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: 10642824
• 负责人: Lawrence Yang Zhu
• 金额: $ 2.99万
• 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 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10642824
• 关键词: Age; Allosteric Site; Binding; Binding Sites; Biochemical; Biological Assay; Biology; Biophysics; Catalytic Domain; Cell Line; Cells; Chemicals; Clinical; 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; Leucine-Rich Repeat; Libraries; Mass Spectrum Analysis; Measures; Mediating; Methods; Modality; Molecular; Molecular Conformation; Mutation; Nature; Nerve Degeneration; Neurodegenerative Disorders; North African; 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; Symptoms; System; Techniques; Testing; Therapeutic; Therapeutically Targetable; Toxic effect; Training; Work; alpha synuclein; autosomal dominant mutation; biophysical techniques; chemical genetics; cohort; dopaminergic neuron; effective therapy; experimental study; genetic approach; genetic association; genome wide association study; improved; 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.

项目概要/摘要 帕金森病是一种常见的神经退行性疾病，至少在临床上已有描述 200 年。虽然治疗方法已在控制患者症状方面取得了进展，但对其的基本了解 生理基础仍然难以捉摸，目前还没有治愈性或进展调节治疗 可用的。自人类基因组计划完成以来，LRRK2（亮氨酸重复激酶2）已 据了解，它与某些帕金森病家族病例形成了强烈的遗传关联。 LRRK2 的常染色体显性突变具有不同的外显率，已在孤立的队列中得到证实， 和 GWAS 研究表明 LRRK2 SNP 在特发性痴呆发展中的重要性 帕金森病也是如此。因此，LRRK2 的研究可以阐明病理生理学的发现。 帕金森病的整体机制。目前的假设假设 LRRK2 激酶过度活跃是 负责特定的细胞毒性和由此产生的神经变性，导致发展 LRRK2 激酶抑制剂的临床和临床前测试。 LRRK2 的关键催化结构域之一，其 Roc- COR 家族 GTPase 是一个高外显率的常染色体显性突变位点，也表明 激酶活性增加。尽管如此，与激酶结构域相比，该结构域的研究相对较少。 努力了解 LRRK2 的 GTPase 结构域可能会带来另一种治疗方式，因为 人们担心当前测试的激酶抑制剂的毒性机制。我建议研究LRRK2 GTPase 使用化学遗传和化学方法，通过开发工具化合物和 适当的生化和生物物理测定以确定 GTPase 调节对 LRRK2 的影响 介导的生理学。初步数据表明 LRRK2 GTPase 可使用多种方法进行测量 开发了测定技术，并且可以重组表达足够的量以实现大规模 筛选活动。在目标 1 中，我建议通过开发亲电子试剂来研究 LRRK2 GTPase 敏感（ES）方法将结构域锁定在 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 活性和定位。在目标 2 中，我建议执行互补的小分子发现 针对 LRRK2 GTPase 的活动，以发现可以靶向正构或 疾病突变定义了变构位点。然后我将在初级多巴胺能神经元细胞中测试这些化合物 因其对改善 LRRK2 突变体介导的细胞毒性的作用。最终，这些研究结果 研究将产生小分子工具化合物和潜在的治疗线索，可用于更好地治疗 了解帕金森病的分子基础及其治疗途径。在此下训练 奖学金将得到包括小分子发现中心（SMDC）在内的多项合作的支持 在加州大学旧金山分校。