种，重复扩增障碍在C9 FTD/ALS和大多数其他重复扩增障碍中， 扩增被转录成含有扩增的串联重复序列的RNA，或xtrRNA，其介导DNA的扩增。 疾病的分子机制。尽管导致C9 FTD/ALS的分子链事件 病理学尚不清楚，但广泛接受的是，减少或阻断xtrRNA的产生将 导致可以阻止疾病进展的有效治疗性治疗。关键在于识别 可以选择性抑制大重复扩增的转录而不影响正常的 基因表达。 一种称为Supt 4 h1（酵母中的Spt 4）的蛋白质作为核心RNA聚合酶II的持续合成因子 酶，以提高跨重复的，结构复杂的，或基因组的大区域的转录。 以前的研究表明，Supt 4 h1主要在酵母中表达，在酵母中被敲低。 C9 FTD/ALS模型生物和患者来源的细胞显著减轻疾病病理学。 Supt 4 h1通过与核心转录因子Sup 5 h的二聚化与RNA聚合酶II相互作用。 这种二聚体的晶体结构揭示了精确的分子接触。因此，我们将开发一个Supt 4 h1- Supt 5 h二聚化测定，可快速报告二聚化状态并适用于高通量 药物筛选该方法具有显色、快速等特点，可用于高通量的化学文库筛选 可访问且具有成本效益。我们将寻找抑制二聚化的小分子。在柜台- 筛选和滴定最好的抑制剂，我们将进一步表征顶级分子的能力， 阻断患者中C9 ORF 72 xtrRNA转录和疾病病理学的下游分子标志物- 衍生细胞 该项目将有可能提供先导小分子，以进一步开发为候选药物， C9FTD/ALS。该项目还可能有助于填补一些疾病治疗方法的紧迫空白 包括一整类神经系统疾病开发的Supt 4 h1-Supt 5 h二聚化测定 这将有助于筛选其他小分子或活性生物制剂的文库。

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.