Lim, Siew Pheng, Noble, Christian Guy, Seh, Cheah Chen, Soh, Sherryl, Julien, Lescar, Shahul, Nilar, Manjunatha, Ujjini Havaldar, Arora, Rishi, Benson, Timothy, Wan, Kah Fei, Dong, Hongping, xie, Xuping, Pei-Yong, Shi and Yokokawa, Fumiaki (2016) Potent allosteric dengue virus NS5 polymerase inhibitors: mechanism of action and resistance profiling. PLOS Pathogens, 12 (8). e1005737. ISSN 1553-7374; 1553-7366

Abstract

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 M against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses.

Item Type:	Article
Date Deposited:	12 Aug 2016 00:45
Last Modified:	12 Aug 2016 00:45
URI:	https://oak.novartis.com/id/eprint/28481