The Substitutions L50F, E166A, and L167F in SARS-CoV-2 3CLpro Are Selected by a Protease Inhibitor In Vitro and Confer Resistance To Nirmatrelvir

Abstract

The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with a >20× increase in 50% effective concentration (EC₅₀) values for ALG-097161, nirmatrelvir (PF-07321332), PF-00835231, and ensitrelvir. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6× to 72×). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. IMPORTANCE Paxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug-resistant viruses. In order to guide the use of novel antivirals, it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid in vitro. The characteristics of in vitro antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next-generation SARS-CoV-2 3CLpro inhibitors.

Keywords: antiviral agents; coronavirus; drug resistance mechanisms; protease inhibitors.

FIG 1

Chemical structures of different 3CLpro inhibitors.

FIG 2

Passaging SARS-CoV-2-GHB (Wuhan) in VeroE6 cells in the presence of increasing concentrations of ALG-097161 (and the efflux inhibitor CP-100356). Selection was initiated at 0.4 μM. At every passage, several new cultures were started with the same concentration as well as a lower and a higher concentration. The passage with the highest compound concentration that could be maintained was selected for the following passage. At passages 5, 8, and 12, vRNA in the cell culture medium was sequenced. Substitutions in the 3CLpro that were found at these passages are indicated.

FIG 3

Enzymatic activity of WT and mutated SARS-CoV-2 3CLpro. (A) The enzymatic activity of WT 3CLpro was measured in a FRET assay. Three independent experiments were performed. The figure shows the results of one representative experiment. (B) Comparison of enzymatic activity between WT 3CLpro and mutated enzymes. Each data point represents the average of three independent experiments, and the mean and standard deviations are shown.

FIG 4

Effect of mutations on 3CLpro dimerization. (A) Initial velocities of enzyme titration of 3CLpro WT and mutant proteins were fitted as described (see Materials and Methods) to calculate the monomer dimer equilibrium dissociation constant. Three independent experiments were performed. The figure shows the results of one representative experiment. (B) 3CLpro dimerization binding affinity for each enzyme. Each data point represents the average of three independent experiments, and the mean and standard deviations are shown.

FIG 5

Predicted binding mode of ALG-097161 covalently bound to the catalytic site of WT 3CLpro and the effect of the L50F, E166A, and L167F substitutions. (A) ALG-097161 (carbon, cyan; nitrogen, navy; oxygen, red) covalently docked to WT 3CLpro (gray cartoon and surface; key side chains shown with carbon, green; nitrogen, navy; oxygen, red). (B) ALG-097161 binds covalently to the catalytic C145 and forms seven hydrogen bonds with 3CLpro: warhead to C145 in the oxyanion hole; P1 lactam to H163, E166, and F140; peptide backbone to H164 and E166; P3 indole to E166. The fused bicyclic P2 substitution maximizes Van der Waals interactions in the S2 subpocket. (C) Interaction diagram for ALG-097161 in triple mutant (L50F, E166A, L167F) 3CLpro. Interaction between the lactam moiety and the side chain of residue 166 is lost due to mutation, while other direct H-bond interactions are conserved (>90% occurrence) over a 100-ns MD run.

FIG 6

Proposed effect of the L167F substitution. Accommodation of the bulkier F167 residue results in some distortion of the binding site and in a possibly suboptimal fit. (A) Overlay of representative MD frames for WT and mutant 3CLPro. WT protein is displayed as a pale green cartoon, and the L50F E166A L167F mutant displayed as a pale blue cartoon. (B) Average distances between F185 (Cα), L167/F167 (Cα), P168 (Cα), and inhibitor. Distances were collected over 2 × 380 frames (5 ns → 100 ns simulated time). Distances between F185 and L/F167+P168 are increased following the L167F change.