Probing the Role of the LRRK2 GTPase in Parkinson's Disease

探讨 LRRK2 GTPase 在帕金森病中的作用

• 批准号: 10412970
• 负责人: Lawrence Yang Zhu
• 金额: $ 3.89万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10412970
• 关键词: African; Allosteric Site; Binding Sites; Biochemical; Biological Assay; Biology; Biophysics; Catalytic Domain; Cell Line; Cells; Chemicals; Clinical; Cohort Studies; Collaborations; Data; Development; Disease; Dopaminergic Cell; Dose; Engineering; Exhibits; Family; Fellowship; Functional disorder; Future; GTP Binding; Genetic; Genetic Determinism; Goals; Guanosine; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; High Prevalence; Human Genome Project; Hyperactivity; Idiopathic Parkinson Disease; LRRK2 gene; Lead; Leucine-Rich Repeat; Libraries; Mass Spectrum Analysis; Measures; Mediating; Methods; Modality; Molecular; Molecular Conformation; Mutation; Nature; Nerve Degeneration; Neurodegenerative Disorders; Nucleotides; Other Genetics; Output; Parkinson Disease; Pathogenesis; Patients; Penetrance; Phosphotransferases; Physiological; Physiology; Positioning Attribute; Preclinical Testing; Prevention; Probability; Recombinants; Risk Factors; Role; Route; Scanning; Secondary to; Site; Surveys; Symptoms; System; Techniques; Testing; Therapeutic; Toxic effect; Training; Work; alpha synuclein; autosomal dominant mutation; biophysical techniques; chemical genetics; dopaminergic neuron; effective therapy; experimental study; genetic approach; genetic association; genome wide association study; in vivo; induced pluripotent stem cell; inhibitor; insight; kinase inhibitor; mutant; neurotoxicity; novel; research clinical testing; screening; small molecule; small molecule inhibitor; success; targeted treatment; therapeutic target; tool; tool development

Project Summary/Abstract Parkinson’s disease is a common neurodegenerative disorder that has been described clinically for at least 200 years. While treatments have advanced to manage patient symptoms, a fundamental understanding of its physiological underpinnings remains elusive, and no curative or progression modifying treatment is currently available. Since the completion of the Human Genome Project, LRRK2 (Leucine Rich Repeat Kinase 2) has been understood to form a strong genetic association with certain familial cases of Parkinson’s disease. Autosomal dominant mutations in LRRK2 of variable penetrance have been demonstrated in isolated cohorts, and GWAS studies have indicated the importance of LRRK2 SNPs in the development of idiopathic Parkinson’s disease as well. Thus, the study of LRRK2 could elucidate findings in the pathophysiological mechanisms of Parkinson’s as a whole. Current hypotheses postulate that LRRK2 kinase hyperactivity is responsible for specific cellular toxicity and resultant neurodegeneration, resulting in the development of LRRK2 kinase inhibitors in clinical and pre-clinical testing. One of the key catalytic domains of LRRK2, its Roc- COR family GTPase, is a site of autosomal dominant mutations of high penetrance that also demonstrate increased kinase activity. Despite this, this domain is relatively understudied compared to the kinase domain. Efforts to understand the GTPase domain of LRRK2 may lead to an alternative modality of therapy, as there are concerns about toxicity mechanisms in currently tested kinase inhibitors. I propose to study the LRRK2 GTPase using chemical genetic and chemical methods via the development of tool compounds and appropriate biochemical and biophysical assays to determine the effect of GTPase modulation on LRRK2 mediated physiology. Preliminary data indicates that the LRRK2 GTPase is surveyable using a variety of developed assay techniques, and can be recombinantly expressed in sufficient amounts to enable large scale screening campaigns. In Aim 1, I propose to study the LRRK2 GTPase via the development of an electrophile sensitive (ES) approach to conformationally lock the domain into either GDP- or GTP-bound states. I will then introduce this system into iPSC-derived dopaminergic neurons and measure the effects of G nucleotide on LRRK2 activity and localization. In Aim 2, I propose to execute complementary small molecule discovery campaigns against the LRRK2 GTPase to uncover tool compounds that can target either the orthosteric or disease mutation defined allosteric sites. I will then test these compounds in primary dopaminergic neuron cells for their effects on ameliorating LRRK2 mutant-mediated cellular toxicity. Ultimately, the findings from these studies will result in small molecule tool compounds and potential therapeutic leads that can be used to better understand the molecular basis of Parkinson’s disease and its avenues for treatment. Training under this fellowship will be supported by several collaborations including the Small Molecule Discovery center (SMDC) at UCSF.

项目摘要/摘要 帕金森氏病是一种常见的神经退行性疾病，至少在临床上被描述 200年。虽然治疗已进步以管理患者症状，但对其症状的基本了解 物理基础仍然难以捉摸，目前尚无现代或进展的修改治疗 可用的。自从人类基因组项目完成以来 据了解，我们与帕金森氏病的某些家庭病例形成了牢固的遗传关联。 在孤立的队列中已证明了可变渗透率的LRRK2中的常染色体显性突变， GWAS研究表明LRRK2 SNP在特发性发展中的重要性 帕金森氏病也是如此。这，LRRK2的研究可以阐明病理生理学的发现 帕金森的整体机制。当前的假设假设LRRK2激酶多动症是 负责特定的细胞毒性和导致的神经变性，从而发展 LRRK2激酶抑制剂在临床和临床前测试中。 LRRK2的关键催化域之一，其ROC- Cor家族GTPase是一个高渗透率的常染色体显性突变的位置，也证明 激酶活性增加。尽管如此，与激酶结构域相比，该域相对理解。 了解LRRK2的GTPase领域的努力可能会导致替代的治疗方式，因为 是当前测试激酶抑制剂中毒性机制的担忧。我建议研究LRRK2 通过开发工具化合物的GTPase使用化学遗传和化学方法 适当的生化和生物物理测定法，以确定GTPase调制对LRRK2的影响 介导的生理学。初步数据表明LRRK2 GTPase可使用多种 开发的测定技术，可以以足够的量重组表达以使大规模表达 筛选活动。在AIM 1中，我建议通过开发电力研究LRRK2 GTPase 敏感的（ES）方法可以将域锁定到GDP-或GTP结合状态。然后我会 将该系统引入IPSC衍生的多巴胺能神经元，并测量G核苷酸对 LRRK2活性和本地化。在AIM 2中，我建议执行完整的小分子发现 针对LRRK2 GTPase的广告系列，以发现可以针对正构剂或的工具化合物 疾病突变定义了变构部位。然后，我将在原发性多巴胺能神经元细胞中测试这些化合物 它们对改善LRRK2突变体介导的细胞毒性的影响。最终，这些发现 研究将导致小分子工具化合物和潜在的治疗铅可用于改善 了解帕金森氏病及其治疗途径的分子基础。在此下进行培训 奖学金将得到包括小分子发现中心（SMDC）在内的几项合作的支持 在UCSF。