Identification of SARS-CoV-2 Main Protease Inhibitors from a Library of Minor Cannabinoids by Biochemical Inhibition Assay and Surface Plasmon Resonance Characterized Binding Affinity

The replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its main protease (M^pro), which is a plausible therapeutic target for coronavirus disease 2019 (COVID-19). Although numerous in silico studies reported the potential inhibitory effects of natural products including cannabis and cannabinoids on SARS-CoV-2 M^pro, their anti-M^pro activities are not well validated by biological experimental data. Herein, a library of minor cannabinoids belonging to several chemotypes including tetrahydrocannabinols, cannabidiols, cannabigerols, cannabichromenes, cannabinodiols, cannabicyclols, cannabinols, and cannabitriols was evaluated for their anti-M^pro activity using a biochemical assay. Additionally, the binding affinities and molecular interactions between the active cannabinoids and the M^pro protein were studied by a biophysical technique (surface plasmon resonance; SPR) and molecular docking, respectively. Cannabinoids tetrahydrocannabutol and cannabigerolic acid were the most active M^pro inhibitors (IC₅₀ = 3.62 and 14.40 μM, respectively) and cannabigerolic acid had a binding affinity KD=2.16×10−4 M). A preliminary structure and activity relationship study revealed that the anti-Mpro effects of cannabinoids were influenced by the decarboxylation of cannabinoids and the length of cannabinoids’ alkyl side chain. Findings from the biochemical, biophysical, and computational assays support the growing evidence of cannabinoids’ inhibitory effects on SARS-CoV-2 M^pro.