Smith Thomas M, Lim Siew Pheng, Yue Kimberley, Busby Scott A, Arora Rishi, Seh Cheah Chen, Wright S Kirk, Nutiu Razvan, Niyomrattanakit Pornwaratt, Wan Kah Fei, Beer David, Shi Pei-Yong, Benson Timothy E
Center for Proteomic Chemistry, Novartis Institutes for BioMedical Research, Inc., Cambridge, MA, USA
Novartis Institute for Tropical Diseases, Singapore.
J Biomol Screen. 2015 Jan;20(1):153-63. doi: 10.1177/1087057114551141. Epub 2014 Sep 24.
Dengue virus (DENV) is the most significant mosquito-borne viral pathogen in the world and is the cause of dengue fever. The DENV RNA-dependent RNA polymerase (RdRp) is conserved among the four viral serotypes and is an attractive target for antiviral drug development. During initiation of viral RNA synthesis, the polymerase switches from a "closed" to "open" conformation to accommodate the viral RNA template. Inhibitors that lock the "closed" or block the "open" conformation would prevent viral RNA synthesis. Herein, we describe a screening campaign that employed two biochemical assays to identify inhibitors of RdRp initiation and elongation. Using a DENV subgenomic RNA template that promotes RdRp de novo initiation, the first assay measures cytosine nucleotide analogue (Atto-CTP) incorporation. Liberated Atto fluorophore allows for quantification of RdRp activity via fluorescence. The second assay uses the same RNA template but is label free and directly detects RdRp-mediated liberation of pyrophosphates of native ribonucleotides via liquid chromatography-mass spectrometry. The ability of inhibitors to bind and stabilize a "closed" conformation of the DENV RdRp was further assessed in a differential scanning fluorimetry assay. Last, active compounds were evaluated in a renilla luciferase-based DENV replicon cell-based assay to monitor cellular efficacy. All assays described herein are medium to high throughput, are robust and reproducible, and allow identification of inhibitors of the open and closed forms of DENV RNA polymerase.
登革病毒（DENV）是世界上最重要的蚊媒病毒病原体，是登革热的病因。DENV RNA依赖性RNA聚合酶（RdRp）在四种病毒血清型中保守，是抗病毒药物开发的一个有吸引力的靶点。在病毒RNA合成起始过程中，聚合酶从“封闭”构象转变为“开放”构象以容纳病毒RNA模板。锁定“封闭”构象或阻断“开放”构象的抑制剂将阻止病毒RNA合成。在此，我们描述了一项筛选活动，该活动采用两种生化测定法来鉴定RdRp起始和延伸的抑制剂。使用促进RdRp从头起始的DENV亚基因组RNA模板，第一种测定法测量胞嘧啶核苷酸类似物（Atto-CTP）的掺入。释放的Atto荧光团允许通过荧光定量RdRp活性。第二种测定法使用相同的RNA模板，但无需标记，可通过液相色谱-质谱法直接检测RdRp介导的天然核糖核苷酸焦磷酸的释放。在差示扫描荧光法测定中进一步评估了抑制剂结合并稳定DENV RdRp“封闭”构象的能力。最后，在基于海肾荧光素酶的DENV复制子细胞测定中评估活性化合物以监测细胞疗效。本文所述的所有测定法都是中到高通量的，稳健且可重复，并且允许鉴定DENV RNA聚合酶开放和封闭形式的抑制剂。
