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 > 20x increase in EC50 values for ALG-097161, nirmatrelvir (PF-07321332) and PF-00835231. 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-fold). 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.
Competing Interest Statement
The authors declare the following financial interests / personal relationships which may be considered as potential competing interests: - Koen Vandyck and Pierre Raboisson are employees of Aligos Belgium BV - Cheng Liu, Antitsa Stoycheva, Sarah K Stevens, Chloe De Vita, Andreas Jekle, Lawrence M Blatt, Leonid Beigelman, Julian A Symons and Jerome Deval are employees of Aligos Therapeutics, Inc. - A patent application on ALG-097161 is pending
Footnotes
↵* Shared Last authorship
