Regulation of Exosome Secretion as a novel therapeutic approach for Alzheimer's Disease

项目摘要 目前阿尔茨海默病(AD)的现有治疗方法仅能适度改善认知功能 和行为衰退。最近针对淀粉样蛋白(Aβ，Aβ)产生或清除的临床试验没有显示 疗效促使人们重新审视AD的治疗方法。阿尔茨海默病患者的大脑已经 显示神经酰胺的积累，神经酰胺是信号分子和外切体的一个组成部分 膜。神经酰胺的一个主要来源是通过中性酶催化的神经鞘蛋白的水解。 鞘磷脂酶2(NSmase2)。即使神经酰胺通过nSMase2短暂增加 上调是正常大脑功能的一部分，实验证据表明慢性nSMase2 上调会导致负面影响，包括神经炎症和氧化应激。最新研究 NSMase2也参与Aβ聚集和通过外体分泌的tau蛋白增殖 来自神经元和神经胶质细胞。此外，遗传或药物对外切体合成的抑制 抑制nSMAse2显著降低体内外Aβ聚集和tau增殖 从而为AD治疗开辟了一条新的途径。虽然nSmase2正在成为AD领域的重要参与者 病因学方面，目前的nSMase2抑制剂不足以开发潜在的治疗方法。 目前可用的缓蚀剂存在效力低(IC50‘S在微米级)、溶解性差、以及 大脑渗透率有限。为了解决这些限制，我们开发了一种人类nSMase2高 全量筛选试验，筛选了超过350,000种化合物，从而鉴定了 几次命中属于不同的化学系列。对其中两个不同的命中进行早期优化 化学系列导致了两个有效的化合物，其IC50分别为50和300 nm。这两种化合物都是 被确认为外切体释放的抑制剂，并显示出良好的药代动力学和脑部 渗透率(AUC脑/AUC血浆=0.27和0.6)。这项建议的目标是进一步优化这些 NSMase2抑制剂用于确定有效的、选择性的、可穿透大脑的候选药物以进行概念验证 在未来的IND启用研究之前，并最终用于AD患者的治疗。目标1是进行 结构-活性关系研究，以提高效力。目标2是表征来自AIM的化合物 1用于功能性抑制外切体释放、代谢稳定性、选择性和体内药代动力学。 目标3是通过在3xTg小鼠身上评估选定的化合物来进行体内概念验证研究 AD的模型。拟议中的工作涉及一种新的治疗靶点，该靶点在机理上与 之前在AD治疗方面的努力，具有解决疾病进展的潜力，并利用了两个 新发现的类药物nSMase2抑制剂的化学系列。

Project abstract Current available treatments for Alzheimer's disease (AD) only provide modest amelioration of cognitive and behavioral decline. Recent clinical trials targeting amyloid-β (Aβ) production or clearance did not show efficacy prompting a reexamination of approaches to AD treatment. Brains from AD patients have been shown to exhibit accumulation of ceramide, a signaling molecule and an integral component of exosomal membranes. One major source of ceramide is through the hydrolysis of sphingomyelin catalyzed by neutral sphingomyelinase 2 (nSmase2). Even though transient increases in ceramide through nSMase2 upregulation are part of normal brain functioning, experimental evidence indicates that chronic nSMase2 upregulation results in negative effects including neuroinflammation and oxidative stress. Recent studies also implicate nSMase2 in both Aβ aggregation and tau protein propagation through exosome secretion from neurons and glial cells. Moreover, inhibition of exosome synthesis by genetic or pharmacological inhibition of nSMAse2 significantly reduced Aβ aggregation and tau propagation both in vitro and in vivo thus opening a new avenue for AD therapeutics. While nSmase2 is emerging as an important player in AD etiology, the current armamentarium of nSMase2 inhibitors is inadequate to develop potential treatments. Currently available inhibitors have limitations including low potency (IC50's in µM level), poor solubility, and limited brain penetration. In order to address these limitations, we developed a human nSMase2 high throughput screening assay and screened over 350,000 compounds which led to the identification of several hits belonging to different chemical series. Early optimization of two of these hits from different chemical series led to two potent compounds with IC50s of 50 and 300 nM. Both of these compounds were confirmed as inhibitors of exosome release and exhibited good pharmacokinetic profiles and brain penetration (AUCbrain/AUCplasma = 0.27 and 0.6). The objective of this proposal is to further optimize these nSMase2 inhibitors to identify a potent, selective, brain penetrable candidate to carry out proof of concept before future IND enablement studies and ultimately for treatment of patients with AD. Aim 1 is to conduct structure-activity relationship studies to improve potency. Aim 2 is to characterize the compounds from aim 1 for functional inhibition of exosome release, metabolic stability, selectivity and in vivo pharmacokinetics. Aim 3 is to carry out in vivo proof of concept studies by evaluating selected compounds in the 3xTg mouse model of AD. The work proposed involves a novel therapeutic target which is mechanistically distinct from previous efforts in AD treatment, has the potential of addressing disease progression, and exploits two newly discovered chemical series of drug-like nSMase2 inhibitors.