Solution structure of a benzimidazole inhibitor in complex with domain iIA of the hepatitis C virus internal ribosome entry site

The solution structure of domain IIa of the hepatitis C virus internal ribosome entry site in complex with a racemic benzimidazole inhibitor was determined by NMR spectroscopy with corroborating fluorescence data. A 38 base RNA construct representing the inhibitor-binding region of domain IIa was synthesized by T7 RNA polymerase. Fully and selectively 13C and 15N labeled and isotopically unlabeled RNA samples were produced and studied in complex with the inhibitor. The inhibitor was previously shown to have inhibitory activity in an HCV replicon assay. It was also previously found to bind in the bulge region of domain IIa. In the free RNA, this five base bulge region introduces a bend in the extended domain II that situates the terminus of the domain over the mRNA cleft in the ribosomal E site. This hinge-like bulge region is not a known binding site for any host or viral translational cofactors, but domain II has been shown to be critical for IRES function, and the bulgeinduced bend in domain IIa has been shown to be important for IRES function in mutagenesis assays. Molecular dynamics refinement in explicit solvent and subsequent free energetic analysis indicate that the inhibitor enantiomers bind with comparable affinity and equivalent binding modes. The structure of this inhibitor/RNA complex suggests that the small molecule rearranges the base stacking in the bulge and introduces a significant conformational change that eliminates the bent RNA helical trajectory. This suggests a iv possible mechanism of inhibition involving the displacement of the domain II terminus from the mRNA cleft on the ribosomal E site. Perhaps most importantly, this structure may serve as a guide in the development of second-generation higher affinity inhibitors of the hepatitis C IRES as well as provide general insights into small molecule inhibitor interactions with RNA.