Cerebrovascular involvement in Alzheimer's Disease: PAR4 Antagonism

摘要 阿尔茨海默病（AD）是痴呆症最常见的原因;越来越明显的是， 脑血管机制与AD病理学密切相关。脑血管病常合并- 发生AD。这种疾病的一个驱动因素是血管系统中的病理性纤维蛋白沉积（PFD） 由抵抗纤溶酶介导的纤维蛋白分解引起。PFD是AD的一个危险因素， β淀粉样蛋白（Aβ）修饰纤维蛋白凝块，使其具有耐药性， 纤溶酶裂解和恶化的PFD。凝血酶将纤维蛋白原裂解成纤维蛋白， 切割并激活蛋白酶激活的受体PAR 1和PAR 4。我们已经发现血小板PAR 4是 导致更大的血小板活化，凝血酶生成和促凝血微粒形成， 血小板PAR 1。PAR 4也在内皮细胞、白细胞、小胶质细胞和神经元上表达，在那里它是 凝血酶激活，导致小胶质细胞激活、血脑屏障（BBB）破坏和微出血。 淋巴细胞PAR 4是归巢至炎症部位所必需的，并且PAR 4表达在炎症条件下是可诱导的。 炎症状况，导致局部损伤加剧。F2 RL 3是编码PAR 4的基因， 在炎症条件下低甲基化。我们的假设是PAR 4拮抗剂可以使PFD最小化 通过1）减少凝血酶产生和纤维蛋白沉积，和2）阻断淋巴细胞浸润和局部 组织损伤为了支持我们的假设，我们已经在初步数据中发现，PAR 4在大肠杆菌中过表达。 具有5种家族性AD突变的5XFAD小鼠的脑血管系统。此外，我们还表明，在老年人中， 人类前额叶皮层中F2 RL 3表达水平较高与认知能力的快速增长有关。 下降作为一种GPCR，PAR 4是一个有吸引力的治疗靶点，我们已经取得了重大进展， 产生PAR 4拮抗剂。然而，它们对内源性栓系的 人或小鼠血小板中的DMPK配体，或DMPK特征在体内有用。目标1： 我建议使用一种计算方法，从小鼠和人PAR 4的比较模型开始 使用DOCK和ROSETTA筛选超大文库，结合按需小分子 图书馆基于Shoichet和Roth实验室使用这种方法生成pM的成功， 我们希望通过对GPCR的高效、高选择性化合物的研究，丰富我们SAR的支架多样性。我们将 反复筛选小鼠和人血小板中的化合物，然后重新进行计算筛选， 改善SAR。在目标2和3中，传统药物化学将再次迭代地改善性质 拮抗剂对束缚配体的效力、选择性和体内生物利用度。最佳拮抗剂 将在5XFAD小鼠中测试小鼠PAR 4的量，以确定其是否可以减缓或阻断AD症状的进展。 这些目标的完成将导致体内工具化合物，用于确定PAR 4的拮抗作用是否可以 在这种严重的AD动物模型中阻止或减缓AD的发展。

ABSTRACT Alzheimer’s disease (AD) is the most common cause of dementia; it is increasingly evident that cerebrovascular mechanisms are critically involved in AD pathology. Cerebrovascular disease frequently co- occurs with AD. One driver of this disease is pathologic fibrin deposition (PFD) in the cerebrovasculature caused by resistance to plasmin-mediated fibrin breakdown. PFD is a risk factor for AD, both non genetic (traumatic brain injury) and genetic (ApoE4), and amyloid beta (Aβ) modifies the fibrin clot, making it resistant to plasmin cleavage and worsening PFD. Thrombin cleaves fibrinogen to fibrin and it also activates platelets by cleaving and activating protease-activated receptors PAR1 and PAR4. We have found that platelet PAR4 is responsible for greater platelet activation, thrombin generation, and procoagulant microparticle formation than platelet PAR1. PAR4 is also expressed on endothelial cells, leukocytes, microglia and neurons, where it is activated by thrombin, leading to activation of microglia, blood brain barrier (BBB) breakdown and microbleeds. Lymphocyte PAR4 is required for homing to sites of inflammation and PAR4 expression is inducible under inflammatory conditions, leading to exaggerated local damage. F2RL3, the gene encoding PAR4, is hypomethylated under inflammatory conditions. Our hypothesis is that PAR4 antagonism may minimize PFD by 1) decreasing thrombin generation and fibrin deposition, and 2) blocking lymphocyte infiltration and local tissue damage. In support of our hypothesis, we have found in preliminary data that PAR4 is overexpressed in the cerebrovasculature of 5XFAD mice with 5 familial AD mutations. In addition, we have shown that in aged humans higher levels of F2RL3 expression in the prefrontal cortex are associated with a faster rate of cognitive decline. As a GPCR, PAR4 is an attractive therapeutic target, and we have made significant progress on generating PAR4 antagonists. However, they do not yet have potent activity against the endogenous tethered ligand in either human or mouse platelets, or the DMPK characteristics to be useful in vivo. In Aim 1, we propose to use a computational approach starting with comparative models of both mouse and human PAR4 to screen an ultra-large library using DOCK and ROSETTA, combined with a make-on-demand small molecule library. Based on the success of the Shoichet and Roth laboratories using this approach to generate pM potency, highly selective compounds for GPCRs, we expect to enrich the scaffold diversity of our SAR. We will iteratively screen compounds in mouse and human platelets, then redo the computational screening to further improve SAR. In Aims 2 and 3, traditional medicinal chemistry will then again iteratively improve the properties of antagonists for potency against the tethered ligand, selectivity and in vivo bioavailability. The best antagonist of mouse PAR4 will be tested in 5XFAD mice to determine if it can slow or block progression of AD symptoms. Completion of these aims will result in in vivo tool compounds for determination if antagonism of PAR4 can arrest or slow the development of AD in this severe animal model of AD.