Molecular Mechanism-Driven Discovery of Novel Small Molecule Inhibitors against Drug-Resistant SARS-CoV-2 Mpro Variants.

Under the selective pressure of nirmatrelvir, a peptidomimetic covalent drug targeting SARS-CoV-2 M^pro, various drug-resistant mutations on M^pro have been acquired in vitro. Among the mutations, L50F and E166V, along with the combination of L50F and E166V, are particularly representative and pose considerable obstacles to the effective treatment of COVID-19. Our previous study identified NMI-001 and NMI-002 as novel nonpeptide inhibitors that target SARS-CoV-2 M^pro, possessing unique scaffolds and binding modes different from those of nirmatrelvir. In view of these findings, we proposed a drug design strategy aimed at rapidly identifying inhibitors that can combat mutation-induced drug resistance. Initially, molecular dynamics (MD) simulation was employed to investigate the binding mechanisms of NMI-001 and NMI-002 against the three drug-resistant mutants (M^pro_L50F, M^pro_E166V, and M^pro_L50F+E166V). Then, we conducted two phases of high-throughput virtual screening. In the first phase, NMI-001 served as a template to perform scaffold hopping-based similarity search in a library of 15,742,661 compounds. In the second phase, 968 compounds exhibiting similarity to NMI-001 were evaluated via molecular docking and MD simulations. Six compounds that may be effective against at least one mutant were identified, and five compounds were procured for conducting in vitro assays. Finally, the compound Z1557501297 (NMI-003) exhibiting inhibitory effects against the E166V (IC₅₀ = 27.81 ± 2.65 μM) and L50F+E166V (IC₅₀ = 8.78 ± 0.74 μM) mutants was discovered. The binding modes referring to NMI-003-M^pro_E166V and NMI-003-M^pro_L50F+E166V were further elucidated at the atomic level. In summary, NMI-003 reported herein is the first compound with activity against E166V and L50F+E166V, which provides a good starting point to design novel antiviral drugs for the treatment of drug-resistant SARS-CoV-2.