Inhibitor-induced structural change in the HCV IRES domain IIa RNA
Ryan B. Paulsen,a, Punit P. Seth,b, Eric E. Swayze,b, Richard H. Griffey,b, Jack J. Skalicky,c, Thomas E. Cheatham III,a,d, and Darrell R. Davis,a,c,1
- Author Affiliations
aDepartment of Medicinal Chemistry,
cDepartment of Biochemistry, and
dDepartment of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112;
bIsis Pharmaceuticals Inc., 1891 Rutherford Road, Carlsbad, CA 92008
Edited by Dinshaw J. Patel, Memorial Sloan-Kettering Cancer Center, New York, NY, and approved March 1, 2010 (received for review October 14, 2009)
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pnas.org
Abstract
Translation of the hepatitis C virus (HCV) RNA is initiated from a highly structured internal ribosomal entry site (IRES) in the 5' untranslated region (5' UTR) of the RNA genome. An important structural feature of the native RNA is an approximately 90° helical bend localized to domain IIa that positions the apical loop of domain IIb of the IRES near the 40S ribosomal E-site to promote eIF2-GDP release, facilitating 80S ribosome assembly. We report here the NMR structure of a domain IIa construct in complex with a potent small-molecule inhibitor of HCV replication. Molecular dynamics refinement in explicit solvent and subsequent energetic analysis indicated that each inhibitor stereoisomer bound with comparable affinity and in an equivalent binding mode. The in silico analysis was substantiated by fluorescence-based assays showing that the relative binding free energies differed by only 0.7 kcal/mol. Binding of the inhibitor displaces key nucleotide residues within the bulge region, effecting a major conformational change that eliminates the bent RNA helical trajectory, providing a mechanism for the antiviral activity of this inhibitor class. |
