Flunarizine prevents hepatitis C virus membrane fusion in a genotype‐dependent manner by targeting the potential fusion peptide within E1

Abstract

To explore mechanisms of hepatitis C virus (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action. Conclusion: These observations reveal novel details about HCV membrane fusion. Moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as cost-effective component of HCV combination therapies.

Clinically Approved Ion Channel Inhibitors Close Gates for Hepatitis C Virus and Open Doors for Drug Repurposing in Infectious Viral Diseases

“…In fact, administration of flunarizine only during a 5-min-long wash with low-pH buffer was necessary and sufficient to inhibit HCV infection (7). Moreover, using singleparticle-tracking live-cell imaging, we observed that, in the presence of flunarizine, HCV particles readily reached cellular tight junctions and were internalized into endosomes but that only membrane fusion was inhibited (7). Since viral flunarizine resistance mutations, which map to residues M267 and Q289 of E1 and M405 of E2 (J6 isolate, GT2a), confer cross-resistance to pimozide, fluphenazine, and trifluoperazine, it can be assumed that these related diphenyl-piperidines and phenothiazines share a mode of action, which, given the findings described above, is unlikely to be inhibition of endocytosis (7).…”

“…Moreover, pimozide inhibits T-type Ca 2ϩ channels much more effectively than flunarizine (38) but inhibits HCV about as much as flunarizine (7). Beyond this, other potent Ca 2ϩ channel inhibitors, like mibefradil (41), penfluridol (42), and NiCl 2 (43), do not inhibit HCV, and chelation of intracellular or extracellular Ca 2ϩ does not influence the antiviral activity of flunarizine (7). Taken together, these observations indicate that the anti-HCV activities of chlorcyclizine and flunarizine (and likely also of compounds related to these molecules) are independent of the inhibition of H 1 histamine receptors, dopamine receptors, and Ca 2ϩ ion channels.…”

“…called the NCGC Pharmaceutical Collection (NPC) (6), and a small group of clinically approved ion channel inhibitors used to treat cardiac or neurologic conditions (7). These independent studies identified several structurally related compounds as potent HCV cell entry inhibitors both in vitro and in vivo.…”

“…Along the same lines, dopamine receptors and T-type Ca 2ϩ ion channels (CACNA1G, CACNA1H, CACNA1I) are not (or are poorly) expressed in Huh-7.5 cells (Ͻ0.18 reads per kilobase per million [RPKM]) (data not shown; T. Pietschmann, unpublished observation), in primary hepatocyte transcriptional profiles, and in human livers (the Human Protein Atlas [http://www.proteinatlas.org]). Moreover, pimozide inhibits T-type Ca 2ϩ channels much more effectively than flunarizine (38) but inhibits HCV about as much as flunarizine (7). Beyond this, other potent Ca 2ϩ channel inhibitors, like mibefradil (41), penfluridol (42), and NiCl 2 (43), do not inhibit HCV, and chelation of intracellular or extracellular Ca 2ϩ does not influence the antiviral activity of flunarizine (7).…”

“…Endocytosis is thought to be critical for HCV infection across all strains and genotypes (10,11). However, the latter drugs inhibit HCV in a highly straindependent manner, with 50% inhibitory concentration (IC 50 ) values varying more than 10-fold between individual strains (7). Moreover, at least flunarizine was shown to inhibit HCV cell entry independently of endocytosis.…”