Abstract
HCV is a small positive-strand RNA virus responsible for a considerable proportion of acute and chronic hepatitis in humans. Although all HCV enzymes are, in theory, equally appropriate for therapeutic intervention, the NS3-NS4A serine protease and the NS5B RNA-dependent RNA polymerase are the most popular targets from a drug-discovery perspective. A number of active-site inhibitors of the NS3 protease as well as allosteric inhibitors of the NS5B polymerase are being developed. We determined the crystal structures of complexes of NS3/NS4A/active-site inhibitor as well as NS5B/allosteric inhibitor to permit structure-based drug design and the efficient optimization of leads. The methods for obtaining such structures by crystal soaking procedures are described.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
De Francesco, R., and Migliaccio, G. (2005). Challenges and successes in developing new therapies for hepatitis C. Nature 436, 953–960.
Urbani, A., Bazzo, R., Nardi, M. C., Cicero, D. O., De Francesco, R., Steinkuhler, C., et al. (1998). The metal binding site of the hepatitis C virus NS3 protease. A spectroscopic investigation. J. Biol. Chem. 273, 18760–18769.
Bianchi, E., Urbani, A., Biasiol, G., Brunetti, M., Pessi, A., De Francesco, R., et al. (1997). Complex formation between the hepatitis C virus serine protease and a synthetic NS4A cofactor peptide. Biochemistry 36, 7890–7897.
Kim, J. L., Morgenstern, K. A., Lin, C., Fox, T., Dwyer, M. D., Landro, J. A., et al. (1996). Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell 87, 343–355.
Yan, Y., Li, Y., Munshi, S., Sardana, V., Cole, J. L., Sardana, M., et al. (1998). Complex of NS3 protease and NS4A peptide of BK strain hepatitis C virus: a 2.2 Å resolution structure in a hexagonal crystal form. Protein Sci. 7, 837–847.
Bressanelli, S., Tomei, L., Roussel, A., Incitti, I., Vitale, R. L., Mathieu, M., et al. (1999). Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Proc. Natl. Acad. Sci. USA 96, 13034–13039.
Lesburg, C. A., Cable, M. B., Ferrari, E., Hong, Z., Mannarino, A. F., and Weber, P.C. (1999). Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat. Struct. Biol. 6, 937–943.
Orsatti, L., Di Marco, S., Volpari, C., Vannini, A., Neddermann, P., and Bonelli, F. (2002). Determination of the stoichiometry of noncovalent complexes using reverse-phase high-performance liquid chromatography coupled with electrospray ion trap mass spectrometry. Anal. Biochem. 309, 11–18.
Di Marco, S., Rizzi, M., Volpari, C., Walsh, M. A., Narjes, F., Colarusso, S., et al. (2000). Inhibition of the hepatitis C virus NS3/4A protease. The crystal structures of two protease-inhibitor complexes. J. Biol. Chem. 275, 7152–7157.
Ontoria, J. M., Di Marco, S., Conte, I., Di Francesco, M. E., Gardelli, C., Koch, U., et al. (2004). The design and enzyme-bound crystal structure of indoline based peptidomimetic inhibitors of hepatitis C virus NS3 protease. J. Med. Chem. 47, 6443–6446.
Di Marco, S., Volpari, C., Tomei, L., Altamura, S., Harper, S., Narjes, F., et al. (2005). Interdomain communication in hepatitis C virus polymerase abolished by small molecule inhibitors bound to a novel allosteric site. J. Biol. Chem. 280, 29765–29770.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Marco, S.D., Carfí, A. (2009). Preparation and Handling of Hepatitis C Viral Proteins NS3 and NS5B for Structural Studies. In: Tang, H. (eds) Hepatitis C. Methods in Molecular Biology™, vol 510. Humana Press. https://doi.org/10.1007/978-1-59745-394-3_9
Download citation
DOI: https://doi.org/10.1007/978-1-59745-394-3_9
Publisher Name: Humana Press
Print ISBN: 978-1-58829-970-3
Online ISBN: 978-1-59745-394-3
eBook Packages: Springer Protocols