Lichen secondary metabolites as potential inhibitors of viral 3-chymotrypsin-like protease (3CLpro)

Background. Despite advances in managing SARS-CoV-2 infections, the virus continues to spread rapidly, prompting interest in repurposing existing drugs and natural compounds as treatments. Promising are agents that target the SARSCoV-2 3CLpro, an enzyme crucial for viral replication. Lichen secondary metabolites stand out due to their diverse biological properties, offering significant potential for therapeutic use against the virus. Materials and methods. The inhibitory effects of lichens against the 3CLpro are assessed through kinetic analyses by measuring the fluorescence intensity with a microtiter plate-reading fluorimeter using a fluorogenic substrate. Cytotoxicity effects are conducted on murine Sertoli TM4 cells. In-silico analyses are performed to examine the interaction dynamics between lichens and the 3CLpro. Results. Initial screenings have identified several lichen compounds as potential inhibitor of 3CLpro, with inhibition rates from 67% to 99% and Ki values between 0.67μM to 22.98μM. These compounds act as slow-binding inhibitors, exhibiting both competitive and noncompetitive inhibition mechanisms, targeting the substrate-binding site and the enzyme dimerization interface, respectively. Molecular docking confirms the biochemical data. Protocetraric, salazinic and fumarprotocetraric acids fit well within the 3CLpro active site, forming stable covalent bonds with the catalytic cysteine145. Perlatolinic acid, identified as noncompetitive inhibitor, can bind both the free enzyme and the enzyme-substrate complex, disrupting crucial interactions necessary for enzyme dimerization and activity. Cell culture and viability assays on TM4 cells confirm the safety profile of lichens. Discussions and conclusion. Our findings not only enrich the existing body of knowledge on therapeutic strategies against SARS-CoV-2 underscoring the utility of lichen-derivatives in developing safe and effective treatments, but also add an important piece to the mosaic of their already numerous biological activities, highlighting their promise as a source of novel antiviral agents. A hallmark of this study is the employment of a dual inhibition strategy, an innovative approach to antiviral drug development. The identification of perlatolinic acid as a potent noncompetitive inhibitor targeting the protease dimer interface represents a pivotal advancement in developing innovative therapeutic strategies against SARS-CoV-2, offering a promising strategy to overcome viral mutation-induced drug resistance. Based on the outcomes of kinetic, computational and cytotoxicity studies, it can be concluded that some examined lichen secondary metabolites serve as suitable scaffolds for developing effective inhibitors targeting the cysteine enzyme of SARS-CoV-2.