Evaluation of in vitro and in vivo efficacy of glycan-based compounds against flavivirus endothelial permeability and vascular leak

摘要 登革热 (DENV) 和寨卡 (ZIKV) 病毒是蚊媒病毒，是主要的医疗和公共卫生问题 世界范围内的问题。 DENV 引起人类最流行的蚊媒病毒性疾病，并且严重 出现血管渗漏的病例可能是致命的。相关的寨卡病毒（ZIKV）最近引起爆炸 美洲流行病，与先天性出生缺陷和吉兰-巴利综合征有关 综合症。尽管它们在世界范围内发病率和死亡率很高，但尚无治疗药物 治疗登革热或寨卡病毒。非结构蛋白 1 (NS1) 是一种参与病毒 RNA 的黄病毒蛋白 复制及其分泌形式在宿主免疫逃避和病毒发病机制中发挥着重要作用。我们 等人最近描述了 NS1 在直接触发内皮屏障功能障碍中的新作用， 诱导人体免疫细胞产生炎症细胞因子，导致体内血管渗漏。在这个 提案中，我们将评估聚糖对 NS1 介导的发病机制的体外和体内功效，如 以及体内对抗 DENV 和 ZIKV 感染。我们开发了多种方法来研究 DENV 和 ZIKV 发病机制基于体外 NS1 诱导的内皮屏障功能障碍的特征（例如，过度 渗透性、内皮糖萼样层 [EGL] 的破坏）和体内，使用病毒的鼠模型 NS1 诱发的血管渗漏疾病。我们的初步结果表明，β-的硫酸化衍生物 巴西蘑菇 (FR-S) 中的葡聚糖对 DENV NS1- 具有体外和体内保护作用 诱导内皮细胞通透性过高是有希望的。我们还表明 FR-S 具有抗 DENV 和抗 ZIKV 体外活性。与 K. Godula 博士（加州大学圣地亚哥分校）合作，我们确定了特定的合成 GAG 模拟分子可有效结合黄病毒 NS1。鉴于 NS1 对黄病毒发病机制的贡献 和我们的初步结果，我们假设聚糖抑制 NS1 诱导的 EGL 降解和血管 体内的眼球渗漏以及病毒感染，并有可能作为新的治疗方式 登革热和寨卡病毒。该方法的创新之处在于我们的目标是抑制严重疾病表现， 除了抗病毒活性。目标 1 将选择 NS1 诱导的最有前途的抑制剂 基于体外预防内皮功能障碍的 DENV 和 ZIKV 病理生理学途径。 我们将筛选 27 种聚糖，包括 FR 和 FR-S、4 种 GAG 模拟物、舒洛地昔和 20 种环糊精类似物 （与 CycloLab 的 T. Sohajda 博士合作），表彰其预防 NS1 诱导的通透性过高的能力。目标 2 将研究所选药物预防内皮功能障碍的体外作用机制 化合物。目标 3 将评估最活跃的化合物针对 DENV 的治疗潜力， ZIKV NS1 和病毒引起的体内血管渗漏、发病率和死亡率。总体而言，该提案解决了—— 迫切需要通过开发针对两种主要黄病毒疾病的新治疗策略 基于聚糖的化合物，针对病毒和感染的病理生理后果。

ABSTRACT Dengue (DENV) and Zika (ZIKV) viruses are mosquito-borne viruses that are major medical and public health problems worldwide. DENV causes the most prevalent mosquito-borne viral disease of humans, and severe cases manifesting vascular leakage can be fatal. The related Zika virus (ZIKV) recently caused explosive epidemics across the Americas and has been associated with congenital birth defects and Guillain-Barré syndrome. Despite their substantial worldwide morbidity and mortality, no therapeutic agents exist for treatment of dengue or Zika. Nonstructural protein 1 (NS1) is a flaviviral protein that participates in viral RNA replication and in its secreted form plays important roles in host immune evasion and viral pathogenesis. We and others recently described novel roles for NS1 in directly triggering endothelial barrier dysfunction and inducing inflammatory cytokine production from human immune cells, contributing to vascular leak in vivo. In this proposal, we will evaluate the in vitro and in vivo efficacy of glycans against NS1-mediated pathogenesis, as well as against DENV and ZIKV infection in vivo. We have developed multiple methods to study DENV and ZIKV pathogenesis based on characterization of NS1-induced endothelial barrier dysfunction in vitro (e.g., hyper- permeability, disruption of the endothelial glycocalyx-like layer [EGL]) and in vivo, using murine models of virus- and NS1-induced disease with vascular leakage. Our preliminary results showing that a sulfated derivative of β- glucan from Agaricus brasiliensis fungus (FR-S) has a protective effect in vitro and in vivo against DENV NS1- induced endothelial hyperpermeability are promising. We have also shown that FR-S has anti-DENV and anti- ZIKV activity in vitro. In collaboration with Dr. K. Godula (UC San Diego) we determined that specific synthetic GAG-mimetic molecules efficiently bind to flavivirus NS1. Given the contribution of NS1 to flavivirus pathogenesis and our preliminary results, we hypothesize that glycans inhibit NS1-induced EGL degradation and vas- cular leakage in vivo as well as viral infection and have potential as novel treatment modalities for dengue and Zika. The approach is innovative in that we target inhibition of severe disease manifestations, in addition to antiviral activity. Aim 1 will select the most promising inhibitor(s) of NS1-induced pathophysiological pathways of DENV and ZIKV based on prevention of endothelial dysfunction in vitro. We will screen 27 glycans, including FR and FR-S, 4 GAG-mimetics, sulodexide, and 20 cyclodextrin analogues (in collaboration with Dr. T. Sohajda, CycloLab), for their ability to prevent NS1-induced hyperpermeability. Aim 2 will investigate the in vitro mechanism of action of prevention of endothelial dysfunction by the selected compounds. Aim 3 will evaluate the therapeutic potential of the most active compounds against DENV and ZIKV NS1- and virus-induced vascular leak, morbidity, and mortality in vivo. Overall, this proposal address- es a critical need, identifying novel therapeutic strategies against two major flaviviral diseases, by developing glycan-based compounds that target both the virus and pathophysiological consequences of infection.