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进程，特别是对于罕见、低流行率疾病和快速传播的传染病，如寨卡病毒。 我们已经开发了TR_FRET测定形式的ZIKV NS-1测定，用于化合物集合的高通量筛选。该测定可用于测量宿主细胞中的ZIKV复制，因为NS-1蛋白在ZIKV复制期间表达。通过使用这种ZIKV NI-1测定的药物再利用筛选，我们已经鉴定并确认了134种抑制人细胞中ZIKV复制的选择性抑制剂。在这134种ZIKV复制抑制剂中，33种是FDA批准的药物，4种在加拿大和日本批准，5种是研究药物。其余的是具有已知作用机制的生物活性化合物。这些化合物可以在体内ZIKV模型中进一步研究，并且具有在临床试验中进一步开发用于治疗ZIKV感染的潜力。此外，我们开发了一种高通量ZIKV滴度测定，其可用于快速筛选在其他筛选测定中鉴定的化合物。 我们还研究了ZIKV与宿主细胞的蛋白质-蛋白质相互作用网络。这已经确定了对ZIKV复制和发病机制至关重要的细胞途径。我们使用蛋白质阵列分析了10种单独的ZIKV蛋白质与大约10，000种人类蛋白质之间的直接相互作用，确定了几种细胞途径和蛋白质复合物。可药用人类基因组的RNAi筛选鉴定了一组参与ZIKV复制的宿主蛋白。已经发现宿主细胞中的蛋白酶体复合物积极参与ZIKV复制。这项研究产生了大量的数据，包括蛋白质-蛋白质相互作用，药物再利用筛选和RNAi筛选，这些数据对其他研究人员研究ZIKV发病机制和揭示药物开发的新靶点具有潜在的实用性。 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）抑制ZIKV复制（Xu等人，Nature Medicine 2016）。 然而，这种批准的药物在水中的溶解度差，口服生物利用度低。我们的药物化学家已经使用前药方法来改善氯硝柳胺的生物利用度问题。 化学努力导致TRND 00507788，一种氯硝柳胺前药，具有优异的水溶性（在pH 7.4缓冲液中>43 ug/mL）和良好的小鼠口服生物利用度。其他药代动力学和体内活性研究正在进行中。最近，这种氯硝柳胺前药在SARS-CoV-2的细胞病变效应测定中进行了测试，证明了抑制病毒感染在人类细胞中的细胞病变效应的能力。

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 have developed ZIKV NS-1 assay in the TR_FRET assay format for high throughput screening of compound collections. This assay can be used to measure ZIKV replication in host cells as the NS-1 protein is expressed during ZIKV replication. Through a drug repurposing screen using this ZIKV NI-1 assay, we have identified and confirmed 134 selective inhibitors that suppressed ZIKV replication in human cells. Among these 134 inhibitors of ZIKV replication, 33 are FDA approved drugs, 4 are approved in Canada and Japan, and 5 are investigational drugs. The rest are bioactive compounds with known mechanisms of action. These compounds can be further studied in the in vivo ZIKV models and have potential to be further developed in clinical trials for treatment of ZIKV infection. In addition, we developed a high-throughput ZIKV titer assay that can be used for rapid screening of compounds identified in other screening assays. We have also studied the protein-protein interaction networks of ZIKV with host cells. This has identified cellular pathways critical to ZIKV replication and pathogenesis. We used a protein array analysis of direct interactions between 10 individual ZIKV proteins and approximately 10,000 human proteins, which identified several cellular pathways and protein complexes. An RNAi screen of the druggable human genome identified a set of host proteins involved in ZIKV replication. The proteasome complex in host cells has been found to be actively involved in ZIKV replication. This study yielded a large set of data including protein-protein interaction, drug repurposing screening, and RNAi screening that have potential utility for other researchers to study ZIKV pathogenesis and reveal new targets for drug development. From our previous drug repurposing screen, niclosamide, an anthelminthic drug, was found to inhibit ZIKV replication (Xu et al, Nature Medicine 2016). However, this approved drug has poor solubility in water and low oral bioavailability. Our medicinal chemists have used a prodrug approach to improve the bioavailability issue of niclosamide. The chemistry effort led to TRND00507788, a Niclosamide prodrug that has excellent water solubility (>43 ug/mL at pH 7.4 buffer) and good oral bioavailability in mice. Additional studies of pharmacokinetics and in vivo activity are ongoing. Recently, this niclosamide prodrug was tested in a cytopathic effect assay of SARS-CoV-2, demonstrating an ability to suppress the cytopathic effect of viral infection in human cells.