Screening for Compounds that Lower Intracellular Alpha-Synuclein Levels

项目摘要/摘要 这项提议的目标是确定能够降低α-突触核蛋白水平和积累的药物 (α)是帕金森氏病(PD)发病机制的基础。这项建议的前提是得到支持的 通过大量报告，包括SNCA(突触核蛋白α)基因的点突变或复制/三倍体 这导致α-SYN增加，导致常染色体显性早发性帕金森病。发现发起人 在帕金森病患者中，SNCA基因低甲基化导致表达增加，而 基因是PD2-4发病的一个因素提示降低α-Syn水平是一个有希望的靶点 治疗性干预5.此外，使用α2-肾上腺素受体等药物降低βSYN水平 据报道，(β2AR)激动剂在细胞系和啮齿动物模型中都具有神经保护作用。一个 对挪威人群的流行病学分析显示，个人使用β2AR激动剂沙丁胺醇治疗 哮喘患者患PD6的风险较低。路易相关病理(LRP)，主要包括 αSYN不仅限于帕金森氏病，而且已在阿尔茨海默病(AD)尸检的脑组织中发现7-9 和路易体痴呆症患者的大脑。载脂蛋白E4是阿尔茨海默病的危险因素，也是一种风险 是帕金森病的致病因素，并与PD10-12的早期发病有关。最近的一项研究也表明两者之间存在联系 脑脊液中较高水平的αSYN与AD13年认知功能减退的早期发展阶段之间的关系。 目前帕金森病的治疗方法只通过调节多巴胺能神经传递来缓解症状， 但不停止或延缓帕金森病发病机制背后的退变过程，迫切需要 能够减缓或阻止帕金森病进展的疾病修饰化合物的开发。致信地址 PD中的这一需求以及AD和DLB中的LRP，在本提案中，我们将执行高通量筛选 (HTS)加州大学洛杉矶分校200K化合物文库，以确定降低细胞内αSYN水平的HITS；这些HITS将 根据效力、类药物特性和脑渗透性进行优先排序，以便进一步分析。化合物 此前报道的较低的αSYN，如β2AR激动剂，也将接受评估和优先排序。在……里面 目标1，可识别大范围α同步构象的AlphaLISA将被优化并用于高温超导 确定减少SK-N-MC人神经上皮瘤细胞内αSYN的化合物6；HITS将 然后在二次分析和小鼠原代多巴胺能神经元中进行验证和剂量反应测定。 在目标2中，化合物的脑渗透性，如β2AR激动剂，从目标1优先命中，以及类似物 将通过体外平行人工膜通透性分析和体内药代动力学进行评估 (PK)分析。In Silo StarDrop分析确定具有良好类药物性能的最佳化合物和 体外ADME-T分析将在机制研究中进行评估。在目标3中，最好的化合物是 帕金森病患者、阿尔茨海默病患者和正常患者多巴胺能神经元的多巴胺能神经元的研究 供体；以及来自Thy1-αSyn14和Thy1-APP小鼠15-17的体外脑器官型切片培养。

PROJECT SUMMARY/ABSTRACT The goal of this proposal is to identify agents that reduce the levels and accumulation of alpha-synuclein (αSyn) that underlies the pathogenesis of Parkinson's disease (PD). The premise of this proposal is supported by numerous reports including point mutations or duplication/triplication of the SNCA (Synuclein Alpha) gene that results in αSyn increases leading to autosomal-dominant early-onset PD. The finding that the promoter for the SNCA gene is hypomethylated in PD resulting in increased expression1 and that over-expression of the gene is a factor contributing to onset of PD2-4 suggests reducing the levels of αSyn is a promising target for therapeutic intervention5. Furthermore, reduction of αSyn levels using agents such as β2-adrenoreceptor (β2AR) agonists has been reported to be neuroprotective in both cell line and rodent models6. An epidemiological analysis of a Norwegian population revealed individuals using β2AR agonist, salbutamol for asthma, have a reduced risk of developing PD6. The Lewy-related pathology (LRP), primarily comprised of αSyn, is not restricted to PD and has been found in a subset of autopsied Alzheimer's disease (AD) brains7-9 and brains of Dementia with Lewy Bodies (DLB) patients. Apolipoprotein E4, a risk factor for AD, is also a risk factor for PD and is associated with earlier onset of PD10-12. A recent study also suggests there is a link between higher levels of αSyn in the CSF and early stages of development of cognitive decline in AD13. Current therapeutics for PD provide only symptomatic relief by modulating dopaminergic neurotransmission, but do not stop or slow the degenerative processes underlying PD pathogenesis, there is an urgent need for the development of disease-modifying compounds capable of slowing or halting PD progression. To address this need in PD as well as LRP in AD and DLB, in this proposal we will perform high throughput screening (HTS) of the UCLA 200K compound library to identify hits that lower intracellular αSyn levels; these hits will be prioritized by potency, drug-like properties, and brain permeability for further analysis. Compounds previously reported to lower αSyn such as β2AR agonists, will also undergo evaluation and prioritization. In Aim 1, an AlphaLISA that recognizes a broad range of αSyn conformations will be optimized and used for HTS to identify compounds that reduce intracellular αSyn in SK-N-MC human neuroepithelioma cells6; hits would then validated and dose-response determined in secondary assays and mouse primary dopaminergic neurons. In Aim 2, brain permeability of compounds such as β2AR agonists, hits prioritized from Aim 1, and analogs would be assessed by in vitro Parallel Artificial Membrane Permeability Analysis and in vivo pharmacokinetics (PK) analysis. Optimal compounds with good drug-like properties determined by in silico StarDrop analysis and in vitro ADME-T assays would be evaluated in mechanistic studies. In Aim 3, the best compounds would be evaluated in induced pluripotent stem cell (iPSC)-derived dopaminergic neurons from PD, AD, and normal donors; and in ex vivo brain organotypic slice cultures from Thy1-αSyn14 and from Thy1-APP mice15-17.