Cerebrovascular involvement in Alzheimer's Disease: PAR4 Antagonism

摘要 阿尔茨海默病(AD)是导致痴呆症的最常见原因；越来越明显的是 脑血管机制在AD的病理过程中起着至关重要的作用。脑血管疾病频繁合并 与AD一起发生。导致这种疾病的一个原因是脑血管中的病理性纤维蛋白沉积(PFD)。 由对纤溶酶介导的纤维蛋白分解产生抵抗所致。PFD是AD的危险因素，两者都是非遗传性的 (创伤性脑损伤)和遗传性(ApoE4)，以及淀粉样β蛋白(Aβ)修饰纤维蛋白凝块，使其具有抵抗力 纤溶酶的裂解和恶化的PFD。凝血酶将纤维蛋白原分解为纤维蛋白，它还通过以下方式激活血小板 裂解和激活蛋白酶激活的受体PAR1和PAR4。我们发现，血小板PAR4是 对更大的血小板活化、凝血酶生成和促凝剂微粒的形成负责 血小板PAR1。PAR4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 被凝血酶激活，导致小胶质细胞激活，血脑屏障(BBB)破裂和少量出血。 淋巴细胞PAR4是归巢到炎症部位所必需的，PAR4在 炎症状态，导致局部损害被夸大。编码PAR4的基因F2RL3是 在炎症条件下发生低甲基化。我们的假设是，PAR4拮抗剂可能使PFD最小化。 通过1)减少凝血酶的产生和纤维蛋白的沉积，以及2)阻断淋巴细胞的渗透和局部 组织损伤。为了支持我们的假设，我们在初步数据中发现，PAR4在 5个家族性AD突变XFAD小鼠的脑血管系统此外，我们已经表明，在老年人中 人类前额叶皮质F2RL3表达水平较高与认知速度较快相关 拒绝。作为一种gpr，PAR4是一个有吸引力的治疗靶点，我们已经在以下方面取得了重大进展 产生PAR4拮抗剂。然而，它们还没有针对内源性栓系的有效活性。 无论是人类还是小鼠的血小板上的配体，或DMPK的特性在体内都是有用的。在目标1中，我们 建议使用一种计算方法，从比较小鼠和人类的PAR4模型开始 使用DOCK和Rosetta结合按需制作的小分子筛选超大型文库 图书馆。基于Shoichet和Roth实验室使用这种方法生成PM的成功经验 作为一种高效、高选择性的GPCRs化合物，我们希望丰富香港特别行政区的支架多样性。我们会 反复筛选小鼠和人类血小板中的化合物，然后重新进行计算筛选以进一步 提高搜救效率。在目标2和目标3中，传统药物化学将再次迭代地改进特性 拮抗剂的效力，选择性和体内生物利用度。最好的对手 将在5XFAD小鼠身上测试PAR4，以确定它是否可以减缓或阻止AD症状的进展。 这些目标的完成将导致体内工具化合物的测定，如果PAR4的拮抗作用 在这种严重的阿尔茨海默病动物模型中，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.