Identification and characterization of coumestans as novel HCV NS5B polymerase inhibitors.
The hepatitis C virus (HCV) NS5B is essential for viral RNA replication and is therefore a prime target for development of HCV replication inhibitors. Here, we report the identification of a new class of HCV NS5B inhibitors belonging to the coumestan family of phytoestrogens. Based on the in vitro NS5B RNA-dependent RNA polymerase (RdRp) inhibition in the low micromolar range by wedelolactone, a naturally occurring coumestan, we evaluated the anti-NS5B activity of four synthetic coumestan analogues bearing different patterns of substitutions in their A and D rings, and observed a good structure-activity correlation. Kinetic characterization of coumestans revealed a noncompetitive mode of inhibition with respect to nucleoside triphosphate (rNTP) substrate and a mixed mode of inhibition towards the nucleic acid template, with a major competitive component. The modified order of addition experiments with coumestans and nucleic acid substrates affected the potencies of the coumestan inhibitors. Coumestan interference at the step of NS5B-RNA binary complex formation was confirmed by cross-linking experiments. Molecular docking of coumestans within the allosteric site of NS5B yielded significant correlation between their calculated binding energies and IC(50) values. Coumestans thus add to the diversifying pool of anti-NS5B agents and provide a novel scaffold for structural refinement and development of potent NS5B inhibitors.
Kaushik-Basu
Neerja
N
Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA. kaushik@umdnj.edu
Bopda-Waffo
Alain
A
Talele
Tanaji T
TT
Basu
Amartya
A
Costa
Paulo R R
PR
da Silva
Alcides J M
AJ
Sarafianos
Stefan G
SG
Noël
François
F
D000998:Antiviral Agents / Q000737:chemistry* / Q000494:pharmacology*; D001667:Binding, Competitive; D003374:Coumarins / Q000737:chemistry / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005419:Flavonoids / Q000737:chemistry / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D048789:Phytoestrogens / Q000737:chemistry* / Q000494:pharmacology*; D012313:RNA / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
D000998:Antiviral Agents; D003374:Coumarins; D004791:Enzyme Inhibitors; D005419:Flavonoids; C527703:LQB34 compound; D048789:Phytoestrogens; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D012313:RNA; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase