Development Of A Fluorescent Method For Screening Inhibitors Of The Influenza M2 Proton Channel

Objective: Seasonal and pandemic influenza A virus infections remain major causes of severe morbidity and mortality worldwide. Since influenza A virus is under continuous antigenic mutation, adaptation, and reassortment, resulting in epidemics locally or pandemics worldwide such as 2005 H5N1(bird flu) and more recently H7N9(bird flu) of2013 have revealed a high morbidity and mortality rate.However,due to anti-influenza drug abuse, the adamantane derivatives amantadine(amt) and rimantadine(rmt)have been limited because of the rapid emergence of drug resistance. The recently emerged H7N9 was reported to show resistance against the most frequently used NA inhibitor, oseltamivir and zanamivir. Faced with this dilemma, we should speed up the development of new anti-influenza drugs against resistant influenza A virus.In this study, we selected influenza A virus M2 proton channel as a research direction and design model of M2 proton channel target in vitro.To aid in the search of new anti-flu drugs targeting the resistant viruses, we are developing a fluorescent based screening method. Drug binding promotes M2 tetramerization in detergents; while change in the M2 association states has been proven to be trackable with the fluorescence polarization change of its properly labeled fluorescent tag. Currently we are optimizing this method in order to develop a high-throughput screen platform eventually.Method:A fluorescent method for screening inhibitors of the influenza M2 proton channel. In our study, M2 TM peptide(residues22-46, C-terminally amidated) were synthesized on Rink Amide resin using Fmoc-amino acids with protected side chain. The NBD fluorophore was labeled to N-terminal of M2 TM peptide when M2 TM peptide were finished.Firstly, the analysis byanalytical ultracentrifugation(AUC) proved NBD fluorophoreaffect the formation M2 TM tetramer and M2 TM monomer-tetramer equilibrium. Then, NBD-M2 TM monomer-tetramer equilibrium was determined by fluorescent screening method in seven detergents and best Kd value of detergent was selected for titration of amt in NBD-M2 TM.Results: 1) AUC results demonstrated NBD fluorophore did not affect the formationof M2 TM tetramerization 2) Fluorescent screening method could determine NBD-M2 TM monomer-tetramer equilibrium.3) amt binding promoted NBD-M2 TM equilibrium change from monomer to tetramer.Conclusions:1) We develop a fluorescent method that can monitor association states of transmembrane peptides/proteins.2) This method can be used to monitor drug binding to M2 TM tetramer. We hope this method can develop a high-throughput screen platform eventually.