Joseph Rollie

Following the COVID19 pandemic’s onset, the oral drug PAXLOVID (Food and Drug Administration, 2021), with the active ingredient nirmatrelvir, was developed (Owen et al., 2021). Nirmatrelvir works by inhibiting the main protease (Mpro) of SARS-CoV-2 (Owen et al., 2021), which is needed for the virus to advance in its life cycle (Arya et al., 2021). Naturally, there are concerns that the virus could evolve resistance to this drug; the goal of this experiment was to assess a set of mutations suspected of granting nirmatrelvir resistance to Mpro. Kinetics assays using a polypeptide substrate bound to a fluorophore and a quencher were used to measure the catalytic activity of Mpro variants; Differential scanning fluorimetry (DSF) was used to assess the melting temperature and stability of Mpro variants. With these techniques, the suspected resistance mutations deletion of P168 (ΔP168) and E166V, along with suspected stabilizing mutations T21I, L50F, and S144A, were investigated. The kinetics results indicated that the Mpro variants ΔP168/S144A and E166V/T21I/L50F/S144A display some nirmatrelvir resistance at higher drug concentrations, and DSF results indicated that E166V/T21I/L50F/S144A displays essentially equal stability to wild type Mpro. The hope for this research is to identify possible ways SARS-CoV-2 could evolve in the aftermath of the PAXLOVID roll-out in order to find measures to prepare for the possibility of resistance mutations arising in the circulating viral strains.