Pharmacophore-Based Discovery of Viral RNA Conformational Modulators

Figures

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  Figure 1. (A) Schematic representation of the HCV IRES, highlighting the flexible bending induced by the 5-nt AACUA bulge of subdomain IIa. (B) Chemical structure of benzimidazole inhibitor Isis-11 with a depiction of the features used to define the pharmacophore: an aromatic moiety stacking between the G52:C111 pair and A53 bulge base, two in-plane hydrogen bonding donors interacting with the Hoogsteen edge of G110, and an out-of-plane positive charge located close to the A109 phosphate group. The protonation state depicted in the figure is predicted for 80% of Isis-11 molecules at pH 7.0. (C) Secondary structure of the subdomain IIad duplex used for FRET assays [12] and the subdomain IIah hairpin utilized in NMR spectroscopy and fluorescence intensity experiments. The position of the cy3 and cy5 fluorophores of IIad is indicated. For fluorescence intensity assays a fluorescein probe was linked to bulge residue C55 of IIah (indicated with F).

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  Figure 2. Scheme showing the pharmacophore-based screening process applied to identify new conformational modulators of HCV RNA subdomain IIa.

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  Figure 3. Chemical structure of the five pharmacophore screening hits evaluated in this study. The molecules are oriented according to the pharmacophore features depicted in Figure 1B and represented in the protonation states likely adopted when binding to RNA. In isolation and at pH 7.0, gn1 is mostly neutral; qn1, qz2, and bz1 predominantly bear one positive charge; and qn2 two charges. Unlike gn1, qz2 is predicted to form two equally stable tautomers, depicted in the figure.

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  Figure 4. Interaction between HCV IRES subdomain IIa and selected pharmacophore hit gn1. (A) IIad FRET curves in the absence (black) or presence of a 100-fold molar excess of unlabeled competitor tRNA (red). (B) IIah-F55 fluorescence intensity curves in the absence (black) and presence (red) of 2 mM MgCl2. (C) Titration of IIah monitored by 1H NMR spectroscopy in the presence of 2 mM MgCl2. The H5–H6 region of the TOCSY spectrum of unbound IIah (blue) is superposed on the spectra of complexes with increasing RNA:gn1 molar ratios, color-coded as indicated in the graph. A map of the gn1 binding site in the IIah hairpin is shown on the right. nt whose aromatic protons undergo chemical shift variations greater than two and three standard deviations from the mean perturbation (0.009 ppm) upon the addition of six equivalents of gn1 are highlighted in orange and red, respectively. nt with overlapped aromatic resonances are black-colored, and residues with aromatic signals not affected by ligand binding are colored grey. The terminal and capping UUCG tetraloop nt added to the viral sequence are indicated in blue and numbered differently.