A Robust Proton Flux (pHlux) Assay for Studying the Function and Inhibition of the Influenza A M2 Proton Channel

Abstract

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.

Potential Viroporin Candidates From Pathogenic Viruses Using Bacteria-Based Bioassays

“…The final bacterial assay that was used measured H + conductivity. Bacteria that constitutively express a pH-sensitive green fluorescent protein—pHluorin [44], can be used to analyze the membrane permeation to H + s [46,47]. Specifically, the emission at 520 nm of pHluorin has two excitation maxima: 390 nm and 466 nm, whose ratio changes as a function of pH [44].…”

“…Specifically, the emission at 520 nm of pHluorin has two excitation maxima: 390 nm and 466 nm, whose ratio changes as a function of pH [44]. Consequently, using a calibration curve enables one to relate the fluorescent quotient directly to the H + concentration [46,47].…”

“…All of these assays had already been tested before on the best-characterized viroporin—the M2 channel from Influenza A [46,47,58,59,60]. This particular channel has the advantage that it has an effective blocker that can negate its channel activity [10], thereby demonstrating the assays’ specificity.…”

“…A liquid handling system (Tecan) was used to add 70 μL of 300 mM citric acid with 0.9% NaCl to the bacteria. The fluorescence emission of each well after the addition of acid was measured by alternate read out of the two filter pairs for 30 s. The ratio for the two differently excited emissions, scriptF=F390 nm/F466 nm was calculated and translated into proton concentration using the following equation according to [46,47]:[H+]=0.132·scriptF−1.75·scriptF0.51.…”

Put a cork in it: Plugging the M2 viral ion channel to sink influenza

“…These reports demonstrate that growth restoration assays are a sensitive, economical and technically simple technique for high throughput screening for inhibitors of M2 and presumably other viroporins. A similar expression system using E. coli has also been used to assess the properties of random M2 mutations (Santner et al, 2018b), although results from these assays do not consistently agree with results obtained by TEVC (Musharrafieh et al, 2019;Santner et al, 2018a).…”