Table 1.

Alanine scanning mutagenesis with FEP (kcal/mol) identifies nirmatrelvir resistance mutations.^a

Mutation	3CLpro-nirmatrelvir ΔGBound statec	3CLpro ΔGFree statec	ΔΔGFEPb
M49A	2.35 ± 0.09	2.24 ± 0.10	0.11
Y54A	12.37 ± 0.17	9.41 ± 0.10	2.96
F140A	−9.13 ± 0.15	−7.62 ± 0.04	−1.52
L141A	12.17 ± 0.07	11.71 ± 0.07	0.46
N142A	77.32 ± 0.04	77.57 ± 0.03	−0.25
S144A	−2.85 ± 0.08	−5.77 ± 0.08	2.92
H164A	18.92 ± 0.10	21.10 ± 0.12	−2.18
M165A	2.32 ± 0.09	2.63 ± 0.06	−0.31
E166A	109.68 ± 0.10	108.38 ± 0.12	1.30
L167A	14.81 ± 0.10	14.81 ± 0.11	0.002
H172A	21.16 ± 0.13	21.11 ± 0.16	0.06
D187A	131.50 ± 0.11	133.39 ± 0.18	−1.88
R188A	267.10 ± 0.12	267.47 ± 0.18	−0.36
Q189A	56.59 ± 0.07	56.15 ± 0.07	0.44
T190A	18.08 ± 0.05	18.51 ± 0.04	−0.42
Q192A	56.53 ± 0.06	57.02 ± 0.06	−0.50

^aWhen ${\text{ΔΔG}}_{\text{FEP}}>0$ mutations to alanine are less favorable in the bound state than in the free state.

^b${\text{ΔΔG}}_{\text{FEP}}$ is calculated where ${\text{ΔΔG}}_{\text{FEP}}={\text{ΔG}}_{\text{Bound}\text{state}}-{\text{ΔG}}_{\text{free}\text{state}}$. All units are in kcal/mol.

^cStatistical uncertainties calculated for bidirectional (i.e., forward and backward transformation) FEP calculations using the BAR estimator are all between ± 0.03 to ± 0.18 kcal/mol.