Novel AR-12 derivatives, P12-23 and P12-34, inhibit flavivirus replication by blocking host de novo pyrimidine biosynthesis

The genus Flavivirus contains many important pathogens, including dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV). AR-12 is a celecoxib-derived anticancer agent that possesses antiviral activity against a broad range of viruses. We pharmacologically exploited this unique activity to develop additional antiviral agents, resulting in the production of the AR-12 derivatives P12-23 and P12-34. At nanomolar concentrations, these compounds were effective in suppressing DENV, ZIKV and JEV replication, exhibiting 10-fold improvements in the efficacy and selectivity indices as compared to AR-12. Regarding the mode of antiviral action, P12-23 and P12-34 inhibited viral RNA replication but had no effect on viral binding, entry or translation. Moreover, these AR-12 derivatives co-localized with mitochondrial markers, and their antiviral activity was lost in mitochondria-depleted cells. Interestingly, exogenous uridine or orotate, the latter being a metabolite of the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH), abolished the antiviral activity of AR-12 and its derivatives. As DHODH is a key enzyme in the de novo pyrimidine biosynthesis pathway, these AR-12 derivatives may act by targeting pyrimidine biosynthesis in host cells to inhibit viral replication. Importantly, treatment with P12-34 significantly improved the survival of mice that were subcutaneously challenged with DENV. Thus, P12-34 may warrant further evaluation as a therapeutic to control flaviviral outbreaks.黄病毒属（Flavivirus）包含诸多重要病原体，包括登革病毒（DENV）、寨卡病毒（ZIKV）以及日本脑炎病毒（JEV）。AR-12是一种由塞来昔布（celecoxib）衍生而来的抗癌制剂，具备广谱抗病毒活性。我们通过药理学手段利用这一独特活性开发新型抗病毒制剂，最终获得了AR-12的衍生物P12-23与P12-34。在纳摩尔浓度下，这类化合物可有效抑制登革病毒、寨卡病毒及日本脑炎病毒的复制，其效力与选择性指数较AR-12提升了10倍。关于其抗病毒作用机制，P12-23与P12-34可抑制病毒RNA复制，但对病毒的吸附、入侵及翻译过程无影响。此外，这些AR-12衍生物可与线粒体标志物发生共定位，且在线粒体缺失细胞中其抗病毒活性完全丧失。值得注意的是，外源性尿苷或乳清酸——其中乳清酸是线粒体酶二氢乳清酸脱氢酶（DHODH）的代谢产物——可抵消AR-12及其衍生物的抗病毒活性。由于二氢乳清酸脱氢酶是从头嘧啶生物合成通路中的关键酶，这类AR-12衍生物或通过靶向宿主细胞的嘧啶生物合成途径以抑制病毒复制。经P12-34处理的小鼠在皮下接种登革病毒后，存活率显著提升。因此，P12-34有望作为治疗药物进一步评估，用于防控黄病毒属病毒的暴发流行。