Molecular Mechanisms of Drug Resistance and Compensation in SARS-CoV-2 Main Protease: The Interplay Between E166 and L50
Sarah N Zvornicanin, Ala M Shaqra, Julia Flynn, Heidi Carias Martinez, Weiping Jia, Stephanie Moquin, Dustin Dovala, Daniel N Bolon, Nese Kurt Yilmaz, Celia A Schiffer
doi:10.1101/2025.01.24.634813
The SARS-CoV-2 main protease (M pro ) is essential for viral replication, and a primary target for COVID-19 antivirals. Direct-acting antivirals such as nirmatrelvir, the active component of Paxlovid, target the M pro active site to block viral polyprotein cleavage and thus replication. However, drug resistance mutations at the active site residue Glu166 (E166) have emerged in in vitro selection studies, raising concerns about the durability of current antiviral strategies. Here, we investigate the molecular basis of drug resistance conferred by E166A and E166V mutations against nirmatrelvir and the related PF-00835231, individually and in combination with the distal mutation L50F. We found that E166 mutations reduce nirmatrelvir potency by up to 3000-fold while preserving substrate cleavage, with catalytic efficiency reduced by only up to 2fold. This loss of catalytic efficiency was compensated for by the addition of L50F in the doublemutant variants. We have determined three cocrystal structures of the E166 variants (E166A, E166V, and E166V/L50F) bound to PF-00835231. Comparison of these structures with wildtype demonstrated that E166 is crucial for dimerization and for shaping the substrate-binding S1 pocket. Our findings highlight the mutability of E166, a prime site for resistance for inhibitors that leverage direct interactions with this position, and the potential emergence of highly resistant and active variants in combination with the compensatory mutation L50F. These insights support the design of inhibitors that target conserved protease features and avoid E166 sidechain interactions to minimize susceptibility to resistance.
Enzyme inhibition assays Inhibition assays were performed in the same assay buffer (50 mM Tris pH 7.5, 50 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), but with only 1% DMSO. To determine the inhibition constant K i , enzyme was incubated at room temperature with increasing concentrations of nirmatrelvir or PF-00835231 for 1 hour in assay buffer. The enzymatic reaction was initiated with 40 μM protease FRET substrate and monitored using a PerkinElmer Envision plate reader. At least three replicates were performed for each inhibitor concentration. The initial velocity for each reaction was calculated by linear regression. The K i was calculated by plotting the initial velocity (RFU/s) at each inhibitor concentration (µM) and then fit to the Morrison equation, using each enzyme's respective K M , in GraphPad Prism 10 28 software. Enzyme levels were adjusted according to the K i estimates from initial experiments to maintain [E]/K i <100 to ensure confidence in K i determinations. In cases of very potent inhibition where the enzyme concentration could not be decreased further due to loss of signal, [E]/100 is reported as the upper estimate for K i as the assay detection limit.
Native Mass Spectrometry Each protein was purified in SEC buffer (25 mM HEPES pH 7.5, 150 mM NaCl, and 1 mM TCEP) and then diluted to 2-4 mg/mL in 50-100 µL in the same buffer. The protein was dialyzed for three hours into 200 mM ammonium acetate (pH 6.8) using a..
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doi:10.3390/v15030781DOI record:
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