Small-Molecule Pharmacological Chaperones to Prevent Synuclein Transmission

 描述（由申请人提供）：我们的总体目标是发现靶向β-突触核蛋白（β-Syn）的小分子伴侣，其防止错误折叠的β-Syn的朊病毒样传递，并且可以开发成治疗神经退行性疾病如帕金森病（PD）、弥漫性路易体和阿尔茨海默病的药物。这些药物将通过一种新的作用机制，直接与β-Syn相互作用，以防止β-Syn在肿瘤细胞中的扩散。 大脑，最终目标是减少疾病进展。我们设想了一种创新的策略，通过这种策略，小分子可以结合并稳定天然或其他非致病状态的HIS-Syn，从而阻止病理事件。在聚集测定中发现了影响β-Syn聚集的大多数小分子，并与β-Syn的聚集途径形式相互作用。相比之下，我们将测试结合到Hissyn天然状态并更广泛地影响Hissyn功能障碍的化合物。从生物物理筛选，我们以前确定了61种化合物，结合到天然的α-突触素（α-突触素-PC），其中一些防止α-突触素介导的功能障碍，如α-突触素聚集和α-突触素诱导的囊泡功能障碍。基于这些有希望的结果，我们建议扩大开发的caseSyn-PC作为新的caseSyn分子伴侣药物。在本申请中，我们将确定β-Syn-PC是否是限制β-Syn细胞间传播的可行策略，并探索此类活性化合物的作用机制。 我们将首先鉴定出能够阻止β-Syn的细胞间传播的β-Syn-PC，并在此过程中鉴定出具有开发成治疗药物潜力的分子。在目的1中，我们将在β-Syn传递的细胞测定中筛选β-Syn-PC，包括基于FRET的测定，其中传递被测量为供体β-Syn与在不同细胞中表达的受体β-Syn的直接缔合。根据初步数据，我们希望找到活性化合物。将通过测试活性化合物的市售类似物的活性来探索结构活性关系。在目标2中，我们将进一步确定哪些传输步骤是由不同的调制器Syn-PC调制的。不同的化合物可能会通过不同的机制影响α-Syn的传递（例如，改变的细胞水平、位置、排出、摄取、错误折叠），因此可能使用β-Syn-PC鉴定新的机制。探讨这些新的机制将为进一步的治疗优化提供重要信息。我们将分析活动的MPSSyn-PC，以确定传输中的哪个阶段被阻塞。将应用生物化学、显微镜和细胞生物学技术来探索培养基、供体和受体细胞中的细胞和细胞外位置和状态。这些分析将阐明活跃的Bissyn-PC如何调节Bissyn的传输，并将阐明可能的检测方法，这些检测方法将有助于将来将这些分子开发成治疗患者的药物。 这些研究将提供第一个证据，证明PD-Syn分子伴侣在阻断参与PD进展和病理学的关键蛋白质的传递方面的功效，并将为开发用于治疗PD-Syn相关神经退行性疾病的新型药物提供基础。

 DESCRIPTION (provided by applicant): Our overall goal is to discover small molecule chaperones targeting -synuclein (Syn) that prevent the prion-like transmission of misfolded Syn and can be developed into drugs to treat neurodegenerative diseases such as Parkinson's (PD), Diffuse Lewy Body, and Alzheimer's diseases. These drugs will act via a novel mechanism of action by directly interacting with Syn to prevent spreading of Syn in the brain, with the ultimate goal of reducing disease progression. We envision an innovative strategy by which small molecules bind to and stabilize native or other non-pathogenic states of Syn and thereby block pathological events. Most small molecules affecting Syn aggregation were found in aggregation assays and interact with aggregation pathway forms of Syn. By contrast, we will test compounds that bind to Syn native states and affect Syn dysfunction more broadly. From a biophysical screen we previously identified 61 compounds that bind to native Syn (Syn-PCs) some of which prevented Syn-mediated dysfunctions such as Syn aggregation and Syn-induced vesicular dysfunction. Based on these promising results, we propose to expand the development of Syn-PCs as novel Syn chaperone drugs. In this application, we will determine if Syn-PCs are a viable strategy to limit Syn cell-to-cell transmission and explore the mechanism of action of such active compounds. We will first identify Syn-PCs capable of preventing the cell-to-cell transmission of Syn and in so doing, identify molecules having potential to be developed into therapeutic drugs. In Aim 1 we will screen the Syn-PCs in cellular assays of Syn transmission including a FRET-based assay in which transmission is measured as a direct association of donor Syn with acceptor Syn expressed in different cells. Based on preliminary data we expect to find active compounds. Structure activity relationships will be explored by testing activity of commercially available analogues of active compounds. In Aim 2 we will further identify which steps of transmission are modulated by different Syn-PCs. Different compounds will likely affect Syn transmission by different mechanisms (e.g., altered cellular levels, location, expulsion, uptake, misfolding) and thus novel mechanisms are likely to be identified using Syn-PCs. It will be greatly informative for further therapeutic optimization to explore these novel mechanisms. We will analyze the active Syn-PCs to identify which stage in transmission is blocked. Biochemical, microscopic and cell biological techniques will be applied to explore the cellular and extracellular location and state of Syn in media and donor and recipient cells. These analyses will elucidate how active Syn-PCs modulate the transmission of Syn and will shed light on possible assays that would be useful for future development of these molecules into drugs to treat patients. These studies will provide the first evidence for efficacy of Syn chaperones in blocking the transmission of a critical protein involved in PD progression and pathology and will provide the foundation for the development of a novel class of drugs for treating Syn related neurodegenerative diseases.