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

项目总结/摘要 帕金森氏病是一种常见的神经退行性疾病，至少在临床上已有描述。 两百年了虽然治疗方法已经发展到控制患者症状，但对其症状的基本理解仍然存在。 生理学基础仍然难以捉摸，并且目前没有治愈性或进展调节治疗。 available.自人类基因组计划完成以来，LRRK 2（富含亮氨酸重复激酶2）已被发现， 据了解，它与帕金森病的某些家族病例形成了强烈的遗传关联。 LRRK 2中可变突变率的常染色体显性突变已在孤立的队列中得到证实， 和GWAS研究表明LRRK 2 SNP在特发性阿尔茨海默病的发生中的重要性。 帕金森氏症也是。因此，LRRK 2的研究可以阐明在病理生理学方面的发现。 帕金森病的发病机制目前的假设假定LRRK 2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 LRRK 2激酶抑制剂的临床和临床前测试。LRRK 2的关键催化结构域之一，其Roc- COR家族GT3是一个常染色体显性高突变位点， 增加激酶活性。尽管如此，与激酶结构域相比，该结构域相对研究不足。 努力了解LRRK 2的GT3结构域可能会导致一种替代的治疗方式， 是目前测试的激酶抑制剂的毒性机制的关注。我建议研究LRRK 2 通过开发工具化合物，使用化学遗传学和化学方法进行GTcycle， 适当的生物化学和生物物理测定以确定GT3调节对LRRK 2的影响 介导生理学初步数据表明，LRRK 2 GTSTAY可以使用各种 开发的测定技术，并且可以以足够的量重组表达， 筛查活动。在目标1中，我建议通过开发亲电试剂来研究LRRK 2 GTdR 敏感（ES）的方法，以构象锁定到GDP或GTP结合状态的结构域。然后我将 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 LRRK 2活性和定位。在目标2中，我建议执行互补小分子发现 针对LRRK 2 GTdR的运动，以发现可以靶向正构或 疾病突变定义的变构位点。然后我将在初级多巴胺能神经元细胞中测试这些化合物 因为它们对改善LRRK 2突变体介导的细胞毒性的作用。最终，这些发现 研究将产生小分子工具化合物和潜在的治疗先导物，可用于更好地 了解帕金森病的分子基础及其治疗途径。在此培训 该奖学金将得到包括小分子发现中心（SMDC）在内的几个合作项目的支持 在UCSF

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.