Evolutionary and Structural Insights about Potential SARS-CoV-2 Evasion of Nirmatrelvir

Abstract

The U.S. FDA approval of PAXLOVID, a combination therapy of nirmatrelvir and ritonavir has significantly boosted our morale in fighting the COVID-19 pandemic. Nirmatrelvir is an inhibitor of the main protease (M^Pro) of SARS-CoV-2. Since many SARS-CoV-2 variants that resist vaccines and antibodies have emerged, a concern of acquired viral resistance to nirmatrelvir naturally arises. Here, possible mutations in M^Pro to confer viral evasion of nirmatrelvir are analyzed and discussed from both evolutionary and structural standpoints. The analysis indicates that those mutations will likely reside in the whole aa45-51 helical region and residues including M165, L167, P168, R188, and Q189. Relevant mutations have also been observed in existing SARS-CoV-2 samples. Implications of this analysis to the fight against future drug-resistant viral variants and the development of broad-spectrum antivirals are discussed as well.

Figure 1:

M^Pro regions that interact with nirmatrelvir and YH-53. (A) The overlay of five published M^Pro-nirmatrelvir complex structures. PDB entries are 7RFS (gray), 7RFW (blue), 7SI9 (yellow), 7TE0 (green), and 7VH8 (purple). Residues that display large variations are labeled. (B) Five M^Pro regions and the residue H172 that interact directly with nirmatrelvir. (C) Six M^Pro regions and the residue H172 that interact directly with YH-53. The structure is based on the pdb entry 7E18.

Figure 2:

Sequence alignment of M^Pro proteins from the betacoronavirus genus. The Smith-Waterman alignment was conducted using the program SnapGene. The sequence conservation is shown in colored bars where high dark colored bars indicate high conservation, low light colored bars low conservation, and black flat lines close to no conservation. Regions that interact directly with nirmatrelvir and YH-53 are labeled. The hinge that connects the NTD and the CTD and the CTD are labeled as well.

Figure 3:

Residues involved in direct interactions between M^Pro and two ligands nirmatrelvir and S-217622. (A) M^Pro residues involved in the recognition of the P1 residue and the covalent warhead of nirmatrelvir. (B) M^Pro residues that interact with the P2 residue of nirmatrelvir. (C) M^Pro residues that interact with the P3 residue and the N-trifluoroacetamide cap of nirmatrelvir. Images in A-C are based on the PDB entry 7TE0. (D) M^Pro residues that form a recognition pocket for the benzothiazole compound of YH-53.

Figure 4:

Mutation counts at different M^Pro sites from sequenced SARS-CoV-2 genomes that have been deposited in GISAID. 800,721 mutations were identified in 6,242,921 sequenced M^Pro entries. (A) Distribution of mutations on all M^Pro sites. Three sites with most mutations are labeled. (B) Distribution of mutations on aa1–60. (C) Distribution of mutations on aa135–195. In B and C, M^Pro regions and site that interact directly with nirmatrelvir and YH-53 are colored in red.

Scheme 1:

Structures of nirmatrelvir and YH-53