Development of novel therapeutics to block Alzheimer's Disease progression

项目摘要 阿尔茨海默氏症(AD)困扰着600多万美国人，非洲裔美国人患阿尔茨海默氏症的可能性是非美国人的两倍 西班牙裔白人发展成AD。阿尔茨海默病患者脑组织蛋白质组学研究显示， 酪蛋白激酶1 epsilon(CK1ε)是一种丝氨酸/苏氨酸蛋白激酶。CK1ε的过度表达已经被证明 与AD的多种病理特征相关，包括神经原纤维缠结的形成 (NFTS)，tau蛋白亚型表达的改变，以及昼夜节律的失调。NFTS是 由微管结合蛋白tau的不可溶形式的聚集体组成。而tau通常结合到 并稳定神经元轴突中的微管阵列，tau的磷酸化导致其解离和 增加了NFT形成的概率。以前的研究表明，CK1ε使tau磷酸化，导致tau 从微管中解离并聚集在胞浆中。由于tau是一种本质上无序的蛋白质，它的 胞液积累增加了其聚集的倾向--形成NFTs的第一步。此外 除了ck1ε在tau从微管解离中的这种直接作用外，还有一个额外的间接作用，即 CK1ε的过度表达与Tau的选择性剪接形式的增加直接相关，该剪接形式降低了 微管结合重复次数从4次到3次。Tau异构体比例的改变是一个标志。 包括阿尔茨海默病在内的多种变态反应。最后，CK1ε活性的增加会导致磷酸化的增加 周期蛋白(PER1/2)在昼夜节律上改变其核心调节活动。这些发现表明 CK1ε是减缓几种病理进展性质的治疗干预的最佳候选者 与AD关联。利用计算对接研究，我们的研究实验室发现了一系列 作为双激酶CK1δ/ε抑制剂和选择性CK1ε抑制剂的萘醌类化合物。 鉴定了与其最接近的同系物相比给予CK1ε选择性的萘醌上的结构特征， CK1δ(对2-氯-5，8-二羟基苯二酚的抑制率为90%~13%，对2-溴-5，8的抑制率为96%~1% 10μM浓度下的二羟基萘醌)。该项目的目标之一是优化我们的领先优势 化合物，以提高其效力和特异性的细胞角蛋白1ε。我们将使用细胞和重组生化 测定它们阻断细胞角蛋白1ε依赖的tau蛋白的磷酸化的有效性 Tau转录本和周期蛋白的磷酸化。

Project Summary Alzheimer’s Disease (AD) afflicts over 6 million Americans, with African Americans being twice as likely as non- Hispanic whites to develop AD. Proteomic studies of AD patient brain tissue revealed increased expression of casein kinase 1 epsilon (CK1ε), a serine/threonine protein kinase. This overexpression of CK1ε has been shown to be correlated with multiple pathological features of AD, including the formation of neurofibrillary tangles (NFTs), alternation of tau protein isoform expression, and dysregulation of the circadian rhythm. NFTs are comprised of aggregates of an insoluble form of the microtubule binding protein tau. While tau normally binds to and stabilizes the microtubule array in neuronal axons, phosphorylation of tau leads to its dissociation and increases the probability of NFT formation. Previous studies show that CK1ε phosphorylates tau, causing tau to dissociate from microtubules and accumulate in the cytosol. As tau is an intrinsically disordered protein, its cytosolic accumulation increases its propensity to aggregate – the first step in the formation of NFTs. In addition to this direct role for CK1ε in the dissociation of tau from microtubules, there is an additional indirect role, in that CK1ε overexpression directly correlates with the increase of an alternatively spliced form of tau that reduces the number of microtubule binding repeats from four to three. The alteration of the ratio of tau isoforms is a hallmark of multiple tauopathies, including AD. Finally, increased CK1ε activity causes increased phosphorylation of period proteins (PER1/2) altering their core regulatory activity on the circadian rhythm. These findings suggest that CK1ε is a prime candidate for therapeutic intervention to slow the progressive nature of several pathologies associated with AD. Using computational docking studies, our research laboratory found a series of naphthoquinones to act as dual kinase CK1δ/ε inhibitors as well as those that are selective inhibitors of CK1ε. Structural features were identified on naphthoquinones that impart selectivity for CK1ε over its closest homolog, CK1δ (90% to 13% inhibition for 2-chloro-5,8-dihydroxynaphthoquinone and 96% to 1% for 2-bromo-5,8 dihydroxynaphthoquinone at 10μM concentration). One of the goals of the project is to optimize our lead compounds to increase their potency and specificity for CK1ε. We will use cellular and reconstituted biochemical assays to determine their efficacy in blocking the CK1ε-dependent phosphorylation of tau, the alternate splicing of the tau transcript, and the phosphorylation of period proteins.