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: 10218979
• 负责人: Varghese John
• 金额: $ 42.34万
• 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/10218979
• 关键词: Address; Affect; Age; Agonist; Albuterol; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease patient; Alzheimer' s disease risk; Amyloid beta-Protein; Applications Grants; Artificial Membranes; Asthma; Autopsy; Biological Assay; Brain; Cell Culture Techniques; Cell Line; Cell Membrane Permeability; Cells; Characteristics; Data; Dementia; Dementia with Lewy Bodies; Development; Disease; Disease Progression; Dose; Drug Kinetics; Epidemiology; Evaluation; Functional disorder; Genes; Genetic Polymorphism; Goals; Histocytochemistry; Human; Immunoblotting; Immunotherapy; Impaired cognition; In Vitro; Individual; Lewy Bodies; Libraries; Link; Mediating; Modeling; Molecular Conformation; Mus; Mutation; Neuroblastoma; Neurodegenerative Disorders; Neuroepithelioma; Neurons; Onset of illness; Parkinson Disease; Pathogenesis; Pathology; Pathway interactions; Patients; Permeability; Pharmaceutical Preparations; Plasma; Play; Point Mutation; Population; Process; Production; Progressive Disease; Promoter Regions; Property; Proteins; Proteomics; Public Health; Quantitative Reverse Transcriptase PCR; Reducing Agents; Reporting; Risk; Risk Factors; Rodent; Rodent Model; Role; Slice; Substantia nigra structure; Testing; Therapeutic; Tissues; Validation; age related; alpha synuclein; alpha synuclein gene; analog; apolipoprotein E-4; base; blood-brain barrier permeabilization; cognitive development; disorder risk; dopaminergic neuron; early onset; efficacy testing; glucosylceramidase; high throughput screening; in silico; in vivo; induced pluripotent stem cell; laboratory development; lead candidate; motor disorder; mouse model; neurotransmission; overexpression; promoter; research clinical testing; response; screening; small molecule; targeted treatment; transcription factor

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.

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。该提案的前提是得到支持 通过大量报告，包括 SNCA（突触核蛋白 Alpha）基因的点突变或重复/三倍化 导致 αSyn 增加，导致常染色体显性早发性 PD。研究发现，发起人为 SNCA 基因在 PD 中低甲基化，导致表达增加 1 以及 SNCA 基因的过度表达 基因是导致 PD2-4 发病的一个因素，表明降低 αSyn 水平是一个有希望的目标 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，(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6。一个 对挪威人群的流行病学分析显示，个体使用 β2AR 激动剂沙丁胺醇 哮喘，患 PD6 的风险降低。路易相关病理学 (LRP)，主要包括 αSyn 不仅限于 PD，并且已在尸检阿尔茨海默氏病 (AD) 大脑的子集中发现7-9 以及路易体痴呆（DLB）患者的大脑。载脂蛋白E4是AD的危险因素，也是一种危险因素 PD 的因素，并与 PD10-12 的早期发作相关。最近的一项研究还表明存在联系 脑脊液中较高水平的 αSyn 与 AD13 认知能力下降的早期发展阶段之间的关系。 目前帕金森病的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 发病机制中的退行性过程，因此迫切需要 开发能够减缓或阻止帕金森病进展的疾病缓解化合物。致地址 PD以及AD和DLB中的LRP的这种需要，在本提案中我们将进行高通量筛选 (HTS) UCLA 200K 化合物库，用于识别降低细胞内 αSyn 水平的命中；这些点击将 根据效力、类药物特性和脑通透性进行优先排序，以进行进一步分析。化合物 先前报道的降低 αSyn 的药物，如 β2AR 激动剂，也将接受评估和优先排序。在 目标 1，识别广泛 αSyn 构象的 AlphaLISA 将被优化并用于 HTS 鉴定可减少 SK-N-MC 人神经上皮瘤细胞细胞内 αSyn 的化合物6；点击率会 然后在二次测定和小鼠原代多巴胺能神经元中进行验证和确定剂量反应。 在目标 2 中，β2AR 激动剂等化合物的脑通透性、优先考虑目标 1 的命中以及类似物 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 分析和确定具有良好类药特性的最佳化合物 体外 ADME-T 测定将在机制研究中进行评估。在目标 3 中，最好的化合物是 在 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进行评估 捐助者；以及 Thy1-αSyn14 和 Thy1-APP 小鼠 15-17 的离体脑器官切片培养物。