Small-Molecule Pharmacological Chaperones to Prevent Synuclein Transmission

 描述(申请人提供)：我们的总体目标是发现针对-突触核蛋白(Syn)的小分子伴侣蛋白，可以防止错误折叠的Syn蛋白的Pron样传递，并可开发成治疗神经退行性疾病的药物，如帕金森氏症(PD)、弥漫性路易体和阿尔茨海默病。这些药物将通过一种新的作用机制发挥作用，直接与SYN相互作用，以防止SYN在 大脑，最终目标是减少疾病的进展。我们设想了一种创新的策略，通过小分子与Syn的自然或其他非致病状态结合并稳定，从而阻止病理事件。影响-Syn聚集的小分子大多在聚集实验中被发现，并与-Syn聚集途径形式相互作用。相比之下，我们将测试与SYN原生状态结合并更广泛地影响SYN功能障碍的化合物。通过生物物理筛选，我们已经鉴定出61个化合物可以与天然的突触蛋白(Synn-PC)结合，其中一些化合物可以预防突触蛋白介导的功能障碍，如突触蛋白聚集和突触蛋白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同步PC作为新型同步伴侣药物的开发。在这项应用中，我们将确定Syn-PC是否是限制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。我们将首先鉴定能够阻止Synn在细胞间传播的Syn-PC，并在这样做的过程中，鉴定具有开发成治疗药物的潜力的分子。在目标1中，我们将在Syn传播的细胞分析中筛选Syn-PC，包括基于FRET的分析，其中传输被测量为供体Syn与在不同细胞中表达的受体Syn的直接关联。根据初步数据，我们预计会发现活性化合物。将通过测试商业上可获得的活性化合物类似物的活性来探索结构和活性关系。在目标2中，我们将进一步确定哪些传输步骤由不同的同步PC调制。不同的化合物可能会通过不同的机制(如改变细胞水平、位置、排出、摄取、错误折叠)影响Syn的传递，因此利用Synn-PC可能识别新的机制。探索这些新的作用机制，将为进一步的治疗优化提供重要的信息。我们将分析活动的同步PC，以确定传输中的哪个阶段被阻止。将应用生化、显微镜和细胞生物学技术来探索Syn在介质和供受体细胞中的细胞和细胞外位置和状态。这些分析将阐明活性SYN-PC如何调节SYN的传递，并将有助于未来将这些分子开发为治疗患者的药物的可能分析。这些研究将首次提供Syn伴侣在阻断与PD进展和病理有关的关键蛋白的传递方面的有效性的证据，并将为开发治疗Syn相关神经退行性疾病的新型药物奠定基础。

 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.