Profiling the in vitro drug-resistance mechanism of influenza A viruses towards the AM2-S31N proton channel blockers

Rami Musharrafieh, Chunlong Ma, Jun Wang

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

The majority of human influenza A viruses currently in circulation carry the amantadine-resistant AM2-S31N channel mutation. We previously discovered a series of AM2-S31N inhibitors with potent antiviral activity against both oseltamivir-sensitive and -resistant influenza A viruses. To understand the drug-resistance mechanism of AM2-S31N inhibitors, we performed serial viral passage experiments using the influenza virus A/California/07/2009 (H1N1) to select drug-resistant AM2 mutations against two representative AM2-S31N channel blockers (1 and 2). Unlike amantadine, which gives rise to resistance after a single passage, compounds 1 and 2 selected for partially resistant viruses at passages 05 and 04 with a V27I and L26I mutation, respectively. This appears to suggest compounds 1 and 2 have a higher genetic barrier to resistance than amantadine at least in cell culture. Passage with a higher drug concentration of compound 2 selected higher level resistant viruses with a double mutant L26I + A30T. The mechanism of resistance and replication fitness for mutant viruses were evaluated by electrophysiology, reverse genetics, growth kinetics, and competition assays. AM2-S31N/V27I and AM2-S31N/L26I channels achieved similar specific proton conductance as AM2-S31N, but the AM2-S31N/L26I/A30T triple mutant had drastically reduced specific proton conductance. Viral replication fitness of AM2-S31N/V27I and AM2-S31N/L26I double mutant viruses were similar to AM2-S31N containing viruses in cell culture. However, AM2-S31N/L26I/A30T viruses displayed attenuated growth as well as inability to compete with AM2-S31N viruses. The results herein offer insight regarding the resistance mechanism of AM2-S31N inhibitors, and may help guide the design of the next-generation of AM2-S31N inhibitors with a higher genetic barrier to drug resistance.

Original languageEnglish (US)
Pages (from-to)10-22
Number of pages13
JournalAntiviral Research
Volume153
DOIs
StatePublished - May 1 2018

Keywords

  • AM2-S31N
  • Influenza
  • Isoxazole
  • Proton channel
  • Resistance

ASJC Scopus subject areas

  • Pharmacology
  • Virology

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