Structural basis of nirmatrelvir and ensitrelvir resistance profiles against SARS-CoV-2 Main Protease naturally occurring polymorphisms

Abstract

SARS-CoV-2 is the causative agent of COVID-19. Mpro is the main viral protease, with a critical role in replication and, therefore, an attractive target for antiviral drug discovery. The clinically approved drug nirmatrelvir from Pfizer, and the clinical candidate ensitrelvir from Shionogi Pharmaceuticals had so far showed great potential for treatment of viral infections. Despite the importance of new therapeutics, the broad use of antivirals is often associated with mutation selection and resistance generation. Herein, we characterized 14 naturally occurring polymorphisms that are already in circulation and are within the radius of action of these two antivirals. Nirmatrelvir retained most of its in vitro activity against most polymorphism tested, while mutants G143S and Q189K were associated with higher resistance. For ensitrelvir, higher resistance was observed for polymorphisms M49I, G143S and R188S, but not for Q189K, suggesting a distinct resistance profile difference between the two inhibitors. The crystal structures of selected polymorphism reveal the structural basis for resistance generation. Our data will assist the monitoring of potential resistant strains, support the design of combined therapy to avoid resistance, as well as assist the development of a next generation of Mpro inhibitors.

Transmissible SARS-CoV-2 variants with resistance to clinical protease inhibitors

“…Furthermore, the selection of D48G also substantiates our hypothesis that disrupting hydrogen bonds between T45 and D48 to destabilize the structure of the helix above the S2 subsite can contribute to ensitrelvir resistance (indicated by our data for T45I and D48Y). Another recent report has also identified M49I as conferring selective resistance to ensitrelvir and elegantly demonstrates the structural basis of this phenotype being due to the bulky isoleucine reorienting H41 and disrupting a base stacking interaction with the inhibitor ( 36 ). This is consistent with our finding that M49L causes greater resistance than M49I due to branching of the leucine side chain at the gamma carbon, which is closer to H41.…”

Transmissible SARS-CoV-2 variants with resistance to clinical protease inhibitors

“…Furthermore, the selection of D48G also substantiates our hypothesis that disrupting hydrogen bonds between T45 and D48 to destabilize the structure of the helix above the S2 subsite can contribute to ensitrelvir resistance (indicated by our data for T45I and D48Y). Another recent report has also identified M49I as conferring selective resistance to ensitrelvir and elegantly demonstrates the structural basis of this phenotype being due to the bulky isoleucine reorienting H41 and disrupting a base stacking interaction with the inhibitor ( 35 ). This is consistent with our finding that M49L causes greater resistance than M49I due to branching of the leucine sidechain at the gamma carbon which is closer to H41.…”