c Academy of Integrative Medicine, Shanghai
University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai,
201203, China.
*Corresponding author:
E-mail address: geguangbo@shutcm.edu.cn (G.-B. Ge)
Phone: +86-024-51323184
**Corresponding author:
E-mail address: xyzhuang59@foxmail.com (X.-Y. Z)
Abstract
The main proteases (Mpro) are highly conserved
cysteine-rich proteins that can be covalently modified by numerous
natural and synthetic compounds. Herein, we constructed an integrative
approach to efficiently discover covalent inhibitors of
Mpro from complex herbal matrices. This work begins
with biological screening of sixty clinically used antiviral herbal
medicines, among which Lonicera japonica (LJ) demonstrated the
strongest anti-Mpro effect (IC50 =
37.82 μg/mL). Mass
spectrometry-based chemical analysis and chemoproteomic profiling
revealed that LJ extract contains at least 50 constituents, of which 22
exhibited the capability to covalently modify Mpro. We
subsequently verified the anti-Mpro effects of these
covalent binders. Gallic acid and quercetin were found to potently
inhibit SARS-CoV-2 Mpro in dose- and time- dependent
manners, with the IC50 values below 10 µM. The
inactivation kinetics, binding affinity and binding mode of gallic acid
and quercetin were further characterized by fluorescence resonance
energy transfer, surface plasmon resonance, and covalent docking
simulations. Overall, this study
established a practical approach for efficiently discovering the
covalent inhibitors of Mpro from herbal medicines by
integrating target-based high-throughput screening and mass
spectrometry-based assays, which would greatly facilitate the discovery
of key anti-viral constituents from medicinal plants.
Keywords: SARS-CoV-2
Mpro, Lonicera japonica (LJ), anti-viral
agents, covalent inhibitors, inhibitory mechanism
1.
Introduction
The Coronavirus disease 2019
(COVID-19) pandemic caused by the
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has a
significant impact on global health and the economy1.
Emerging
evidence highlights the potential value of herbal medicine in the
treatment of viral infections including COVID-19, due to their
potentials to block viral replication
and relieve virus-induced systemic inflammation. For instance,Lonicera japonica, Pomegranate peel, and Bergenia ligulata have
been shown to be capable of impede
influenza virus
replication2-4,Isatis tinctoria, Laggera
pterodonta and Forsythia
suspensa have been reported to both
inhibit virus replication and
suppress inflammatory responses5-7.
Herbal
medicines are undoubtedly a rich source of tool compounds and drug leads
for developing novel antiviral
drugs8,9. However, unlike Western medicines that
commonly formulated with a single active ingredient,
herbal medicines contain complex
mixtures of chemically diverse
natural
compounds10. This complexity poses a challenge when it
comes to identifying and isolating the specific active constituents
responsible for their therapeutic effects. Furthermore, the availability
of commercially purified compounds for certain constituents is often
limited, further hindering in-depth characterization of their
pharmacological effects and mechanisms of action.
Several
virus-based and host-based targets have been validated for the discovery
and development of antiviral agents to combat COVID-19. Among these
therapeutic targets for β-coronaviruses (CoVs), the
Mpro has garnered significant attentions from
medicinal chemists due to its
critical role in the viral
replication and assembly11. There is increasing
evidence indicating that inhibiting or
disrupting Mprocan prevent the formation of replication-essential enzymes, thus
impeding viral multiplication and
replication12,13. The SARS-CoV-2
Mpro, also known as 3-chymotrypsin-like proteases
(3CLpro), is a canonical
cysteine protease that contains
12 cysteine residues per
monomer14. Among these, Cys145 and Cys44, located in
the catalytic
pocket of the enzyme, are
particularly important for its function15-17. They
form the catalytic dyad that cleaves the viral polyproteins into smaller
functional proteins necessary for viral replication. In addition, Cys156
and Cys300 play important roles in forming enzymatically active dimeric
forms of the protein17-19.
Targeting
these cysteine residues is a promising avenue of research for developing
effective inhibitors against SARS-CoV-2 Mpro.
The
scientific community has dedicated to discovering new covalent
inhibitors for Mpro. Commonly employed approaches in
this endeavor include
high-throughput screening and
structure-based virtual screening, both relying on known
compound libraries or
databases20,21. In this context, several natural
compounds derived from traditional medicinal plants, such as myricetin,
oridonin and isojacareubin, have been identified. These compounds
possess catechol or Michael receptors that have the ability to
covalently bind to the cysteine residues in SARS-CoV-2
Mpro16,17,22,23.
In this work, a practical strategy for rapidly discovering irreversible
inhibitors of SARS-CoV-2 Mpro from complex components
(such as herbal extracts or compound prescriptions) was demonstrated.
Our approach integrates target-based high-throughput screening and mass
spectrometry (MS)-based approaches, enabling efficient screening of the
herbal medicines with potent time-dependent inhibition on SARS-CoV-2
Mpro and to rapidly identify the covalent
Mpro inhibitors from crude herbal extract
(Fig.
1 ).
Firstly, fluorescence resonance energy transfer (FRET) technique was
employed for screening the anti-Mpro effects of
clinically used anti-viral herbal medicines and the results showed thatLonicera japonica (LJ) significantly inhibited SARS-CoV-2
Mpro in a time-dependent manner. After then,
ultra-high performance liquid chromatography-high resolution mass
spectrometry (UHPLC-HRMS) was used for global chemical profiling of the
constituents in LJ extract, while the Mpro-compound
conjugated were carefully characterized, which provided key information
to decipher the key constituents in LJ extract with the capability to
covalently modify Mpro, as well as the key modified
site(s) of Mpro by small molecules. This strategy
enables both multiplexed screening and direct identification of small
molecule binders from complex herbal mixtures, eliminating
the
need for fractionation.
Furthermore,
inactivation kinetics, binding kinetics and covalent docking simulations
were conducted to gain further insights into the interactions between
the target protein and the newly identified Mproinhibitors from LJ extract.