Drug Repurposing Screening for Zika Virus Infection

根据现有的临床前和临床数据重新定位已批准的药物和临床阶段化合物可以大大加快这一过程，特别是对于罕见的低发病率疾病和快速传播的传染病，如寨卡病毒。 我们开发了一种TR-FRET格式的ZIKV NS-1检测方法，用于高通量筛选化合物集合。该筛选鉴定并确认了134种选择性抑制ZIKV在人类细胞中复制的抑制剂。由于来自ZIKV NS-1药物再利用筛选的大多数HITS的作用机制和/或临床适应症是已知的，我们将这些确认的化合物与我们在约翰·霍普金斯大学的合作者确定的蛋白质网络联系起来，从而识别出对ZIKV复制和致病至关重要的细胞通路。这种蛋白质网络分析使用了10个单独的ZIKV蛋白质与大约10,000个人类蛋白质之间直接相互作用的蛋白质阵列分析。我们还整合了来自可药物人类基因组的RNAi筛选的结果，该结果确定了一组参与ZIKV复制的宿主蛋白。我们发现宿主细胞中的蛋白酶体复合体积极参与ZIKV的复制(Guang等人。2021年)。 在我们建立化合物筛选方法的过程中，我们还观察到ZIKV感染人神经干细胞和SNB-19胶质母细胞瘤细胞系后线粒体膜电位的破坏和线粒体的碎裂。我们发现，这些变化的严重程度与感染细胞中ZIKV蛋白的数量有关。进一步的实验发现，在ZIKV感染后，丝裂原蛋白2水平的降低调节了线粒体的融合。我们还发现，线粒体分裂抑制因子1(Mdivi-1)是一种抑制线粒体分裂的小分子，它可以减少ZIKV感染细胞的线粒体破坏和细胞死亡。结果提示线粒体异常断裂与ZIKV诱导的神经细胞死亡有关(Shu Y.et al.2020)。因此，针对ZIKV感染细胞线粒体分裂融合异常可能成为治疗ZIKV感染所致神经细胞凋亡的新的治疗策略。 从我们之前的药物再利用筛选中，发现氯硝柳胺，一种驱虫药，可以抑制ZIKV的复制(Xu等人，《自然医学》2016)。但这种批准的药物在水中的溶解度较差，口服生物利用度较低。为了优化氯硝柳胺的结构，以改善氯硝柳胺类似物作为治疗寨卡病毒的候选药物的全身暴露，我们进行了结构-活性关系(SAR)和结构-性质关系(SPR)研究(Shamim等人，2021)。

Repositioning of approved drugs and clinical-stage compounds with existing preclinical and clinical data can greatly expedite the process, particularly for rare, low-prevalence diseases and for rapidly spreading infectious diseases, such as Zika virus. We developed a ZIKV NS-1 assay in the TR-FRET format for high throughput screening of compound collections. The screen identified and confirmed 134 selective inhibitors that suppressed ZIKV replication in human cells. Because the mechanisms of action and/or clinical indications of the most hits from the ZIKV NS-1 drug repurposing screen are known, we linked these confirmed compounds to the protein network identified by our collaborators at the Johns Hopkins University leading to the identification of cellular pathways critical to ZIKV replication and pathogenesis. This protein network analysis used a protein array analysis of direct interactions between 10 individual ZIKV proteins and approximately 10,000 human proteins. We also integrated the results from the RNAi screen of the druggable human genome that identified a set of host proteins involved in ZIKV replication. We found that the proteasome complex in host cells to be actively involved in ZIKV replication (Guang et al. 2021). During our assay development for compound screening, we also observed disrupted mitochondrial membrane potential and mitochondrial fragmentation after ZIKV infection in human neural stem cells and the SNB-19 glioblastoma cell line. We found that the severity of these changes correlated with the amount of ZIKV proteins in the infected cells. Further experiments found that the decreased levels of mitofusin 2 modulate mitochondria fusion after ZIKV infection. We also found that mitochondrial division inhibitor 1 (Mdivi-1), a small molecule inhibiting mitochondria fission, decreased mitochondria disruptions and cell death in ZIKV-infected cells. The results suggested a relationship between the abnormal mitochondrial fragmentation and ZIKV-induced neuronal cell death (Shu Y. et al. 2020). Thus, a new therapeutic strategy for treatment of neuronal apoptosis caused by ZIKV infection could target the abnormality of mitochondrial fission-fusion in ZIKV infected cells. From our previous drug repurposing screen, Niclosamide, an anthelminthic drug, was found to inhibit ZIKV replication (Xu et al, Nature Medicine 2016). But this approved drug has poor solubility in water and low oral bioavailability. To optimize the structure of niclosamide to improve systemic exposure for application of niclosamide analogs as drug lead candidates for treating Zika, we conducted structure-activity relationship (SAR) and structure-properties relationship (SPR) studies (Shamim et al., 2021).