No abstract

Current interferon-based therapy for hepatitis C virus (HCV) infection is inadequate, prompting a shift toward combinations of direct-acting antivirals (DAA) with the first protease-targeted drugs licensed in 2012. Many compounds are in the pipeline yet primarily target only three viral proteins, namely, NS3/4A protease, NS5B polymerase, and NS5A. With concerns growing over resistance, broadening the repertoire for DAA targets is a major priority. Here we describe the complete structure of the HCV p7 protein as a monomeric hairpin, solved using a novel combination of chemical shift and nuclear Overhauser effect (NOE)-based methods. This represents atomic resolution information for a full-length virus-coded ion channel, or “viroporin,” whose essential functions represent a clinically proven class of antiviral target exploited previously for influenza A virus therapy. Specific drug-protein interactions validate an allosteric site on the channel periphery and its relevance is demonstrated by the selection of novel, structurally diverse inhibitory small molecules with nanomolar potency in culture. Hit compounds represent a 10,000-fold improvement over prototypes, suppress rimantadine resistance polymorphisms at submicromolar concentrations, and show activity against other HCV genotypes. Conclusion: This proof-of-principle that structure-guided design can lead to drug-like molecules affirms p7 as a much-needed new target in the burgeoning era of HCV DAA. (Hepatology 2014;59:408–422)

A series of bivalent ligands for a Shiga-like toxin have been synthesized, their experimentally determined inhibitory activities were compared with a simplified thermodynamic model, and computer simulations were used to predict the optimal tether length in bivalent ligands. The design of the inhibitors exploits the proximity of the C-2' hydroxyl groups of two P(k)-trisaccharides when bound to two different, neighboring carbohydrate recognizing binding sites located on the surface of Shiga-like toxin. NMR studies of the complex between the toxin and bivalent ligands show that site 2 and site 1 of a single B subunit are simultaneously occupied by a tethered P(k)-trisaccharide dimer. A simplified thermodynamic treatment provides the intrinsic affinities and binding energies for the intermolecular and intramolecular association events and permits the deconvolution of the contributions to the relative binding energies for the set of bivalent ligands. Conformational analysis based on MD simulations for bivalent galabioside dimers containing different tethers demonstrated that the calculated local concentrations of the pendant ligand at the second binding site correlate with the experimentally determined relative affinity values of the respective bivalent ligands, thereby providing a predictive method to optimize tether length.

The carbohydrate moieties of Erythrina cristagalli lectin were released as oligosaccharides by hydrazinolysis, followed by N-acetylation and reduction with NaB3H4. Fractionation of the tritium-labelled oligosaccharide mixture by Bio-Gel P-4 column chromatography and high-voltage borate electrophoresis revealed that it is composed of five neutral oligosaccharides. Structural studies by sequential exoglycosidase digestion in combination with methylation analysis and two-dimensional 'H-NMR showed that the major component was the fucosecontaining heptasaccharide Mancr3(Mancr6)(Xylfl2)Manfl4GlcNAcfl4(Fucc3)GlcNAcol. This is the first report of such a structure in plant lectins. Small amounts of the corresponding afucosyl hexasaccharide were also identified, as well as three other minor components.The structure of the heptasaccharide shows the twin characteristics of a newly established family of N-linked glycans, found to date only in plants. The characteristics are substitution of the common pentasaccharide core [Mana3(Mana6)Manfl4GlcNAcfl4GlcNAc] by (1) a D-xylose residue linked 61 -2 to the P-mannosyl residue and (2) an L-fucose residue linked a 1 4 3 to the reducing terminal N-acetylglucosamine residue.The oligosaccharide heterogeneity pattern for Erythrina cristagalli lectin was also found for the lectins from four other Erythrina species and the lectins of two other legumes, Sophora japonica and Lonchocarpus capassa.All species of the genus Erythrina tested contain Nacetyllactosamine-specific lectins that are remarkably similar in their properties [l, 21. These molecules are all glycoproteins (3 -10% neutral carbohydrate by weight) with M , approximately 60000 and are composed of two identical or nearly identical subunits. Their amino acid composition is very similar and a high degree of homology exists in the N-terminal sequences. [20] and more recently for sycamore cell culture laccase [21] and Caesalpinia pulcherrimu protease inhibitor [22].The simultaneous presence of xylose and fucose therefore appears to be a common feature of many plant glycoproteins. This combination has only been described in glycoproteins of animal origin for Helix pomatiu a-haemocyanin [23].In this paper we describe the isolation of the asparaginelinked oligosaccharides of E. cristagalli lectin, their separation by high-resolution gel permeation chromatography and highvoltage electrophoresis and their characterisation by a combination of methylation analysis, nuclear magnetic resonance and sequential exoglycosidase digestion. We also compare the oligosaccharides of E. cristagalli lectin with those of the lectins from four other species of Erythrina (E. corallodendron, E. caffra, E. latissima and E. lysistemon) and the unrelated legumes Lonchocarpus capassa and Sophora japonica.