Screening for Compounds that Lower Intracellular Alpha-Synuclein Levels

筛选降低细胞内 α-突触核蛋白水平的化合物

• 批准号: 10524695
• 负责人: Varghese John
• 金额: $ 12.99万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-15 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10524695
• 关键词: Address; Agonist; Albuterol; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease risk; Alzheimer’s disease biomarker; Amyloid beta-42; Applications Grants; Asthma; Autopsy; Biological Assay; Brain; Cell Line; Cells; Complement; Data; Dementia; Dementia with Lewy Bodies; Development; Disease; Disease Progression; Epidemiology; Fluorescence; Genes; Goals; Human; Impaired cognition; Individual; Induced pluripotent stem cell derived neurons; Libraries; Link; Neuroepithelioma; Neurons; Parents; Parkinson Disease; Pathogenesis; Pathology; Patients; Permeability; Pharmaceutical Preparations; Point Mutation; Population; Property; Proteins; Public Health; Reducing Agents; Reporting; Risk; Risk Factors; Rodent Model; Therapeutic; Therapeutic Intervention; age related; alpha synuclein; alpha synuclein gene; apolipoprotein E-4; cognitive development; dopaminergic neuron; early onset; efficacy testing; high throughput screening; in vivo; induced pluripotent stem cell; inhibitor; lead candidate; motor disorder; overexpression; parent grant; promoter; screening; targeted treatment

PROJECT SUMMARY/ABSTRACT The goal of this Supplement, as with the parent grant, 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 Supplement proposal is supported by preliminary data from the parent grant and 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 expression and that over-expression of the gene is a factor contributing to onset of PD suggests reducing the levels of αSyn is a promising target for therapeutic intervention. 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 models. An epidemiological analysis of a Norwegian population revealed individuals using β2AR agonist, salbutamol for asthma, have a reduced risk of developing PD. In this Supplement, as part of expansion of the aim 1, we will evaluate the effect of validated hits on αSyn oligomerization and formation of intracellular aggregates using the bimolecular fluorescence complementation (BiFC) assay. Such inhibitors could reduce intracellular αSyn levels by downstream effects enhancing αSyn degradation. 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) brains 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 PD. 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 AD. In this Supplement as part of expansion of the aim 3 we will evaluate prioritized hits in iPSC derived neurons for effect on AD biomarkers. Current therapeutics for PD provide only symptomatic relief, 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 the parent proposal we are performing high throughput screening (HTS) of the UCLA 200K compound library to identify hits that lower intracellular αSyn levels; these hits will be validated and prioritized by potency, drug-like properties, and brain permeability for further analysis. In this Supplement for Aim 1 Expansion, hits identified from HTS by AlphaLISA that reduce intracellular αSyn in SK-N-MC human neuroepithelioma cells and are validated in primary and secondary assays in the parent grant would be evaluated in αSyn oligomerization using the BiFC assay that we will set up during the Supplement proposal. This will enable further elucidation of the mechanism of action (MoA) of the active hits. In Aim 3 Expansion, select compounds from Aim 2 would be evaluated in induced pluripotent stem cell (iPSC)-derived dopaminergic neurons from PD, AD, and normal donors; we will evaluate for both αSyn levels and AD biomarkers including sAPPα, sAPPβ and Aβ42.

项目总结/摘要 与父母补助金一样，该补助金的目标是确定降低水平的药物， α-突触核蛋白（αSyn）的积累是帕金森病（PD）发病机制的基础。的 这一补充提案的前提是支持初步数据，从家长赠款和许多 包括SNCA（突触核蛋白α）基因的点突变或重复/三倍的报告， αSyn增加导致常染色体显性早发性PD。SNCA基因的启动子 在PD中是低甲基化的，导致表达增加，基因的过度表达是一个因素， 有助于PD发作表明，降低αSyn水平是治疗干预的一个有前景的目标。 此外，使用诸如β2-肾上腺素受体（β2AR）激动剂的药物降低αSyn水平已被证实是有效的。 据报道，在细胞系和啮齿动物模型中都具有神经保护作用。一名挪威人的流行病学分析 人群显示，使用β2AR激动剂沙丁胺醇治疗哮喘的个体， 警局在本补充中，作为目标1扩展的一部分，我们将评估验证命中对αSyn的影响 使用双分子荧光互补寡聚化和形成细胞内聚集体 （BiFC）测定。这种抑制剂可以通过下游效应增强αSyn，从而降低细胞内αSyn水平 降解主要由αSyn组成的路易相关病理（LRP）不限于PD， 在阿尔茨海默病（AD）和路易体痴呆症（Dementia with Lewy Bodies）的尸检大脑中发现了 (DLB)患者载脂蛋白E4是AD的一个危险因素，也是PD的一个危险因素，并且与早期PD相关。 PD发作。最近的一项研究还表明，CSF中较高水平的αSyn与早期 AD认知功能下降的发展阶段。在本补充中，作为目标3扩展的一部分，我们将 评估iPSC衍生的神经元中的优先命中对AD生物标志物的影响。PD的当前治疗方法 仅提供症状缓解，因此迫切需要开发改善疾病的化合物 能够减缓或停止PD进展。为了解决PD以及AD和DLB中的LRP中的这一需求， 我们正在对UCLA 200 K化合物库进行高通量筛选（HTS）， 识别降低细胞内αSyn水平的命中;这些命中将通过效价、药物样 性质和脑渗透性以供进一步分析。在目标1扩展的本补充文件中， 通过AlphaLISA从HTS中分离，减少SK-N-MC人神经上皮瘤细胞中的细胞内αSyn， 在母基金的一级和二级试验中验证的，将在αSyn寡聚化中进行评价， 我们将在补充提案期间建立的BiFC测定。这将有助于进一步阐明 活性命中的作用机制（MoA）。在目标3扩展中，目标2中的选定化合物将是 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;我们将评价αSyn水平和AD生物标志物，包括sAPPα、sAPPβ和Aβ42。