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）病毒是蚊子传播的病毒，是主要的医疗和公共卫生问题。 全世界的问题。DENV引起人类最普遍的蚊媒病毒性疾病， 出现血管渗漏的病例可能是致命的。相关的寨卡病毒（ZIKV）最近引起了爆炸性的 流行病在整个美洲，并已与先天性出生缺陷和格林-巴利 综合征尽管它们在世界范围内的发病率和死亡率很高，但对于它们没有治疗剂。 治疗登革热或寨卡病毒。非结构蛋白1（NS 1）是一种黄病毒蛋白，参与病毒RNA 病毒复制及其分泌形式在宿主免疫逃避和病毒致病中起重要作用。我们 和其他人最近描述了NS 1在直接触发内皮屏障功能障碍中的新作用， 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 我们将评估聚糖对NS 1介导的发病机制的体外和体内功效， 以及体内抗DENV和ZIKV感染。我们已经开发了多种方法来研究DENV和ZIKV 基于体外NS 1诱导的内皮屏障功能障碍的表征的发病机制（例如，超 渗透性，破坏内皮糖萼样层[EGL]）和体内，使用病毒- 和NS 1诱导的疾病伴血管渗漏。我们的初步结果表明，β- 来自巴西蘑菇真菌的葡聚糖（FR-S）在体外和体内对DENV NS 1具有保护作用， 诱导的内皮细胞高通透性是有希望的。我们还表明，FR-S具有抗DENV和抗 体外ZIKV活性。与K博士合作。Godula（UC San Diego），我们确定特定的合成 GAG模拟分子有效地结合黄病毒NS 1。考虑到NS 1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NS 1诱导的EGL降解和vas， 以及病毒感染，并有可能作为新的治疗方式， 登革热和寨卡病毒这种方法是创新的，因为我们的目标是抑制严重的疾病表现， 除了抗病毒活性。目的1将选择最有前途的NS 1诱导的抑制剂， 本发明的目的是基于体外预防内皮功能障碍来研究DENV和ZIKV的病理生理学途径。 我们将筛选27种聚糖，包括FR和FR-S，4种GAG模拟物，舒洛地特和20种环糊精类似物 (in与T博士合作Sohajda，CycloLab），因为它们能够防止NS 1诱导的高渗透性。目的 2将研究通过所选的药物预防内皮功能障碍的体外作用机制。 化合物.目的3将评估针对DENV的最具活性的化合物的治疗潜力， ZIKV NS 1和病毒诱导的体内血管渗漏、发病率和死亡率总的来说，这项建议涉及- 是一个迫切的需要，确定新的治疗策略，对两个主要的黄病毒疾病，通过开发 基于聚糖的化合物，其靶向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.