Inhibition of dengue virus polymerase by blocking of the RNA tunnel.
Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)(20)]. Chemical modification of the initial "hit" improved the compound potency to an IC(50) (that is, a concentration that inhibits 50% RdRp activity) of 0.7 microM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC(50) of 5 microM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC(50)s of >100 microM against hepatitis C virus RdRp and human DNA polymerase alpha and beta. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.
Novartis Institute for Tropical Diseases, 10 Biopolis Road, #05-01 Chromos, Singapore.
Pornwaratt
P
Niyomrattanakit
Yen-Liang
YL
Chen
Hongping
H
Dong
Zheng
Z
Yin
Min
M
Qing
J Frasier
JF
Glickman
Kai
K
Lin
Dieter
D
Mueller
Hans
H
Voshol
Joanne Y H
JY
Lim
Shahul
S
Nilar
Thomas H
TH
Keller
Pei-Yong
PY
Shi
D000495:Allosteric Site; D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry* / Q000494:pharmacology; D001665:Binding Sites; D003715:Dengue / Q000188:drug therapy*; D003716:Dengue Virus / Q000201:enzymology; D004353:Drug Evaluation, Preclinical; D020128:Inhibitory Concentration 50; D012367:RNA, Viral / Q000037:antagonists & inhibitors; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors*; D013441:Sulfinic Acids; D062367:ortho-Aminobenzoates / Q000494:pharmacology* / Q000627:therapeutic use
D000998:Antiviral Agents; D012367:RNA, Viral; D013441:Sulfinic Acids; D062367:ortho-Aminobenzoates; D012324:RNA-Dependent RNA Polymerase