Structural and virologic mechanism of the emergence of resistance to Mpro inhibitors in SARS-CoV-2

Significance The present X-ray crystallographic and virologic study demonstrates how SARS-CoV-2's main protease (Mpro) inhibitor resistant variants emerge when SARS-CoV-2 is propagated in VeroE6TMPRSS2 cells under inhibitors’ selection pressure. We found that E166V substitution acquired in Mpro (MproE166V) disrupts the presumed Mpro-protomer-dimerization-initiating Ser1’-Glu166 interactions, loosens nirmatrelvir’s binding to Mpro’s active site, and compromises nirmatrelvir’s anti-SARS-CoV-2 activity. However, TKB245, a fluorinated-benzothiazole-containing Mpro inhibitor, stays bound to MproE166V’s active site and exerts substantial activity against MproE166V-carrying SARS-CoV-2, while that activity is less than that against the wild-type virus. When propagated with another Mpro inhibitor (5h), SARS-CoV-2 acquired A191T, became resistant to 5h, and gained improved replicability. The data shed light on developing more potent agents against SARS-CoV-2 strains including drug-resistant variants.


Fig. S3
. L50F substitution in M pro does not affect the size or shape of M pro 's binding pocket for nirmatrelvir.(A) Superimposition of crystal structures of M pro WT (green), M pro E166V (yellow), and M pro with two substitutions (M pro L50F/E166V in cyan) in complex with nirmatrelvir (cyan).Note that when E166V is present, nirmatrelvir is not complexed with M pro E166V.Nirmatrelvir is illustrated with surrounding residues in stick form.The active site residues occupy nearly identical positions with minimal deviations among the three structures.The PDB IDs are: M pro WT (7VH8), M pro E166V (8H82), and M pro L50F/E166V (8H5P).(B) Superimposition of the three crystal surfaces: M pro E166V (in yellow) and M pro L50F/E166V (cyan) in complex with nirmatrelvir.The surface representation of the binding pocket also exhibits virtually identical features.The L50F substitution, located adjacent to the binding pocket, predominantly interacts closely with Q189 but does not induce any discernible alterations in its position.The PDB IDs are 8H82 for M pro E166V and 8H5P for M pro L50F/E166V.
L50F substitution in M pro compensates for the reduced replication fitness of SCoV2E166V.Replication profiles of rgSCoV2E166V (blue) and rgSCoV2L50F/E166V (red) were examined in CSRA.Viral RNA extracted from supernatants at the end of each passage was subjected to Sanger sequencing, and the proportions of Leu and Phe at position 50 in M pro s were determined.Cell-based antiviral assays were conducted using M P I-selected SCoV2 variants.EC50 values for each compound were determined using % reduction in viral RNA copy numbers in the no-drug controls and represent averages ± one-standard deviations obtained from at least three independent experiments.Target cells used were VeroE6 TMPRSS2 and Hela hACE2/TMPRSS2 cells.In the enzymatic analyses, recombinant M pro preparations were employed.Fold-changes in parentheses denote the values compared to the control values.
Table S2.Enzymatic characterization of recombinant M pro .
Fold-change of kcat/Km is defined as a ratio of kcat/Km for wild-type over kcat/Km for recombinant M pro E166V or M pro A191T.

Fig. S4 .
Fig. S4.A191T substitution does not affect the Ser1' and Glu166 interactions critical for M pro protomer dimerization.(A) X-ray crystal structure of M pro A191T complexed with 5h (yellow sticks) featuring the dimer interface of the enzyme.Note that A191T substitution does not affect the Ser1'-Glu166 interactions.(B) The tight hydrogen bond interaction network (black dashed lines) formed is associated with 5h.The location of Thr191 is distant from the network as shown in (A) and is not seen in this figure.

Fig. S5 .
Fig. S5.Species of M pro or M pro -compound complexes observed by native MS.Species of M pro or M pro -compound complexes observed in native MS seen in Fig. 3 were identified by the comparison between each deconvoluted mass from measured spectra and corresponding theoretical mass.* Mean values of deconvoluted masses determined using at least three charge states.

Table S3 . Antiviral profiles of compounds against recombinant SCoV2 mutants.
EC50 values for each compound were determined using % reduction in viral RNA copy numbers from the no-drug control and represent average and one standard deviation obtained from three independent experiments.The target cells used were VeroE6 TMPRSS2 cells.

Table S4 . Data collection and refinement statistics (molecular replacement)
Values in parentheses are for the highest-resolution shell.