Screening for inhibitors of the hepatitis C virus internal ribosome entry site RNA

Abstract

The highly conserved internal ribosome entry site (IRES) of hepatitis C virus (HCV) regulates translation of the viral RNA genome and is essential for the expression of HCV proteins in infected host cells. The structured subdomain IIa of the IRES element is the target site of recently discovered benzimidazole inhibitors that selectively block viral translation through capture of an extended conformation of an RNA internal loop. Here, we describe the development of a FRET-based screening assay for similarly acting HCV translation inhibitors. The assay relies on monitoring fluorescence changes that indicate rearrangement of the RNA target conformation upon ligand binding. Screening of a small pilot set of potential RNA binders identified a benzoxazole scaffold as a ligand that bound selectively to IIa IRES target and was confirmed as an inhibitor of in vitro viral translation. The screening approach outlined here provides an efficient method to discover HCV translation inhibitors that may provide leads for the development of novel antiviral therapies directed at the highly conserved IRES RNA.

Functional conservation despite structural divergence in ligand-responsive RNA switches

“…Inspired by Yarus' classical study of arginine and G binding to self-splicing group I introns (28), we used a previously established FRET assay (23,29) to test arginine, guanine, and their derivatives for binding to the subdomain IIa RNA. Conformational capture of the dye-labeled RNA switch (SI Appendix, Fig.…”

“…To test for the effect of the guanine interaction on IRES function, we measured luciferase reporter expression levels from a bicistronic construct in an in vitro translation assay (29). The bicistronic construct allows measuring effects on IRES-driven translation of Renilla luciferase while also providing an internal control of a cap-initiated firefly luciferase (SI Appendix, Fig.…”

“…FRET folding and compound screening experiments were performed as described earlier (29). The in vitro transcription-translation assay was performed using HCV bicistronic luciferase constructs, as previously reported (40).…”

2-Aminobenzoxazole ligands of the hepatitis C virus internal ribosome entry site

“…2 Conformational capture of the IIa target had been investigated by a FRET-based assay which also served as a tool for measuring ligand affinity. 3 From crystal structure determination of the RNA target in complex with benzimidazole 1 a detailed picture emerged of the interactions involved in ligand binding (Figure 1C). 4 The 2-aminobenzimidazole scaffold plays a key role in target recognition, by engaging in base stacking interactions with the benzene ring and providing two hydrogen bonds to the Hoogsteen edge of a guanosine residue (G110).…”

“…The activity of compounds was assessed by testing binding affinity for the IRES IIa RNA in a FRET assay as previously described. 3 Target affinity expressed as EC 50 value was determined from fitting single-site binding dose response curves to data obtained by averaging triplicate compound titration experiments (Tables 1–4). …”