The severe acute respiratory syndrome coronavirus 2 main protease (SARS-CoV-2-Mpro)
plays an essential role in viral replication, transcription, maturation, and entry into host cells. Furthermore, its cleavage specificity for viruses, but not humans, makes it a promising drug target for the
treatment of coronavirus disease 2019 (COVID-19). In this study, a fragment-based strategy including potential antiviral quinazolinone moiety and glutamine- or glutamate-derived peptidomimetic
backbone and positioned nitro functional groups was used to synthesize putative Mpro inhibitors.
Two compounds, G1 and G4, exhibited anti-Mpro enzymatic activity in a dose-dependent manner,
with the calculated IC50 values of 22.47 ± 8.93 µM and 24.04 ± 0.67 µM, respectively. The bio-layer
interferometer measured real-time binding. The dissociation kinetics of G1/Mpro and G4/Mpro
also showed similar equilibrium dissociation constants (KD) of 2.60 × 10-5 M and 2.55 × 10-5 M,
respectively, but exhibited distinct association/dissociation curves. Molecular docking of the two
compounds revealed a similar binding cavity to the well-known Mpro inhibitor GC376, supporting
a structure-function relationship. These findings may open a new avenue for developing new
scaffolds for Mpro inhibition and advance anti-coronavirus drug research.