Development of Novel Neuroprotective Agents for Parkinson?s Disease

描述（由申请人提供）：帕金森病（PD）是一种使人衰弱的神经退行性疾病，其特征是黑质致密（SNc）中多巴胺能神经元的进行性和实质性丧失，导致严重的神经症状，包括震颤、运动迟缓和僵硬。现有的PD治疗方法主要集中在通过补偿神经化学缺陷来减轻运动症状，但它们未能阻止神经退行性过程的进展。缺乏有效的PD神经保护药物主要是由于对黑质多巴胺能系统退行性过程的复杂机制了解有限。然而，最近对凋亡通路和氧化应激信号的阐明提供了一些重要的线索，为开发新的PD神经保护策略带来了希望。在这方面，我们发现了PD模型中氧化损伤期间凋亡细胞死亡的关键途径，涉及caspase-3蛋白水解激活一种新的PKC异构体PKC4，以促进多巴胺能神经元的凋亡。使用显性阴性突变体、caspase-裂解位点抗性突变体或sirna抑制PKC4的蛋白水解激活，完全从帕金森毒素MPP+和6-OHDA诱导的凋亡细胞死亡中拯救多巴胺能神经元。有趣的是，我们还发现与其他细胞相比，PKC4在黑质多巴胺能神经元中高度表达。总之，这些发现表明PKC4是多巴胺能细胞中关键的下游促凋亡激酶，可能是PD的一个有希望的治疗靶点。重要的是，在经典mptp诱导的PD动物模型中，PKC4抑制剂rottlerin治疗对行为缺陷、神经化学物质耗损和黑质多巴胺能神经元损伤具有良好的保护作用，证实PKC4是开发PD神经保护剂的可行靶点。下一步，SBIR i期申请将开发一系列新的PKC4小分子抑制剂，并对其进行优化，以鉴定具有高治疗指数的先导化合物。该提案的具体目标是：i)设计和合成一系列新的PKC4抑制剂类似物，ii)利用高通量激酶谱分析系统分析PKC4抑制剂的选择性和抑制效能，iii)确定PKC4抑制剂在PD细胞培养模型中的潜在神经保护作用。有效的治疗靶点、综合研究设计和优秀的专业知识的结合将帮助我们实现这些重要目标。我们期望确定一组先导化合物用于PD动物模型的测试，以用于未来的ii期应用。总体的方法有望导致基于机制的神经保护剂治疗PD的发展。公共卫生相关性：帕金森病是一种主要的进行性神经退行性疾病，其主要运动症状为僵硬、运动迟缓、震颤和姿势不稳定。现有的帕金森病治疗方法只治疗症状，不能阻止神经退行性过程的进展。最近，我们发现PKC4是开发神经保护剂的一个新的治疗靶点，因为该激酶在黑质多巴胺能神经元的凋亡细胞死亡中起关键作用。拟议的研究将开发一系列小分子PKC4抑制剂，并评估其在帕金森病模型中的神经保护特性。总的来说，从这项建议中获得的知识将导致开发一种有效的神经保护药物来治疗帕金森病。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a debilitating neurodegenerative disorder characterized by progressive and substantial loss of dopaminergic neurons in the substantia nigra compacta (SNc), resulting in severe neurological signs including tremors, bradykinesia, and rigidity. The existing approaches to PD treatment are mainly focused on alleviating the motor symptoms by compensating for neurochemical deficits, but they fail to halt the progression of the neurodegenerative process. The lack of effective neuroprotective drugs for PD is primarily attributed to a limited understanding of the complex mechanisms involved in the degenerative processes of the nigral dopaminergic system. However, recent elucidation of apoptotic pathways and oxidative stress signaling has offered some important clues that boost hope for development of novel neuroprotective strategies for PD. In this regard, we uncovered a key apoptotic cell death pathway during oxidative insult in PD models involving the proteolytic activation of a novel PKC isoform, PKC4, by caspase-3 to promote apoptosis in dopaminergic neurons. Suppression of proteolytic activation of PKC4 using dominant negative mutants, a caspase-cleavage site resistant mutant, or siRNAs completely rescued dopaminergic neurons from Parkinsonian toxins MPP+ and 6-OHDA- induced apoptotic cell death. Interestingly, we also found that PKC4 is highly expressed in nigral dopaminergic neurons as compared to other cells. Collectively, these findings indicate that PKC4 is a key downstream proapoptotic kinase in dopaminergic cells and may be a promising therapeutic target for PD. Importantly, treatment with the PKC4 inhibitor rottlerin offered excellent protection against behavioral deficits, neurochemical depletion, and nigral dopaminergic neuronal damage in the classic MPTP-induced animal model of PD, confirming that PKC4 is a viable target for development of neuroprotective agents for PD. As a next logical step, this SBIR phase-I application proposes to develop a series of new PKC4 small molecule inhibitors and optimize them to identify lead compounds with a high therapeutic index. The specific objectives of the proposal are: i) To design and synthesize a series of novel PKC4 inhibitor analogs, ii) To profile the selectivity and inhibitory potency of PKC4 inhibitors using a high throughput kinase profiling system, iii) To determine the potential neuroprotective effects of the PKC4 inhibitors in cell culture models of PD. The combination of a validated therapeutic target, integrative study design, and excellent expertise will help us accomplish these important goals. We anticipate identifying a set of lead compounds for testing in PD animal models for future Phase-II application. The overall approach is expected to lead to development of a mechanism- based neuroprotective agent for treatment of PD. PUBLIC HEALTH RELEVANCE: Parkinson's disease is a major progressive neurodegenerative disorder characterized by the cardinal motor symptoms of rigidity, bradykinesia, tremors, and postural instability. The existing therapeutic approach for Parkinson's disease only treats the symptoms and fails to prevent the progression of the neurodegenerative process. Recently, we identified that PKC4 is a novel therapeutic target for development of neuroprotective agents because the kinase plays a key role in apoptotic cell death of nigral dopaminergic neurons. The proposed studies will develop a series of small molecule PKC4 inhibitors and evaluate their neuroprotective properties in Parkinson's disease models. Overall, the knowledge gained from this proposal will lead to development of an effective neuroprotective drug for treatment of Parkinson's disease.