High throughput screen of 100,000 small molecules in C9orf72 ALS neurons dataset2

Background. An intronic G4C2 repeat expansion in the C9orf72 gene is the major known cause for Amyotrophic Lateral Sclerosis. There is evidence forthree possible disease mechanisms: a pathological gain of function of nuclear repeat RNA foci, repeat associated noncanonical (RAN) translation into toxic dipeptide repeat (DPR) polyproteins as well as C9orf72 haploinsufficiency. Which of these defects need to be restored to prevent or halt disease progression remains a fundamental question for the development of ALS therapeutics. Methods. Here we developed a high content imaging assay for G4C2 RNA foci detection in C9orf72 patient iPS derived neurons miniaturised to the 1536well format. 96’200 small molecules were screened for RNA foci modulation, with counterscreens for cell toxicity and general gene expression inhibition. Hits were validated in relevant cell models from different C9orf72 donors and selected for further characterization of their cellular and molecular mode of action. Results. Among the validated hits we identified analogues of known modulators of SF3B1, a major component of the U2 snRNP spliceosomal subunit. Structure activity relationship and genome wide splice analysis confirmed that compounds to clear nuclear G4C2 RNA foci by targeting SF3B1. A combination of genetically engineered G4C2 reporter systems and RNA-protein interactome studies reveals that pharmacological SF3B1 modulation promotes recruitment of SRSF1 to nuclear G4C2 RNA, mobilizing it from RNA foci into nucleocytoplasmic export. Thisleadsto increased RAN translation and, ultimately, enhanced DPR cell toxicity. Preventing SRSF1 phosphorylation by small molecule inhibition of SR protein kinase (SRPK) conversely results in a build up of nuclear RNA foci, antagonistic to SF3B1 modulation. Conclusion. Together, our data provide a new pharmacological entry point with publically available, antagonistic tool compoundsfor modulating G4C2 repeat expansion pathology in C9orf72 ALS models. This will facilitate the dissection of C9orf72 pathobiology by uncoupling RNA foci from RAN translation. Based on our data we propose that therapeutic RNA foci elimination strategies warrant caution due to a potential storage function, counteracting translation into toxic dipeptide repeat polyproteins. Instead, our data support modulation of nuclear export via SRSF1 or SR protein kinases as possible targets for future pharmacological drug discovery

背景。C9orf72基因的内含子G4C2重复扩增是目前已知的肌萎缩侧索硬化（Amyotrophic Lateral Sclerosis，ALS）的主要致病原因。已有研究支持三种潜在的致病机制：核内重复RNA焦点（RNA foci）的病理性功能获得、重复相关非经典（RAN）翻译产生毒性二肽重复（DPR）多蛋白，以及C9orf72单倍体剂量不足。明确需修复上述哪一种缺陷以阻止或延缓疾病进展，是ALS治疗药物研发的核心科学问题。
方法。本研究建立了适配1536孔板体系的高内涵成像检测法，用于检测C9orf72患者诱导多能干细胞（iPS）衍生神经元中的G4C2 RNA焦点。我们筛选了96200种小分子化合物以调控RNA焦点的形成，并同步开展细胞毒性与广谱基因表达抑制的复筛实验以排除假阳性。命中化合物在不同C9orf72供体的相关细胞模型中得到验证，并进一步表征其细胞与分子作用模式。
结果。在验证后的命中化合物中，我们发现了剪接因子3B亚基1（SF3B1）调节剂的类似物，SF3B1是U2小核核糖核蛋白剪接亚基的核心组分。构效关系分析与全基因组剪接组分析证实，此类化合物可通过靶向SF3B1清除核内G4C2 RNA焦点。结合基因工程改造的G4C2报告基因系统与RNA-蛋白质相互作用组研究，我们发现SF3B1的药理学调控可促进SRSF1向核内G4C2 RNA的招募，将其从RNA焦点动员至核质输出通路。该过程会增强RAN翻译，最终加剧DPR的细胞毒性。反之，通过小分子抑制丝氨酸/精氨酸蛋白激酶（SRPK）以阻断SRSF1磷酸化，则会导致核内RNA焦点堆积，与SF3B1调控的效果相拮抗。
结论。综上，本研究发现了一类可公开获取的拮抗型工具化合物，可作为调控C9orf72相关性ALS模型中G4C2重复扩增病理的全新药理学干预靶点。该工具可通过将RNA焦点与RAN翻译解耦联，助力C9orf72相关病理生物学机制的解析。基于本研究结果，我们提示治疗性RNA焦点清除策略需谨慎评估，因为其可能存在潜在的储存功能，反而会抵消毒性二肽重复多蛋白的翻译过程。与之相反，本研究支持以SRSF1或SR蛋白激酶介导的核输出通路作为未来药物研发的潜在靶点。