Validation and Screening of New RNA Targets - the hepatitis C virus internal ribosome entry site:

Hepatitis C virus (HCV) infection affects ~170 million people worldwide and is a major cause of chronic hepatitis as well as hepatocellular carcinoma (Lancet 372, 321; 2008). Current therapies suffer from low efficiency and serious side effects. Therefore, there is an urgent need for novel antiviral agents for the treatment of HCV infection.

Among the potential targets for HCV inhibitors is a highly conserved untranslated region (UTR) of the viral RNA genome which harbors an internal ribosome entry site (IRES). The HCV IRES binds with high affinity to host cell 40S ribosomal subunits and initiates translation in a cap-independent fashion. The IRES element adopts an ordered structure which is dominated by independently-folding RNA domains, among them the subdomain IIa which is one of the targets we are interested in (Figure 1).

Crystal structure analysis in our laboratory revealed that the subdomain IIa folds into a sharply bent motif (read more) which is required to accurately position the highly conserved hairpin loop IIb at the interface between the ribosomal subunits. Attesting to the functional importance of the subdomain IIa, residues in this region are highly conserved in HCV clinical isolates (Figure 2).

We had previously suggested that specific ligand binding to the IIa RNA might affect the native conformation of the bent subdomain and prevent the correct positioning of the hairpin loop IIb at the ribosome, ultimately blocking viral translation. To test this hypothesis, we have established an assay based on fluorescence resonance energy transfer (FRET) between cyanine dye labels attached at the termini of the RNA target (Figure 3). In the folded native state, the dye labels are at a distance that allows FRET to occur with high efficiency. FRET intensity will be reduced in the presence of ligands that induce a conformation with a widened RNA interhelical angle which translates into increased distance between the dye labels. We use the FRET assay to screen for ligands that induce conformational changes in the IRES subdomain IIa target (Figure 4). Such compounds are potential inhibitors of HCV translations and promising candidates for the development of antiviral drugs.

With the help of the FRET assay we demonstrated that benzimidazole inhibitors of the HCV replicon (J. Med. Chem. 48, 7099; 2005) act by conformational induction of a widened interhelical angle in the IRES subdomain IIa which facilitates the undocking of subdomain IIb from the ribosome and ultimately leads to inhibition of IRES-driven translation in HCV-infected cells. Studies on HCV replicon carrying subdomain IIa mutations confirmed the conformational mechanism of inhibition in the cellular context (Figure 5).

This is the first example of such a conformational mechanism proven for a biologically active small molecule that targets an RNA structure outside the bacterial ribosome.

(supported by the NIH, grant # R01 AI0720)
Conformational inhibition of the HCV IRES RNA (2009) Parsons J, Castaldi MP, Dutta S, Dibrov SM, Wyles DL & Hermann T. Nature Chem. Biol. 5, 823-825. (PDF)

Comment on this work in News and Views by DW Begley & G Varani: (PDF)

A comprehensive review of our work on the HCV IRES target: Hepatitis C virus translation inhibitors targeting the internal ribosome entry site (2014) Dibrov SM, Parsons J, Carnevali M, Zhou S, Rynearson KD, Ding K, Garcia Sega E, Brunn ND, Boerneke MA, Castaldi MP & Hermann T. J. Med. Chem. 57, 1694-1707. (PDF)

We are exploring the molecular recognition of ribonucleic acid (RNA) by small molecules.

Figure 1: The HCV IRES subdomain IIa target.

Figure 2: Sequence conservation of the IIa target in HCV clinical isolates (% occurence).

Figure 3: Dependence of FRET efficiency on interhelical angle in the IIa target.

Figure 4: Result of FRET binding screen of a small biased library against the IIa target.

Figure 5: HCV translation inhibition by an IRES subdomain IIa-binding inhibitor. In cells electroporated with replicon, the inhibitor blocked IRES-driven translation at low micromolar concentrations. The A57U mutation, which lowers affinity of the inhibitor for IIa, reduced reporter expression as well, consistent with weaker inhibitor binding to the mutant in the cellular context.