Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling

作者：Siew Pheng Lim, Christian G. Noble, Cheah C. Seh, Tingjin Sherryl Soh, Abbas El Sahili, Grace Kar Yarn Chan, Julien Lescar, Rishi Arora, Timothy E. Benson, Shahul Nilar, Ujjini H. Manjunatha, Kah Fei Wan, Hongping Dong, Xuping Xie, Pei‐Yong Shi, Fumiaki Yokokawa · 发表于：PLoS Pathogens · 年份：2016 · DOI：10.1371/journal.ppat.1005737 · 被引用次数：187 · 研究领域：Mosquito-borne diseases and control、Malaria Research and Control、Insect symbiosis and bacterial influences

Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a "de novo" initiation mechanism. Crystal structures of the flavivirus RdRp revealed a "closed" conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the "GDD" active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed "N pocket"). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1-2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of ...