These results suggest that chebulagic acid restores growth in yeast cells selectively expressing M2(S31N). 2.3. adamantane-sensitive M2(S31). In contrast, chebulagic acid inhibits Cefadroxil in vitro influenza A replication regardless of M2 sequence, suggesting that it also acts on other influenza targets. Taken together, results implicate chebulagic acid and/or its hydrolysis fragments as new chemical leads for M2(S31N) and influenza-directed antiviral development. strains contain a multicopy plasmid of M2(S31N) or M2(S31) from the Udorn strain of influenza A under the control of the inducible GAL1 promoter. As a result, galactose-induced M2 expression inhibits yeast growth over time, as measured by culture turbidity. However, the co-incubation of galactose-treated cells with non-toxic inhibitors of M2 restores yeast growth. To validate the use of this assay, we induced expression of M2(S31N) in yeast in the presence of the control M2(S31N) inhibitor M2WJ352 (compound 3) or control M2(S31) inhibitor amantadine (1). After 20 hours incubation, expression of M2(S31N) reduced yeast growth Cefadroxil to 30.6 11.6% (mean SD) of the strain treated with glucose, while expression of M2(S31) reduced growth to 24.4 12.9% of glucose-treated cells. However, while the growth of the M2(S31N)-expressing yeast strain was not affected by the addition of up to 30 M amantadine (i.e., restoring < 10% of yeast growth), incubation with 30 M M2WJ352 induced an average of 27.1 8.3% increased growth relative to untreated M2(S31N)-expressing cells (Figure 3A), consistent with the selective inhibition of M2(S31N) by M2WJ352 but not amantadine. Conversely, while M2WJ352 did not substantially restore growth of M2(S31N)-expressing yeast at up to 30 M, amantadine restored growth with clear dose-dependence. For example, 0.3 M amantadine restored an average of 17.8 3.0% yeast growth in 3 independent experiments, while 10 M restored up to 37.9 5.6% growth (Figure 3B). These results are consistent with previously reported growth restoration data  and the inhibitory properties of amantadine and M2WJ352 as measured by electrophysiology [20,25]. Open in a separate window Figure 3 Ability of compounds to restore growth in yeast expressing M2. (A,B) Restoration of yeast growth in M2(S31N) (A) and M2(S31)-expressing cells (B) in the presence of Rabbit Polyclonal to CLIP1 the control M2(S31N) inhibitor M2WJ352 and control M2(S31) inhibitor amantadine. (C,D) restoration of yeast growth in M2(S31N) (C) and M2(S31)-expressing cells (D) in Cefadroxil the presence of natural products shown in Figure 2 (compounds 9C16). Effects of chebulagic acid are highlighted in green. We next assessed Cefadroxil the ability of the 8 compounds identified from the VS to restore growth of M2(S31N)-expressing yeast at 25 g/mL (Figure 3C). Two compounds (12 and 15) resulted in substantially reduced turbidity (22.1 3.9 and 70.4 0.1% reduced growth, respectively) and clear cell death as observed by microscopy and were not considered further. However, three compounds restored at least 10% yeast growth at 25 g/mL including compounds 10/agathisflavone (20.9 4.4%), 13/thiocillin I (16.9 9.2%), and 16/chebulagic acid (29.5 4.4%) (Figure 3C). These results suggest that a subset of compounds identified by VS might counteract the detrimental effects of M2(S31N) expression on yeast growth, where the activity of 25 g/mL (~26.2 M) chebulagic acid is on par with the activity of 30 M M2WJ352. Notably, none of the 8 compounds restored > 10% growth of yeast expressing M2(S31), with all observed activities within the biological noise of the assay (Figure 3D). Cefadroxil These results suggest that chebulagic acid restores growth in yeast cells selectively expressing M2(S31N). 2.3. Molecular Simulation of Chebulagic Acid with Both Wild-Type and Mutant Forms of M2 Viroporin To investigate how chebulagic acid may interact with M2, we next performed molecular docking studies with it and the M2 transmembrane domain tetramer (PDB code: 2LY0, NMR structure of residues 19C49 of M2 (H3N2) in dodecylphosphocholine micelles) . This was computationally modified to include S31 when necessary, as described in the literature . As chebulagic acid was too large to fit within the M2 pore, we assumed that only one or more portions of the molecule were functionally active. We investigated the docking of two predicted hydrolysis reaction products: the galloyl unit (P1) and the chebuloyl unit (P2) (Figure 4). P1 and P2 were separately docked to both M2(S31N) and M2(S31), and the top-ranking poses for each docked complex with the tightest.