Discovery of Nirmatrelvir Resistance Mutations in SARS-CoV-2 3CLpro: A Computational-Experimental Approach

Abstract

The COVID-19 pandemic has emphasized the urgency for effective antiviral therapies against SARS-CoV-2. Targeting the main protease (3CLpro) of the virus has emerged as a promising approach, and nirmatrelvir (PF-07321332), the active component of Pfizer's oral drug Paxlovid, has demonstrated remarkable clinical efficacy. However, the emergence of resistance mutations poses a challenge to its continued success. In this study, we employed alchemical free energy perturbation (FEP) alanine scanning to identify nirmatrelvir-resistance mutations within SARS-CoV-2 3CLpro. FEP identified several mutations, which were validated through in vitro IC 50 experiments and found to result in 8-and 72-fold increases in nirmatrelvir IC 50 values. Additionally, we constructed SARS-CoV-2 omicron replicons containing these mutations, and one of the mutants (S144A/E166A) displayed a 20-fold increase in EC 50 , confirming the role of FEP in identifying drug-resistance mutations. Our findings suggest that FEP can be a valuable tool in proactively monitoring the emergence of resistant strains and guiding the design of future inhibitors with reduced susceptibility to drug resistance. As nirmatrelvir is currently widely used for treating COVID-19, this research has important implications for surveillance efforts and antiviral development.

In Vitro and In Silico Explorations of the Protein Conformational Changes of Corynebacterium glutamicum MshA, a Model Retaining GT-B Glycosyltransferase

“…We performed two simulations with each simulation box containing the wild-type unbound complex and the bound mutant complex. All FEP calculations were run bidirectionally with forward and reverse transformations with the average typically reported as the determined relative free energy difference. − Two copies of MshA were placed in a ∼ 180 Å cubic box, with each protein separated by ∼70 Å. This method ensures that the system is neutral while performing charge-changing mutations .…”

Exploring Cannabinoids as Potential Inhibitors of SARS-CoV-2 Papain-like Protease: Insights from Computational Analysis and Molecular Dynamics Simulations

“…This sets it apart from other clinically evaluated SARS-CoV-2 antivirals like remdesivir and molnupiravir. Nevertheless, Paxlovid is associated with side effects, necessitates administration within the initial five days of symptom onset, and carries the risk of resistance mutations [ 7 , 8 , 9 , 10 , 11 , 12 ]. Additionally, various approaches such as protein minibinders [ 13 , 14 ], peptides [ 15 , 16 , 17 ], decoy ACE2 proteins [ 18 , 19 , 20 , 21 , 22 , 23 ], monoclonal antibodies [ 24 ], and nanobodies [ 25 ] have been devised to develop antiviral therapeutics for COVID-19.…”