Fluorescent IRE sensor for PD drug discovery

摘要 帕金森氏病(PD)是一种衰弱的神经退行性疾病，影响约 60岁以上人口的1%-2%。帕金森病的病理特征是 细胞内蛋白聚合体的存在会导致退化和死亡 黑质中的多巴胺能神经元。突触前蛋白α-突触核蛋白是 被发现是路易小体的主要成分，α-突触核蛋白聚集被认为 是帕金森病发病机制中的关键事件。过去的药物发现工作主要集中在 关于防止α-突触核蛋白聚集或增加α-突触核蛋白清除。然而， 在细胞系和动物模型中的过度表达和敲除研究表明 改变细胞内α-突触核蛋白水平也可以调节PD的病理。这些 结果表明，α-突触核蛋白翻译是一个重要的，但尚未探索的目标 警局的药物发现。一种推测的铁反应元件(IRE)，显示出高序列 在远端5‘-非翻译区发现了与铁蛋白IRE相似的结构 和研究表明，这种IRE在调节α-突触核蛋白的表达中起着关键作用 α-突触核蛋白翻译。到目前为止，还没有高通量的药物发现方法 可以直接询问药物与控制蛋白质翻译的RNA结构的结合。 这是为了绕过通常用于研究的容易出现伪影的蛋白质报告 翻译抑制药。因此，这一阶段的SBIR应用旨在开发一个 监测药物与IRES结合的荧光传感器，可实现高通量 药物筛选针对α-突触核蛋白IRE的化合物并最终结果 在最终可能发展为α-突触核蛋白抑制剂的先导化合物中 综合。

SUMMARY Parkinson's disease (PD) is a debilitating neurodegenerative disease that affects about 1-2% of the population over the age of 60. The pathological hallmark of PD is the presence of intracellular protein aggregates that cause the degeneration and then death of dopaminergic neurons in the substantia nigra. The presynaptic protein α-synuclein was found to be the main component of Lewy bodies and α-synuclein aggregation is believed to be the key event in PD pathogenesis. Past drug discovery efforts have been focused on preventing α-synuclein aggregation or increasing α-synuclein clearance. However, overexpression and knockdown studies in cell lines and animal models showed that altering intracellular α-synuclein protein levels can also regulate PD pathology. These results suggest that α-synuclein translation is a significant yet under explored target for PD drug discovery. A putative iron-responsive element (IRE) that exhibited high sequence and structural similarities with the ferritin IRE was found in the distal 5'-untranslated region of α-synuclein mRNA and studies suggest that this IRE plays a critical role in regulating α-synuclein translation. Thus far, there is no high-throughput drug discovery methods that can interrogate drug binding directly to RNA structures that control protein translation. This is needed to bypass artifact-prone protein reporters that are typically used to study translation inhibitors. Therefore, this phase I SBIR application aims to develop a fluorescence sensor that monitors drug binding to IREs and can enable high-throughput drug screens for compounds that specifically target α-synuclein's IRE and ultimately result in lead compounds that could eventually be developed into inhibitor of α-synuclein protein synthesis.