Screening for Compounds that Lower Intracellular Alpha-Synuclein Levels

项目概要/摘要 该提案的目标是确定降低α-突触核蛋白水平和积累的药物 （αSyn）是帕金森病（PD）发病机制的基础。支持这一建议的前提是 通过大量的报道，包括SNCA（突触核蛋白α）基因的点突变或重复/三倍， 导致αSyn增加，导致常染色体显性早发性PD。这一发现表明， SNCA基因在PD中低甲基化，导致表达增加1和 基因是导致PD 2 -4发病的一个因素，这表明降低αSyn水平是治疗PD 2 -4的一个有希望的靶点。 治疗干预5.此外，使用β2-肾上腺素受体等药物降低αSyn水平， 据报道，（β 2 AR）激动剂在细胞系和啮齿动物模型中都具有神经保护作用6。一个 对挪威人群的流行病学分析显示，使用β2AR激动剂沙丁胺醇的个体 哮喘，患PD 6的风险降低。路易相关病理学（LRP），主要包括 αSyn不限于PD，并且已在尸检的阿尔茨海默病（AD）脑的子集中发现7 -9 和路易体痴呆（DLB）患者的大脑。载脂蛋白E4是AD的一个危险因素， 是PD的危险因素，并与PD 10 -12的早期发作相关。最近的一项研究也表明， CSF中较高水平的αSyn与AD 13中认知能力下降的早期发展阶段之间的关系。 目前的PD治疗方法仅通过调节多巴胺能神经传递来缓解症状， 但不能阻止或减缓PD发病机制下的退行性过程，因此迫切需要 开发能够减缓或阻止PD进展的疾病改善化合物。解决 PD以及AD和DLB中的LRP的这种需求，在本提案中，我们将进行高通量筛选 (HTS)的UCLA 200 K化合物库，以确定降低细胞内αSyn水平的命中;这些命中将 根据效力、药物样性质和脑渗透性进行优先级排序，以供进一步分析。化合物 之前报道的降低αSyn的药物，如β2AR激动剂，也将进行评估和优先考虑。在 目标1，将优化识别广泛αSyn构象的AlphaLISA并用于HTS 鉴定减少SK-N-MC人神经上皮瘤细胞中细胞内αSyn的化合物6;命中将 然后在二级测定和小鼠原代多巴胺能神经元中验证并确定剂量反应。 在目标2中，化合物如β2AR激动剂、目标1优先的命中物和类似物的脑渗透性 将通过体外平行人工膜渗透性分析和体内药代动力学进行评估 (PK)分析.通过计算机StarDrop分析确定具有良好药物样性质的最佳化合物， 体外ADME-T测定将在机理研究中进行评价。在目标3中，最好的化合物是 在来自PD、AD和正常人的诱导多能干细胞（iPSC）衍生的多巴胺能神经元中评价 供体;以及Thy 1-α Syn 14和Thy 1-APP小鼠的离体脑器官型切片培养物15 -17。

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.