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病毒 世界性的问题。登革热病毒导致人类最流行的蚊媒病毒疾病，严重的 表现为血管渗漏的病例可能是致命的。相关的寨卡病毒(ZIKV)最近引发了爆炸 横跨美洲的流行病，并与先天性出生缺陷和格林-巴利综合症有关 综合症。尽管它们在世界范围内的发病率和死亡率很高，但目前还没有治疗药物。 登革热或寨卡病毒的治疗。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致病中起着重要作用。我们 和其他人最近描述了NS1在直接引发内皮屏障功能障碍和 诱导人免疫细胞产生炎性细胞因子，导致体内血管渗漏。在这 建议，我们将评估葡聚糖在体外和体内对抗NS1介导的致病作用的有效性。 对DENV和ZIKV的体内感染也有明显的抑制作用。我们已经开发了多种方法来研究DENV和ZIKV 基于NS1诱导的体外内皮屏障功能障碍的特征的发病机制(例如，高 渗透性，内皮细胞糖萼样层的破坏[EGL])和在体内，使用小鼠病毒模型- NS1诱发的血管渗漏疾病。我们的初步结果显示，β的一种硫酸盐衍生物- 巴西蘑菇葡聚糖(FR-S)对新城疫病毒的体内外保护作用 诱导内皮细胞高通透性是很有前景的。我们还证明了FR-S具有抗DENV和抗-DENV的作用。 ZIKV的体外活性。在与加州大学圣地亚哥分校的K.Godula博士的合作下，我们确定了特定的合成 模拟Gag的分子能有效地与黄病毒NS1结合。鉴于NS1在黄病毒致病中的作用 我们的初步结果是，我们假设多糖抑制NS1诱导的EGL降解和血管生成。 活体内渗漏和病毒感染，并有可能成为新的治疗方法 登革热和寨卡病毒。这种方法是创新的，因为我们的目标是抑制严重疾病的表现，在 除抗病毒活性外。目的1将选出最有希望的NS1诱导的抑制剂(S) DENV和ZIKV体外预防内皮功能障碍的病理生理途径。 我们将筛选27个多糖，包括FR和FR-S，4个Gag-模拟物，舒洛地特和20个环糊精类似物 (与T.Sohajda博士合作，CyclLab)，以表彰其防止NS1诱导的高通透性的能力。目标 2将从体外实验中探讨其预防内皮功能障碍的作用机制。 化合物。目的3将评估最有效的化合物对DENV和 ZIKV NS1-和病毒诱导的体内血管渗漏、发病率和死亡率。整体而言，这项建议针对的是- ES是一项迫切的需求，确定了针对两种主要黄病毒疾病的新治疗策略，通过开发 以多糖为基础的化合物，既针对病毒，也针对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.