Bullard-Feibelman Kristen M, Fuller Benjamin P, Geiss Brian J
Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado, United States of America.
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, United States of America.
PLoS One. 2016 Jul 18;11(7):e0158923. doi: 10.1371/journal.pone.0158923. eCollection 2016.
Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus that causes severe and debilitating disease symptoms. Alarmingly, transmission rates of CHIKV have increased dramatically over the last decade resulting in 1.7 million suspected cases in the Western hemisphere alone. There are currently no antivirals for treatment of CHIKV infection and novel anti-alphaviral compounds are badly needed. nsP1 is the alphavirus protein responsible for the methyltransferase and guanylyltransferase activities necessary for formation of the 5' type 0 cap structure added to newly formed viral RNA. Formation of this cap depends on nsP1 binding GTP and transferring a methylated GMP to nascent viral RNA. We have developed a fluorescence polarization-based assay that monitors displacement of a fluorescently-labeled GTP analog in real time. Determining the relative affinities of 15 GTP analogs for nsP1 GTP revealed important structural aspects of GTP that will inform identification of inhibitors able to outcompete GTP for the nsP1 binding site. Validation of the assay for HTS was completed and a secondary orthogonal assay that measures guanylation activity was developed in order to evaluate hits from future drug screens. This platform provides an avenue for identification of potent nsP1 inhibitors, which would potentially provide compounds capable of treating disease caused by CHIKV infection.
基孔肯雅病毒（CHIKV）是一种由蚊子传播的甲病毒，可引发严重且使人衰弱的疾病症状。令人担忧的是，在过去十年中，CHIKV的传播率急剧上升，仅在西半球就导致了170万例疑似病例。目前尚无用于治疗CHIKV感染的抗病毒药物，因此急需新型抗甲病毒化合物。nsP1是甲病毒蛋白，负责甲基转移酶和鸟苷酸转移酶活性，这些活性是形成添加到新形成的病毒RNA上的5'0型帽结构所必需的。这种帽的形成取决于nsP1结合GTP并将甲基化的GMP转移到新生的病毒RNA上。我们开发了一种基于荧光偏振的检测方法，可实时监测荧光标记的GTP类似物的置换情况。确定15种GTP类似物与nsP1 GTP的相对亲和力，揭示了GTP的重要结构方面，这将为鉴定能够在nsP1结合位点上竞争GTP的抑制剂提供信息。完成了用于高通量筛选（HTS）的检测方法的验证，并开发了一种测量鸟苷化活性的二级正交检测方法，以评估未来药物筛选中的命中物。该平台为鉴定有效的nsP1抑制剂提供了一条途径，这些抑制剂可能会提供能够治疗由CHIKV感染引起的疾病的化合物。
