New Drug Discovery Paradigms for Synucleinopathies

综述：治疗共核病的新药发现范式 细胞蛋白平衡功能障碍导致错误折叠蛋白的异常堆积 几种神经退行性疾病的病理学。α-突触核蛋白(AS)是 被称为路易小体的细胞内包涵体-路易小体痴呆的细胞病理学特征， 帕金森氏病和阿尔茨海默病(AD)的常见病理。AS和tau病理都可能 AD中的重叠是因为这两种蛋白质有可能相互成核和聚集。因此， 联核病是连接几个中枢神经系统蛋白质错误折叠障碍的高优先级靶点。α-突触核蛋白 是一种由140个氨基酸组成的胞内蛋白质。它是一种本质上无序的单体，但可以采用多种- 与脂泡结合的螺旋构象。这种从无序构象到螺旋构象的转变是 被认为导致错误折叠的富含片层的结构的形成，这些结构可以形成可溶的低聚物和 集合体。我们的假设是，由于膜磷脂诱导的二聚化是一种早期的，速率- 限制蛋白质错误折叠途径的一步，最终导致联核病；而这是一种药物抑制 这一步将具有治疗价值。我们已经开发出一种体外裂解荧光素酶蛋白质互补 当AS寡聚和聚集时，检测AS初始二聚的检测方法。我们的建议是利用这个 在两种创新的高通量筛选(HTS)范例中进行分析，以确定新的聚集 抑制剂。一种方法将走标准的漫长道路来识别新的小分子。我们会放映 分析缓冲液中的100,000种化合物与磷脂一起调节，以促进二聚作用。一组 二次化验将用于确定确认的HTS击中的特异性、效力、作用模式和 细胞活动。预期的目标1里程碑是确定一位适合提名为 临床前药物开发候选人。因为小尺寸的类药物之间的不匹配 与涉及蛋白质-蛋白质相互作用(PPI)的膨胀表面积相比，HTS对于 蛋白质错误折叠抑制剂具有挑战性。为了有可能加速将新疗法推向临床，我们 提出一种更非常规的方法，在这种方法中，我们将尝试确定两个FDA的组合 通过变构协同作用起超加成作用的批准药物，以防止自结合和聚集 就像。我们将使用我们开发的用于筛选FDA批准的药物混合物的多重HTS方法来 发现稳定单体结构的组合，防止自缔合。预期目标2 里程碑是鉴定两种先前批准的药物的组合，这些药物可能会进展到 治疗联体核病的概念验证临床测试比单个新的化学实体更快。如果 如果成功，我们开发的发现新型PPI调制器的新方法可能会对 促进对蛋白质错误折叠和聚集引起的其他神经退行性疾病的理解。

Summary: New Drug Discovery Paradigms for Synucleinopathies Dysfunction in cellular proteostasis leads to abnormal accumulation of misfolded proteins implicated in the pathology of several neurodegenerative diseases. Alpha-synuclein (aS) is the primary component of intracellular inclusions known as Lewy bodies—the cytopathological hallmark of Lewy body dementias, Parkinson’s disease and a frequent pathology in Alzheimer’s disease (AD). Both aS and tau pathology may overlap in AD because of the potential for inter-nucleation and aggregation of these two proteins. Thus, synucleinopathy is a high-priority target that bridges several CNS protein-misfolding disorders. Alpha-synuclein is a 140-amino-acid intracellular protein. It is an intrinsically disordered monomer but can adopt multiple - helical conformations on binding to lipid vesicles. This transition from disordered to -helical conformations is thought to lead to the formation of misfolded -sheet-rich structures that can form soluble oligomers and aggregates. Our hypothesis is that aS dimerization induced by membrane phospholipids is an early, rate- limiting step in protein-misfolding pathways that ultimately leads to synucleinopathies; and that a drug inhibiting this step will have therapeutic value. We have developed an ex vivo split-luciferase protein complementation assay to detect the initial dimerization of aS as it oligomerizes and aggregates. Our proposal is to use this assay in two innovative, high throughput screening (HTS) paradigms to identify novel aS aggregation inhibitors. One approach will take the standard long road to identify novel small molecules. We will screen 100,000 compounds in assay buffer conditioned with phospholipids that enhance aS dimerization. A panel of secondary assays will be used to characterize confirmed HTS hits for specificity, potency, mode of action and cellular activity. The expected Aim 1 milestone is identification of a novel lead suitable for nomination as a preclinical drug development candidate. Because of the mismatch between the small sizes of drug-like molecules compared to the expansive surface area involved in protein-protein interactions (PPI), HTS for protein-misfolding inhibitors is challenging. To potentially accelerate getting new therapies to the clinic, we propose a more unconventional approach in which we will attempt to identify a combination of two FDA approved drugs acting supra-additively by allosteric synergy to prevent the self-association and aggregation of aS. We will use a multiplexed-HTS method we developed for screening mixtures of FDA-approved drugs to discover combinations that stabilize monomeric aS structure, preventing self-association. The expected Aim 2 milestone is identification of a combination of two previously approved drugs that can potentially advance to proof-of-concept clinical testing for treating synucleinopathies faster than an individual new chemical entity. If successful, the new approaches we develop to discover novel PPI modulators could have broader utility for advancing understanding of other neurodegenerative diseases caused by protein misfolding and aggregation.