A yeast-based system to study SARS-CoV-2 M pro structure and to identify nirmatrelvir resistant mutations

Abstract

The SARS-CoV-2 main protease (M ^pro ) is a major therapeutic target. The M ^pro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As M ^pro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as a proxy for M ^pro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays showed that an E166R substitution conferred strong nirmatrelvir resistance while an E166N mutation compromised activity. On the other hand, N142A and P132H mutations caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of M ^pro E166R, and M ^pro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of M ^pro that may arise as M ^pro antivirals become more widely used.

Fig. 1.. M^pro confers a significant reduction in growth in yeast caused by decreases in a variety of cellular proteins.

A) The indicated SARS-CoV-2 genes under a galactose inducible promoter were expressed in yeast and conferred growth defects compared to empty vector (EV). B) Bar graph shows the total growth of cultures after 72 hours normalized to EV. C) Expression of the catalytically inactive M^pro C145A mutant does not confer a growth reduction and yeast grow similarly to EV control cells. D) Protein levels of the M^pro C145A mutant and wild-type M^pro (WT) are comparable. Shown are two biological replicates for each form of M^pro. E) Total protein lysates made from yeast expressing the wild-type M^pro (WT) or M^pro C145A mutant (MUT) were subjected to mass spectrometric analyses revealing 153 proteins were higher in abundance in the mutant relative to the wild-type. F) Gene Ontology (GO) analyses indicates an enrichment of proteins with functions in translation that are significantly reduced in the presence of M^pro versus M^pro C145A. Plots in A and B show averages from three biological replicates and error bars are standard deviations.

Fig. 2.. Yeast growth assays identify nirmatrelvir resistant M^pro mutants.

A) Total growth of cultures after 72 hours expressing M^pro in the presence of increasing doses of nirmatrelvir normalized to growth of yeast carrying empty vector (EV) are plotted. Nirmatrelvir is effective at protecting yeast from the toxicity of M^pro as growth is restored to levels comparable to EV. B) Yeast expressing PL^pro show a growth reduction and treatment with nirmatrelvir (nir) does not rescue the growth defect. On the other hand, treating cells with GRL0617 (GRL) restores some growth. C) Yeast expressing substitutions E166D and E166N grow as well as EV but E166R, P132H, and N142A results in significant growth reduction comparable to wild-type M^pro. D) Western analysis shows that mutants and wild-type M^pro are expressed at comparable levels. E - G) Cells expressing P132H and N142A remain sensitive to nirmatrelvir, indicated by growth recovery, but E166R appears to be resistant as there is a lack of growth even when treated with 200μM of nirmatrelvir. H) RC₅₀ measurements of each mutant in response to nirmatrelvir treatment. For all experiments, at least three biological and three technical replicates were performed for EV, wild-type and mutant M^pro. Error bars represent standard deviations.

Fig. 3.. Enzymatic assays demonstrate that M^pro E166R is highly resistant to PF-00835231, nirmatrelvir, and GC-376.

A) Michaelis–Menten plot of M^pro and its mutants with various concentrations of FRET substrate. The K_m, V_max, k_cat, and k_cat/K_m values are shown in the table on the right. B) The IC₅₀ plots of nirmatrelvir, GC-376, and PF-00835231 against M^pro, M^pro E166R, and M^pro N142A. C) K_i plots of nirmatrelvir, GC-376, and PF-00835231 against M^pro, M^pro E166R, and M^pro N142A.

Fig. 4.. Crystal structures of E166R reveals structural basis for resistance and of E166N reveals basis for inactivity.

A) Apo Mpro WT (white, PDB 7JP1) aligned with apo Mpro E166N (green, PDB 8DDI). B) Mpro WT GC376 complex (white, PDB 6WTT) aligned with Mpro E166R GC376 complex (magenta, PDB 8DDM). WT hydrogen bonds are shown as black dashes, and mutant hydrogen bonds are shown as red dashes. GC376 is shown in white for the WT structure and cyan for the mutant structure. Mutations are indicated with red text. Ser1 from an adjacent protomer is indicated with orange text.