The M2 protein is an important target for drugs in the fight against the influenza virus. Because of the emergence of resistance against antivirals directed toward the M2 proton channel, the search for new drugs against resistant M2 variants is of high importance. Robust and sensitive assays for testing potential drug compounds on different M2 variants are valuable tools in this search for new inhibitors. In this work, we describe a fluorescence sensor-based assay, which we termed "pHlux", that measures proton conduction through M2 when synthesized from an expression vector in Escherichia coli. The assay was compared to a previously established bacterial potassium ion transport complementation assay, and the results were compared to simulations obtained from analysis of a computational model of M2 and its interaction with inhibitor molecules. The inhibition of M2 was measured for five different inhibitors, including Rimantadine, Amantadine, and spiro type compounds, and the drug resistance of the M2 mutant variants (swine flu, V27A, and S31N) was confirmed. We demonstrate that the pHlux assay is robust and highly sensitive and shows potential for high-throughput screening.
Bibliographical noteFunding Information:
Christian A. Olsen: 0000-0002-2953-8942 Jakob R. Winther: 0000-0001-6995-9154 Martin Willemoes:̈ 0000-0003-1689-2712 Kresten Lindorff-Larsen: 0000-0002-4750-6039 Funding This research was supported by a Sapere Aude Starting Grant from the Danish Council for Independent Research (12-126214) and grants from the Eva og Henry Frænkels Mindefond and the Lundbeck Foundation to K.L.-L. Notes The authors declare no competing financial interest. Mutant enzymes are abbreviated according to the position of the changed residue, e.g., S31N indicating a single-amino acid alteration at position 31 from serine to asparagine.
Copyright © 2018 American Chemical Society.