Natural compounds as potential inhibitors of viral 3-chymotrypsin-like protease (3CLpro)

Despite the recent advances to control the ongoing SARS-CoV-2 pandemic, the rapid spread of the virus is still challenging to face. Although the scientific community has turned to the development of effective vaccines, the repurposing of approved drugs and natural compounds has emerged as a particularly promising strategy for combating the virus infection. The strategic repurposing of such agents, especially those targeting the SARS-CoV-2 3CL main protease, a critical enzyme in viral replication, holds considerable potential for therapeutic intervention. Within the vast spectrum of natural compounds, lichen secondary metabolites, curcuminoids, and various phenylpropanoid compounds have been identified as potential candidates due to their broad spectrum of biological activities. However, their therapeutic potential has yet to be fully exploited. An initial screening of natural compounds was conducted to identify their inhibitory potential on 3CLpro, focusing subsequently on those demonstrating the most promise. The investigation into their inhibitory activity involved kinetic parameter assessment through fluorescence intensity measurements using a microtiter plate-reading fluorimeter and a fluorogenic substrate designed specifically for this purpose. Additionally, the cytotoxicity of some compounds was evaluated on murine Sertoli TM4 cells to ensure their safety for potential therapeutic use, along with computational studies to elucidate the interaction dynamics between the selected compounds and the enzyme. Our findings reveal that some compounds exhibit no inhibitory effects on 3CLpro, while lichen secondary metabolites and most of the curcuminoids demonstrate significant inhibitory activity, acting as slow-binding inhibitors of the enzyme. Analysis of the mechanism of inhibition reveals the presence of both competitive and noncompetitive inhibitors among the compounds, highlighting a dual approach to inhibit the protease that involves targeting the substrate-binding site and the enzyme's dimerization interface, respectively. None of the tested compounds exhibit cytotoxic effects on murine epithelial cells, corroborating their safety profile and potential for therapeutic application. Computational studies, providing insights into the interaction dynamics between the natural compounds and the protease, reinforce the biochemical data. The findings contribute significantly to the existing knowledge of the biological activities of natural compounds. Based on the outcomes of kinetic analyses, computational studies, and cytotoxicity studies, we can conclude that some examined natural compounds can be considered suitable scaffolds for developing effective inhibitors of the cysteine enzyme of SARS-CoV-2.