Table 2. Effects of M2 Mutants on Drug Binding Investigated by Reverse Genetics, EP, Antiviral Assays, and MD Simulations.

M2 variantsa	% inhibition (2 min, 100 μΜ)b	Kd (μΜ)c	EC50 (μΜ)d	RMSDproteine	RMSDligandf	res27–res27	ΔGeffg
S31N	54.5 ± 2.6	5.5 ± 0.3	0.9 ± 0.1	3.17 ± 0.33	2.37 ± 0.40	5.94 ± 1.19	–51.44 ± ± 2.49
S31N V27A	8.1 ± 2.0	240.7 ± 61.8	>30	3.42 ± 0.38	2.14 ± 0.35	8.29 ± 0.94	–44.02 ± 4.30
S31N V27F	20.4 ± 5.4	>300	>30	3.43 ± 0.58	2.62 ± 0.33	8.71 ± 0.91	–47.40 ± 3.59
S31N I32N	72.2 ± 2.2	20.6 ± 5.7	4.9 ± 2.7	2.28 ± 0.22	2.83 ± 0.56	6.68 ± 1.43	–52.46 ± 4.17
S31N G34E	28.2 ± 27.5	n.a.	>30	4.31 ± 0.54	4.31 ± 0.54	12.69 ± 2.13	–47.10 ± 3.48
S31N R45H	33.7 ± 1.3	118.3 ± 22.2	6.2 ± 2.4	3.52 ± 0.60	3.11 ± 0.59	8.39 ± 3.82	–43.51 ± 3.39

^aRecombinant viruses were generated using A/Udorn/1972 (H3N2) background via reverse genetics.

^bEP studies use A/California/07/2009 (H1N1) M2 (S31N protein) variants; mean ± SEM calculated at 2 min of drug perfusion.

^cCalculated from the ratio, k_off/k_on.

^dPlaque reduction assay.

^eMean ± SD (Å). Protein RMSD is calculated for the C_α atoms of the α-helices, for the last 50 ns of the trajectories. Frame 0 is used as reference structure.

^fMean ± SD (Å). Ligand RMSD is calculated after superposition of each protein–ligand complex to that of frame 0 (reference structure) based on the Cα atoms of the protein, for the last 50 ns of the trajectories.

^gEffective binding free energy calculated as ΔG_eff = ΔE_MM + ΔG_sol (see Methods section).