Fluorescent IRE sensor for synucleinopathy drug discovery

摘要 这一第二阶段提案的目标是通过验证一种高度有效的方法来推进突触核病药物的发现 吞吐量筛选(HTS)检测和建立RNA靶向药物的RNA结构传感器平台 发现号。路易体痴呆是老年人退行性痴呆的第二大常见形式 阿尔茨海默病后的人群；其特点是α-突触核蛋白(SNCA)的异常积聚 集合体。以致病的SNCA蛋白为特征的疾病统称为突触核病，它 还包括帕金森氏症、多系统萎缩和阿尔茨海默病伴杏仁核受限 刘易体。目前还没有针对任何一种共核病的疾病修正疗法。这是众所周知的 SNCA基因复制增加SNCA水平，并与疾病进展和严重程度相关， 导致早期帕金森症和痴呆症。研究表明，降低SNCA水平可以减少聚集， 防止路易体的形成，并赋予神经保护。因此，在疾病期间抑制SNCA的表达 前驱期有可能减缓疾病的进展或阻止疾病的发生。SNCA翻译为 受该基因5‘端非编码区的铁反应元件(IRE)控制。为了证明可行性，我们 开发了概念验证RNA结构传感器，对小分子的结合和 反义寡核苷酸，并论证了高温超导应用的可行性。为了实现这项提案的目标， 我们将完成以下具体目标：1)完成SNCA特定RNA传感器的HTS优化，并 进行中试筛选，2)通过开发HTS-2)建立RNA结构传感器平台的通用性 以另一种致病RNA结构为目标的兼容传感器，3)开发标准操作程序 定制RNA传感器服务商业化，4)进行初步筛选以鉴定SNCA的抑制剂 蛋白质翻译。如果成功，我们将有一个经过验证的HTS检测方法来发现突触核病药物，并 旨在加快当前基于RNA结构的药物发现步伐的RNA结构传感器平台 并使更多针对RNA的药物开发计划针对致病的RNA结构。

ABSTRACT The goal of this Phase II proposal is to advance synucleinopathy disease drug discovery by validating a high- throughput screening (HTS)-ready assay and establish a RNA structure sensor platform for RNA-targeted drug discovery. Dementia with Lewy bodies is the second most common form of degenerative dementia in the elderly population after Alzheimer’s disease; and it is characterized by abnormal accumulation of alpha-synuclein (SNCA) aggregates. Diseases featuring pathogenic SNCA proteins are collectively known as synucleinopathies, which also include Parkinson’s disease, multiple system atrophy, and Alzheimer’s Disease with Amygdala restricted Lewy bodies. There is currently no disease-modifying cure available for any of the synucleinopathies. It is known that SNCA gene duplication increases SNCA levels and is correlated with disease progression and severity, leading to early parkinsonism and dementia. Studies showed reductions in SNCA levels can reduce aggregation, prevent Lewy body formation, and confer neuroprotection. Thus, inhibiting SNCA expression during disease prodromal phase has the potential to slow disease progression or halt disease onset. SNCA translation is controlled by an iron-response element (IRE) in the 5’UTR of the mRNA. To demonstrate feasibility, we developed proof-of-concept RNA structure sensors that were responsive to the binding of small molecules and antisense oligonucleotides, and demonstrated feasibility for HTS use. To accomplish the goal of this proposal, we will complete the following specific aims: 1) Finalize HTS optimization of the SNCA-specific RNA sensor and perform a pilot screen, 2) Establish the generalizability of the RNA structure sensor platform by developing HTS- compatible sensors targeting another pathogenic RNA structure, 3) Develop a standard operating procedure for the commercialization of custom RNA sensor services, 4) Perform a primary screen to identify inhibitors of SNCA protein translation. If successful, we will have a validated HTS assay for synucleinopathy drug discovery and a RNA structure sensor platform that aimed to accelerate the current pace in RNA structure-based drug discovery and to enable more RNA-targeted drug development programs targeting disease-causing RNA structures.