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.