TY - JOUR
T1 - Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors
T2 - Mechanism of Action and Resistance Profiling
AU - Lim, Siew Pheng
AU - Noble, Christian Guy
AU - Seh, Cheah Chen
AU - Soh, Tingjin Sherryl
AU - El Sahili, Abbas
AU - Chan, Grace Kar Yarn
AU - Lescar, Julien
AU - Arora, Rishi
AU - Benson, Timothy
AU - Nilar, Shahul
AU - Manjunatha, Ujjini
AU - Wan, Kah Fei
AU - Dong, Hongping
AU - Xie, Xuping
AU - Shi, Pei Yong
AU - Yokokawa, Fumiaki
N1 - Funding Information:
I have read the journal's policy and the authors of this manuscript have the following competing interests: SPL, CGN, CCS, SN, UM, KFW, HD, XX, PYS, FY are employees of Novartis Institute for Tropical Diseases. RA, TB are employees of Novartis Institute of Biomedical Research. JL is an employee of Nanyang Technological University of Singapore and a consultant of Novartis Institute for Tropical Diseases. TSS is a PhD student funded by the Novartis Institute for Tropical Diseases and Singapore Economic Development Board. SPL and JL are editorial board members for ANti-viral Research Journal. PYS has served as Editor (ACS Infectious Diseases, Journal of General Virology, and Nature Vaccine) and Editorial Board member (Journal of Virology, Virology, and Antiviral Research).
Funding Information:
We thank Thierry Diagana, David Beer, Paul Smith and Francesca Blasco for support and useful discussions, Nicole Tay, Ka Yan Chung, Katherine Chan, Lim Chin Chin, Hao Ying Xu, Agatha Susila and Alexander Theodore Chao, for technical support, and colleagues at Novartis Biomedical Research Institute, Emeryville, CA, USA, for compound testing in human rhinovirus cell-based assays. We thank Shamala Devi for providing the clinical isolates of the four serotypes of dengue virus. JL and AES were supported by National Medical Research Council Grant CBRG14May051 (to JL).
Publisher Copyright:
© 2016 Lim et al.
PY - 2016/8
Y1 - 2016/8
N2 - 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 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.
AB - 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 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.
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U2 - 10.1371/journal.ppat.1005737
DO - 10.1371/journal.ppat.1005737
M3 - Article
C2 - 27500641
AN - SCOPUS:84984824834
VL - 12
JO - PLoS Pathogens
JF - PLoS Pathogens
SN - 1553-7366
IS - 8
M1 - e1005737
ER -