tetano
Editor, Senior Moderator
Biochemistry. 2018 Sep 19. doi: 10.1021/acs.biochem.8b00721. [Epub ahead of print]
[h=1]A robust proton flux (pHlux) assay for studying function and inhibition of the Influenza A M2 proton channel.[/h] Santner P, Martins JMDS, Laursen JS, Behrendt L, Riber L, Olsen CA, Arkin IT, Winther JR, Willemo?s M, Lindorff-Larsen K.
[h=3]Abstract[/h] The M2 protein is an important target for drugs in the fight against the influenza virus. Due to the emergence of resistance against antivirals directed towards the M2 proton channel, the search for new drugs against resistant M2 variants is of high importance. Robust and sensitive assays to test potential drug compounds on different M2 variants are valuable tools in this search for new inhibitors. In the present 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 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 both robust, has high sensitivity and shows a potential for high-throughput screening.
PMID: 30230312 DOI: 10.1021/acs.biochem.8b00721
[h=1]A robust proton flux (pHlux) assay for studying function and inhibition of the Influenza A M2 proton channel.[/h] Santner P, Martins JMDS, Laursen JS, Behrendt L, Riber L, Olsen CA, Arkin IT, Winther JR, Willemo?s M, Lindorff-Larsen K.
[h=3]Abstract[/h] The M2 protein is an important target for drugs in the fight against the influenza virus. Due to the emergence of resistance against antivirals directed towards the M2 proton channel, the search for new drugs against resistant M2 variants is of high importance. Robust and sensitive assays to test potential drug compounds on different M2 variants are valuable tools in this search for new inhibitors. In the present 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 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 both robust, has high sensitivity and shows a potential for high-throughput screening.
PMID: 30230312 DOI: 10.1021/acs.biochem.8b00721