Recent changes in the mutational dynamics of the SARS-CoV-2 main protease substantiate the danger of emerging resistance to antiviral drugs

Abstract

Introduction: The current coronavirus pandemic is being combated worldwide by nontherapeutic measures and massive vaccination programs. Nevertheless, therapeutic options such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main-protease (M^pro) inhibitors are essential due to the ongoing evolution toward escape from natural or induced immunity. While antiviral strategies are vulnerable to the effects of viral mutation, the relatively conserved M^pro makes an attractive drug target: Nirmatrelvir, an antiviral targeting its active site, has been authorized for conditional or emergency use in several countries since December 2021, and a number of other inhibitors are under clinical evaluation. We analyzed recent SARS-CoV-2 genomic data, since early detection of potential resistances supports a timely counteraction in drug development and deployment, and discovered accelerated mutational dynamics of M^pro since early December 2021.

Methods: We performed a comparative analysis of 10.5 million SARS-CoV-2 genome sequences available by June 2022 at GISAID to the NCBI reference genome sequence NC_045512.2. Amino-acid exchanges within high-quality regions in 69,878 unique M^pro sequences were identified and time- and in-depth sequence analyses including a structural representation of mutational dynamics were performed using in-house software.

Results: The analysis showed a significant recent event of mutational dynamics in M^pro. We report a remarkable increase in mutational variability in an eight-residue long consecutive region (R188-G195) near the active site since December 2021.

Discussion: The increased mutational variability in close proximity to an antiviral-drug binding site as described herein may suggest the onset of the development of antiviral resistance. This emerging diversity urgently needs to be further monitored and considered in ongoing drug development and lead optimization.

Keywords: COVID-19; Mpro; Paxlovid; SARS-CoV-2; drug resistance; main protease; nirmatrelvir; viral evolution.

FIGURE 1

Time-resolved mutation dynamics of M^pro. (A) Logarithmic abundance of single-point amino-acid exchanges at position A193, as a representative for the residues in the region of interest, within the total set of 10.5 million M^pro sequences. (B) Logarithmic daily infection numbers (dark blue) reported by WHO (https://covid19.who.int/data, accessed on 30 June 2022) in connection with the increase in total (summed over all positions and possible exchanges) unique amino-acid exchanges (gray), with a 7-day running average in red. (C) Number of unique amino-acid exchanges for all 306 positions in the M^pro sequence. Positions R188-G195 (solid) and adjacent residues V186, D187, T196, and D197 (dotted) are highlighted with bold lines.

FIGURE 2

Mutation dynamics in the M^pro amino-acid sequence. (A) Final distribution of unique amino-acid exchanges for every position within the wild-type M^pro sequence including all sequences with a corresponding collection date before December 2021 (transparent multi-colored bars). Associated surprisal indices and Shannon entropies are superimposed in light and dark gray, respectively. (B) As in panel (A), including all sequences with corresponding collection dates up until 22 June 2022, with associated surprisal indices and Shannon entropies, which were calculated from sequences collected from 1 December 2021 to 22 June 2022. (C) Average rise in unique amino-acid exchanges along the protein sequence including sequences collected before (blue) and starting from (red) 1 December 2021. Insert: Zoom in on positions R188-G195 and adjacent residues V186, D187, T196, and D197. The sequence position, complemented by structural properties partly obtained from PDB entry 7SI9 (43), is displayed on the x-axis.

FIGURE 3

Structural representation of mutation dynamics. (A) Structure of M^pro [PDB entry 7SI9 (43)] represented as a putty figure showing the cumulative number of unique amino-acid exchanges along the protein sequence including sequences collected before 1 December 2021, referring to Figure 2A (#uaae). The active-site cavity is colored by the number of unique amino-acid exchanges of its surrounding residues (color bar). (B) As in panel (A) but including only sequences collected starting from 1 December 2021, referring to Figure 2B (#uaae). (In between panels A, B) Zoom in on the active-site cavity, including the inhibitor nirmatrelvir [PDB 7SI9 (43)]. Additionally, we provide a movie in the Supplementary Movie 1, showing the cumulative unique amino-acid exchanges per day from 24 December 2019 to 22 June 2022.