Random Mutagenesis Analysis of the Influenza A M2 Proton Channel Reveals Novel Resistance Mutants

Paul Santner, Joao Miguel Da Silva Martins, Caroline Kampmeyer, Rasmus Hartmann-Petersen, Jonas S. Laursen, Amelie Stein, Christian A. Olsen, Isaiah T. Arkin, Jakob R. Winther, Martin Willemoës*, Kresten Lindorff-Larsen

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

The influenza M2 proton channel is a major drug target, but unfortunately, the acquisition of resistance mutations greatly reduces the functional life span of a drug in influenza treatment. New M2 inhibitors that inhibit mutant M2 channels otherwise resistant to the early adamantine-based drugs have been reported, but it remains unclear whether and how easy resistance could arise to such inhibitors. We have combined a newly developed proton conduction assay with an established method for selection and screening, both Escherichia coli-based, to enable the study of M2 function and inhibition. Combining this platform with two groups of structurally different M2 inhibitors allowed us to isolate drug resistant M2 channels from a mutant library. Two groups of M2 variants emerged from this analysis. A first group appeared almost unaffected by the inhibitor, M-089 (N13I, I35L, and F47L) and M-272 (G16C and D44H), and the single-substitution variants derived from these (I35L, L43P, D44H, and L46P). Functionally, these resemble the known drug resistant M2 channels V27A, S31N, and swine flu. In addition, a second group of tested M2 variants were all still inhibited by drugs but to a lesser extent than wild type M2. Molecular dynamics simulations aided in distinguishing the two groups where drug binding to the wild type and the less resistant M2 group showed a stable positioning of the ligand in the canonical binding pose, as opposed to the drug resistant group in which the ligand rapidly dissociated from the complex during the simulations.

Original languageAmerican English
Pages (from-to)5957-5968
Number of pages12
JournalBiochemistry
Volume57
Issue number41
DOIs
StatePublished - 16 Oct 2018

Bibliographical note

Publisher Copyright:
Copyright © 2018 American Chemical Society.

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