Nucleic Acids Research Advance Access originally published online on January 18, 2008
Nucleic Acids Research 2008 36(5):1482-1496; doi:10.1093/nar/gkm1178
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Nucleic Acids Research, 2008, Vol. 36, No. 5 1482-1496
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Identification and characterization of coumestans as novel HCV NS5B polymerase inhibitors
1Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA, 2Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St John's University, Jamaica, NY 11439, USA, 3Laboratorio de Quimica Bioorganica (LQB), Nucleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil, 4Department of Molecular Microbiology and Immunology, C. Bond Life Sciences Center, University of Missouri-Columbia, School of Medicine, 1201 E. Rollins Road, Columbia, MO 65211-7310, USA and 5Departamento de Farmacologia Basica e Clinica, Instituto de Ciencias Biomedicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590, Brazil
*To whom correspondence should be addressed. Tel: +1 973 972 8653; Fax: +1 973 972 5594; Email: kaushik{at}umdnj.edu
Received October 29, 2007. Revised December 22, 2007. Accepted December 26, 2007.
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 IC50 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.
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