Discovery of a Novel Compound with Anti-Venezuelan Equine Encephalitis Virus Activity That Targets the Nonstructural Protein 2

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.阿尔法病毒（Alphaviruses）作为新发与再现病毒，对人类健康构成严重威胁。委内瑞拉马脑炎病毒（Venezuelan equine encephalitis virus, VEEV）属于新世界阿尔法病毒，可引发人类与马匹的脑炎，但目前尚无针对性治疗药物。迄今为止，已报道的抗VEEV或抗阿尔法病毒抑制剂类化合物仅表现出中等活性。为发现靶向新型病毒靶点的新型抗VEEV抑制剂类别，本研究基于活VEEV TC-83诱导的细胞病变效应（cytopathic effect, CPE）的细胞保护检测方法，构建高通量筛选（high-throughput screen）模型，对34万种化合物库进行筛选。从中共筛选得到5种新型抗VEEV化合物，并选取其中一种喹唑啉酮类化合物CID15997213（半数抑制浓度IC50=0.84 μM）开展后续表征研究。该化合物的抗病毒作用具有阿尔法病毒特异性，可抑制VEEV与西方马脑炎病毒，但对东方马脑炎病毒无抑制活性。体外实验证实该化合物可抑制病毒RNA、蛋白及子代病毒的合成。针对一组选定的RNA病毒，未检测到该化合物的抗病毒活性。本研究在非结构蛋白2（nonstructural protein 2, nsP2）的N端结构域中发现了赋予该化合物耐药性的突变位点，并通过反向遗传学方法（reverse genetic approach）验证了关键作用残基。加药时序实验显示，该化合物在病毒基因组复制最为活跃的复制中期发挥抑制作用。在小鼠模型中，以50 mg/kg/天的给药剂量，该化合物可完全保护小鼠免受致死性VEEV感染发病。综上，本研究发现了一种强效抗VEEV化合物，其独特靶向病毒nsP2的N端结构域。尽管nsP2的具体功能尚未阐明，但本研究结果提示该蛋白在病毒复制过程中发挥关键作用，同时也为开发阿尔法病毒感染的治疗手段提供了全新的研发方向。