English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

Hepatitis C virus (HCV) infection, which causes hepatitis C and can chronically lead to serious and life-threatening diseases including liver cirrhosis and hepatocellular carcinoma (Lauer and Walker, 2001), is a rising global health problem. More than 170 million people are infected by HCV worldwide and 3–4 million people are infected each year. No effective vaccines are available to prevent HCV infection. Moreover, HCV is a fast mutating RNA virus with seven distinct genotypes and many subtypes within each genotype. The high degree of genetic diversity can lead to further viral resistance to the current therapies within individual patients (Li et al., 2012). Hence, there remains a strong desire in the medical community to explore new therapeutic opportunities. p7, the only viroporin encoded by the HCV genome, is a 63-residue protein that oligomerizes in membrane to form ion channels with cation selectivity (Moradpour and Penin, 2013). The p7 channel has been shown to facilitate efficient assembly and release of infectious virions, thus has been sought after as a potential anti-HCV drug target. Several inhibitors have been found to inhibit p7 channel activity with varying efficacies, including the amantadine and rimantadine (Griffin et al., 2003) that also block the influenza M2 channel (Pielak et al., 2011), hexamethylene amiloride (HMA) (Premkumar et al., 2004), and the long-alkyl-chain iminosugar derivatives (Pavlovic et al., 2005). Another novel small molecule known as BIT225, which has partial structural resemblance to HMA, also inhibits p7 activity and has been pursued as an anti-HCV drug candidate in clinical trials (Luscombe et al., 2010). For many years, the inhibition mechanism of the known p7 inhibitors remained elusive in the absence of a highresolution p7 structure. Previous solution NMR studies revealed that the adamantane compounds bind to six equivalent hydrophobic pockets (due to the 6-fold symmetry of the p7 hexamer) near the kink of the central pore-forming helices, consisting of elements from different helical segments and from different subunits (OuYang et al., 2013). A more recent study showed that insertion of rimantadine into these pockets could function as “molecular wedge” that attenuates conformational breathing needed for cation flow through the N-terminal constriction of the p7 channel (Dev et al., 2015). In addition to the studies on the p7 hexamer, a combined NMR and modeling study modeled the binding of rimantadine and several new compounds in the context of a p7 heptamer complex (Foster et al., 2014). Compared to rimantadine, HMA has a completely different chemical structure and was reported to inhibit p7 channel in different genotypes with stronger inhibition at similar drug concentration (Griffin et al., 2008). Earlier NMR titrations showed that HMA caused larger chemical shift perturbations in the loop region, while amantadine and NN-DNJ have a larger effect on the chemical shifts of the terminal regions (Cook et al., 2013). Therefore, HMA may act on a different site of p7 than rimantadine, and it is of substantial interest to identify this binding site. Here, we capitalized on the NMR system of the p7 channel from genotype 5a, designated p7 (5a) and used a combination of NMR titration and nuclear Overhauser enhancement (NOE) experiments to characterize HMA binding to the p7 channel. We used 100 μmol/L p7 (5a) (monomer concentration) solubilized in 20 mmol/L DPC and recorded good quality H-C HSQC spectra of the methyl groups and H-N TROSY-HSQC spectra of the backbone amine at various drug concentrations. NMR titration experiment showed that titrating the p7 channel with HMA had no effects on the methyl groups of Val25 and Val26 (Fig. 1A), which are key constituents of the rimantadine binding pocket (OuYang et al., 2013). In contrast, rimantadine titration strongly perturbed the Val25 resonance (Fig. 1A). Remarkably, HMA titration induced chemical shift changes at the N-terminal constriction of the channel (marked by Val5 and Ile6), very similar to those caused by rimantadine binding. The methyl HSQC spectra show that the two different compounds perturbed the Cγ1H3 of Val5 (Fig. 1B) and the Cδ1H3 of Ile6 (Fig. 1C) with essentially the same pattern. Moreover, the overall chemical shift change patterns in the H-N TROSY-HSQC spectra induced by HMA and rimantadine are also very similar (Fig. S1). We then performed more direct experiments to accurately pinpoint the HMA binding site by measuring protein-drug

Structural basis of interaction between the hepatitis C virus p7 channel and its blocker hexamethylene amiloride