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

项目摘要/摘要 帕金森氏病是一种常见的神经退行性疾病，至少在临床上已经被描述过 200年了。虽然治疗方法已经进步到管理患者症状，但对其基本理解 生理基础仍然难以捉摸，目前还没有根治或改善进展的治疗方法。 可用。自人类基因组计划完成以来，LRRK2(Leucine Rich Repeat Kinase 2)已经 据了解，帕金森氏症的某些家族病例与基因有很强的关联。 LRRK2的常染色体显性突变在孤立的队列中被证明是可变外显性的， GWAS的研究表明LRRK2 SNPs在特发性心脏病的发生发展中的重要性 还有帕金森氏症。因此，对LRRK2的研究可以阐明其在病理生理学上的发现。 帕金森氏症的整体发病机制。目前的假说假设LRRK2激酶过度活跃是 对特定的细胞毒性和由此产生的神经退化负责，导致 临床和临床前试验中的LRRK2激酶抑制剂。LRRK2的关键催化结构域之一，它的Roc- COR家族GTP酶是常染色体显性高外显性突变的一个位点，也证明了 增加了激酶的活性。尽管如此，与激酶结构域相比，该结构域的研究相对较少。 了解LRRK2的GTPase结构域的努力可能会导致另一种治疗方式，就像 目前测试的激酶抑制剂的毒性机制令人担忧。我建议研究LRRK2 利用化学、遗传和化学方法开发工具化合物和GTP酶 适当的生化和生物物理方法来确定GTP酶对LRRK2的调节作用 中介生理学。初步数据表明，LRRK2 GTP酶可以用各种 开发了检测技术，并可以重组表达足够的量以实现大规模 放映活动。在目标1中，我建议通过开发一种电泳体来研究LRRK2 GTP酶 将结构域构象锁定到GDP或GTP结合状态的敏感(ES)方法。那我会的 将该系统引入IPSC来源的多巴胺能神经元并检测G核苷酸对其的影响 LRRK2的活性和定位。在目标2中，我建议执行互补小分子发现 针对LRRK2 GTP酶的运动，以发现可以靶向邻位构型或 疾病突变定义了变构位点。然后我将在原代多巴胺能神经元细胞中测试这些化合物 它们对改善LRRK2突变介导的细胞毒性的作用。归根结底，这些发现 研究将导致小分子工具化合物和潜在的治疗线索，可以用来更好地 了解帕金森氏症的分子基础及其治疗途径。在此基础上进行培训 奖学金将得到包括小分子发现中心(SMDC)在内的几个合作机构的支持 在加州大学旧金山分校。

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.