Structure‐guided design affirms inhibitors of hepatitis C virus p7 as a viable class of antivirals targeting virion release

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)

Viroporins: structure, function and potential as antiviral targets

“…p7 channels also adopt multiple conductance states and exhibit 'burst activity', with a strong influence afforded by the membrane environment, potentially via effects on the overall channel structure. p7 from a variety of genotypes has also been shown to conduct small molecules, such as the pH-sensitive fluorophore HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid) (Wozniak et al, 2010) and carboxyfluorescein (StGelais et al, 2007), indicative of channel-pore dualism; one study recently questioned the relevance of such behaviour (Gan et al, 2014), yet indirect systems are widely utilized in viroporin studies, including by these same authors , and results faithfully and consistently reproduced those obtained for infectious HCV culture (Foster et al, 2011(Foster et al, , 2014Griffin et al, 2008;Wozniak et al, 2010). In this regard, the ability of p7 to mediate proton conductance within infected Huh7 cells remains the only activity for which a biologically relevant function has been assigned within the HCV life cycle (Wozniak et al, 2010), although roles for other observed conductances cannot be ruled out.…”

“…Viable full-length HCV containing IRES elements inserted between E2 and p7 or p7 and NS2 argued against a functional role for p7 precursors (Jones et al, 2007). Both early-and late-acting defects in virion production have been described where p7 was (partially) deleted, mutated at specific residues or treated with inhibitors (Bentham et al, 2013;Foster et al, 2011Foster et al, , 2014Jones et al, 2007;Steinmann et al, 2007a;Vieyres et al, 2013;Wozniak et al, 2010). This is now known to result from p7 performing multiple functions within infected cells, comprising distinct protein-protein interactions as well as its channel-forming activity.…”

“…p7 channel activity appears to influence a late-acting phase of the HCV life cycle, distinct from that concerning protein-protein interactions; whereas p7 deletions and deleterious point mutations abrogate infectivity in all compartments (Atoom et al, 2013;Bentham et al, 2013;Brohm et al, 2009;Jones et al, 2007;Steinmann et al, 2007a;Wozniak et al, 2010), small-molecule p7 inhibitors prevent the accumulation of secreted, but not intracellular infectivity (Foster et al, 2011(Foster et al, , 2014. Point mutations recapitulating the p7 inhibitor-induced phenotype have not been identified, yet unlike (partial) deletion mutants (Brohm et al, 2009), infectivity of HCV carrying mutations to the basic loop region (to either Ala or the less hydrophobic Glu) can be partially restored by trans-complementation with IAV M2 or by treating cells with the V-type ATPase inhibitor bafilomycin A (Bentham et al, 2013;Wozniak et al, 2010).…”

“…As the majority of intracellular HCV infectivity is known to reside in the pH-neutral ER (Gastaminza et al, 2008), p7 likely controls a secretory 'bottleneck' with relatively few virions passing through acidic compartments at a particular time. Hence, secreted rather than the bulk of cell-associated infectivity is sensitive to p7 inhibitors (Foster et al, 2011(Foster et al, , 2014. However, HCV cell-to-cell spread appears less sensitive to the effects of p7 inhibitors (Meredith et al, 2013), suggesting that this pathway may be less dependent on channel activity, albeit with genotype variability.…”

“…3): two genotype 1b monomeric structures (PDB ID: 3ZD0, 2MTS) (Cook et al, 2013;Foster et al, 2014), and a complete hexameric genotype 5a channel complex (PDB ID: 2M6X) (OuYang et al, 2013). Whilst both monomeric structures formed hairpins, protomers within the 5a structure adopted an unusual i+3 'staple-like' conformation, comprising three helical domains that interacted with three adjacent neighbours.…”