3.6 Binding kinetics of gallic acid and quercetin with SARS-CoV-2 Mpro
The binding affinity of gallic acid and quercetin to Mpro was further investigated using SPR technique, which allowed the analysis of their interactions. As shown inFig. S72 , the observed association and dissociation phases of the gallic acid and quercetin exhibit clear biphasic behavior, which aligns with the anticipated two-step binding interaction of an irreversible inhibitor. Wherein, gallic acid and quercetin were both bound to Mpro in a dose-dependent manner and exhibited moderate affinity with KD values of 0.321 μM and 0.308 μM, respectively (Table S3 ). Notably, the sensorgram plots suggested that gallic acid binds and dissociates more slowly from Mpro than quercetin, which might be caused by the multiple binding sites of gallic acid on Mpro38.
3.7 Covalent docking
The binding modes of gallic acid covalently binding to SARS-CoV-2 Mpro at either Cys85 or Cys128 of were depicted inFig. 6 and Fig. S73 . At the Cys85 site, gallic acid interacts with Asp187 and Arg40 through electrostatic interactions (π-Cation or π-Anion) and hydrogen bonds. At Cys128 site, hydrophobic interactions (π-Sigma, Alkyl and π-Alkyl) are responsible for fixing gallic acid to protein. The binding modes of quercetin covalently binding to SARS-CoV-2 Mpro were also analyzed. While quercetin forms covalent bonds with Cys22, it also forms hydrophobic interactions (π-Sulfur, π-Cation) and hydrogen bond with Lys61. In brief, covalent docking simulation revealed that gallic acid and quercetin inhibited SARS-CoV-2 Mpro through interacting with several key non-cysteine residues and covalently modifying some cysteine residues.
Discussion
Mpro plays a crucial role in the replication process of multiple β-coronaviruses including SARS-CoV-2 virus. Mpro are a class of highly conserved cysteine hydrolases distributed in coronaviruses, no similar mammalian genes are estimated to encode Mpro like proteases in healthy humans39. These characteristics prompted the researchers to discover and develop more efficacious covalent inhibitors of Mpro for completely blocking its catalytic activity. To this end, great efforts have been made to efficiently identify the covalent inhibitors targeting this protease utilizing various approaches. Given that Mpro is a cysteine-rich protease, it offers the potential to identify or design small molecules that can covalently bind to its key cysteine residues through a mechanism of nucleophilic addition, thereby inactivating the enzyme40,41. In the past few years, researchers have demonstrated that many natural ingredients found in medicinal and edible herbal products can covalently bind the key cysteines of SARS-CoV-2 Mpro. Not only do these ingredients possess strong ability to inactivate Mpro, but they also exhibit good safety. Inspired by these findings, we are committed to discover more covalent inhibitors of SARS-CoV-2 Mpro in herbal products. However, discovering covalent inhibitors for Mpro in clinically used herbs poses a significant challenge in antiviral pharmacology research. This is due to the highly complex chemical composition of these products, with many components existing in extremely small amounts (less than 0.1% of the total weight). Efficient and accurate identification of such inhibitors remains a crucial task.
To address this issue, this study presents a practical platform for the rapid discovery of new SARS-CoV-2 Mpro covalent inhibitors from herbal medicines. The process involves screening antiviral herbs with fluorescent labeling techniques, analyzing the herbal medicine composition using HRMS, and characterizing modified peptides with mass spectrometry-based chemoproteomic method to identify compounds with potential covalent inhibitory activity. The comprehensive strategy proposed in this study for the rapid discovery of covalent inhibitors of SARS-CoV-2 Mpro from herbs has a few points that are worth noting. In the screening process, the time-dependent inhibition of Mpro by an herbal extract indicates the presence of covalent inhibitors of Mpro. Despite the complex and diverse nature of chemical components in herbal medicines poses a challenge in fully characterize them, the use of high-resolution mass spectrometry enables the assignment of most chemical structures by matching the LC-MS/MS features with natural compounds in the database or reference standards. Additionally, a high coverage of peptide sequence is critical for identifying all constituents that can covalently bind to the target protein. Identifying isomers individually is also necessary because their presence in the extract is inevitable. This can be done by separately incubating isomers or compounds of similar molecular weights with the target protein. Based on this strategy, we successfully found Lonicera japonica as having significant time-dependent inhibitory potential against SARS-CoV-2 Mpro among 60 herbs. Covalent inhibitors of Mpro were also uncovered from this herbal medicine.
Herbal medicines offer alternative therapies to address the multiple symptoms and complications associated with COVID-19 management42,43. It has been widely employed in the treatment of respiratory infections, and certain herbal components have gained approval as marketed drugs, over-the-counter nutritional supplements, or food additives44. Generally, the long-term use of herbal medicines and the availability of marketed herbal products have demonstrated satisfactory safety profiles, making them suitable for extended prophylactic use. Although the emergency phase of the pandemic may have subsided with the emergence of the less pathogenic omicron variant, the incidence of infection remains high among individuals who have received vaccines or have experienced natural infection45,46. In the post-pandemic period, the utilization of herbal medicines and their bioactive fractions holds great potential for both preventive measures and supportive treatment in relation to COVID-1947. As an extensively used herbal medicine in clinical settings, LJ possesses a wide range of health-promoting effects, such as heat-clearing and detoxifying, anti-inflammatory effects, activating meridians, broad-spectrum anti-bacterial and anti-viral effects48-50. These characteristics make LJ a commonly employed herb for treating respiratory infections, fever, sore throat, and other inflammatory conditions. Considering LJ’s significant ability to inhibit Mpro and its anti-inflammatory effects, it is expected that LJ may exert multifaceted effects in terms of reducing the duration of COVID-19 symptoms, suppressing uncontrolled inflammation (such as cytokine release syndrome), and alleviating long COVID symptoms.
Conclusion
In summary, an integrative approach was constructed to efficiently discover SARS-CoV-2 Mpro covalent inhibitors from anti-viral herbs, including target-based high-throughput inhibition assay and mass spectrometry-based chemoproteomic approaches. By employing this multifaceted analytical approach, we successfully uncovered the covalent inhibitors of SARS-CoV-2 Mprofrom a clinically used anti-viral herb Lonicera japonica that was found with significant time-dependent inhibition against SARS-CoV-2 Mpro. Our findings revealed that 22 constituents in LJ extract could covalently modify the cysteine residues of SARS-CoV-2 Mpro. Among these, gallic acid, quercetin and cynaroside could dose- and time- dependently inhibited SARS-CoV-2 Mpro, with the IC50 values of less than 20 µM. The inhibition mechanisms and binding modes between of two potent inhibitors (gallic acid and quercetin) were comprehensively characterized. Overall, we present the screening, discovering, biochemical and biophysical validation of covalent inhibitors targeting Mpro from antiviral herbal medicines. Beyond providing potential lead compounds for future development of novel anti-SARS-CoV-2 agents, this integrated approach is expected to facilitate the efficient and confident identification of naturally occurring anti-viral constituents derived from medicinal plants.