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

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.

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).…”