key: cord-0683705-d2vawtid
authors: Xiong, Yuan; Zhu, Guang-Hao; Zhang, Ya-Ni; Hu, Qing; Wang, Hao-Nan; Yu, Hao-Nan; Qin, Xiao-Ya; Guan, Xiao-Qing; Xiang, Yan-Wei; Tang, Hui; Ge, Guang-Bo
title: Flavonoids in Ampelopsis grossedentata as covalent inhibitors of SARS-CoV-2 3CL(pro): Inhibition potentials, covalent binding sites and inhibitory mechanisms
date: 2021-07-30
journal: Int J Biol Macromol
DOI: 10.1016/j.ijbiomac.2021.07.167
sha: 8c6d63ed7f12f02ec4b8be83368439497f983434
doc_id: 683705
cord_uid: d2vawtid

Coronavirus 3C-like protease (3CL(pro)) is a crucial target for treating coronavirus diseases including COVID-19. Our preliminary screening showed that Ampelopsis grossedentata extract (AGE) displayed potent SARS-CoV-2-3CL(pro) inhibitory activity, but the key constituents with SARS-CoV-2-3CL(pro) inhibitory effect and their mechanisms were unrevealed. Herein, a practical strategy via integrating bioactivity-guided fractionation and purification, mass spectrometry-based peptide profiling and time-dependent biochemical assay, was applied to identify the crucial constituents in AGE and to uncover their inhibitory mechanisms. The results demonstrated that the flavonoid-rich fractions (10-17.5 min) displayed strong SARS-CoV-2-3CL(pro) inhibitory activities, while the constituents in these fractions were isolated and their SARS-CoV-2-3CL(pro) inhibitory activities were investigated. Among all isolated flavonoids, dihydromyricetin, isodihydromyricetin and myricetin strongly inhibited SARS-CoV-2 3CL(pro) in a time-dependent manner. Further investigations demonstrated that myricetin could covalently bind on SARS-CoV-2 3CL(pro) at Cys300 and Cys44, while dihydromyricetin and isodihydromyricetin covalently bound at Cys300. Covalent docking coupling with molecular dynamics simulations showed the detailed interactions between the orthoquinone form of myricetin and two covalent binding sites (surrounding Cys300 and Cys44) of SARS-CoV-2 3CL(pro). Collectively, the flavonoids in AGE strongly and time-dependently inhibit SARS-CoV-2 3CL(pro), while the newly identified SARS-CoV-2 3CL(pro) inhibitors in AGE offer promising lead compounds for developing novel antiviral agents.

Over the last two decades, the growing occurrences of coronaviruses-related diseases with high mortality have been one of the long-standing and life-threatening issues to the global population [1] . Currently, the newly emerging coronavirus disease 2019 (COVID-19), a globally infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has brought a colossal threat to public health, economic development and society safety [2, 3] . In the past one-year, exhaustive efforts have been made by the scientists to discover efficacious therapeutics for treating COVID-19, via targeting on several validated therapeutic targets. Among all identified therapeutic targets for combating COVID-19, the chymotrypsin-like protease (3CL pro ) has drawn great concerns and has been recognized as a pivotal therapeutic target for fighting this pandemic, due to its high conservative and indispensable role in viral replication [4] [5] [6] . It has been validated that strong inhibition or dysfunction of 3CL pro can successfully block SARS-CoV-2 replication, and further generate benefits in the treatment of COVID-19 [7] .

Although a variety of SARS-CoV-2 3CL pro inhibitors have been identified recently, the majority of them were restricted to the reversible interactions with the target protease [8] [9] [10] .

By contrast, the covalent inhibitors could significantly attenuate the proteolytic activity of SARS-CoV-2 3CL pro via forming a stable chemical bond, which then inactivated this key protease and blocked coronavirus replication. Generally, the covalent inhibitors of target hydrolases bear at least one electrophilic group (such as quinones [11] , Michael receptors [12] , or some metal elements [13] ) that could covalently bind to the nucleophilic residues (such as cysteine). The covalent inhibitors possessed several inherent advantages (such as J o u r n a l P r e -p r o o f high specificity, good inhibition potency, and durable interactions) [14] , which could bring benefits to both anti-COVID-19 and other CoVs-related diseases. Unfortunately, the promising warheads and lead compounds for the development of efficacious SARS-CoV-2 3CL pro covalent inhibitors for treating COVID-19 are rarely reported. Recently, to find more efficacious SARS-CoV-2 3CL pro inhibitors with good safety profile, a high-throughput screening campaign was implemented to screen the herbal products with strong SARS-CoV-2 3CL pro inhibition activities, by using a fluorescence-based biochemical assay [15, 16] . After a large-scale screening of herbal products, we noticed that Ampelopsis grossedentata extract (AGE) strongly inhibited SARS-CoV-2 3CL pro in both time-and dose-dependently inhibition manners, with the apparent IC 50 value of 3.44 μg/mL after 60-min preincubation. This finding suggests that AGE should contain the naturally occurring covalent inhibitors of SARS-CoV-2 3CL pro .

Herein, a practical strategy via integrating bioactivity-guided fractionation and purification, mass spectrometry-based peptide identification and time-dependent inhibition assays, was utilized to recognize and characterize the key constituents in AGE with SARS-CoV-2-3CL pro inhibitory activities. The results clearly showed that the flavonoid-rich fractions (10-17.5 min on reverse phase liquid chromatography) displayed strong inhibitory activities against were optimized as the mobile phases in gradient conditions: 0.01-2 min, 95% A; 15-20 min,70%-25% A; 22-25 min, 95% A. The AGE sample (5 μL, 10 mg/mL) was injected and separated on a Shim-pack VP-ODS C18 column (2.0 × 250 mm, 4.6 μm) with the flow rate of 0.4 mL/min at 40 ºC, and the LC fractions were collected every 2.5 minutes. Ten collected LC fractions were dried under vacuum pressure, and then redissolved in DMSO for assessment of 3CL pro inhibition activity.

An LC-TOF-MS/MS system (Foster City, CA, USA) equipped with a Shimadzu UFLC system (Kyoto, Japan) was used to identify the major constituents in the bioactive fractions (10-17.5 min) of AGE in both positive and negative ion modes. The mass parameters were listed in Table S1 . Meanwhile, five major constituents were isolated by using a preparative HPLC (Waters, USA). The AGE sample (10 mL, 10 mg/mL) was continuously injected and separated on a Acchrom-C18 column (50 × 450 mm, 7 μm), accompanied by 80% of Water-0.1% formic acid and 20% of acetonitrile: methanol (4:1) with the flow rate of 65 mL/min. Five isolated constituents in AGE were enriched and dried in vacuo separately, the solid of each constituent was collected for structural characterization and SARS-CoV-2 3CL pro inhibition assay.

To identify the covalently modified sites for three flavonoids on SARS-CoV-2 3CL pro , the peptides of target enzyme co-incubated with or without each tested flavonoid were analyzed by using a nanoLC-MS/MS system [17, [18] [19] [20] . Firstly, the SARS-CoV-2 3CL pro (147 μg, final content) was co-incubated with inhibitors (400 mM, final concentration) at 37 °C 

The inactivation kinetics for three flavonoids were investigated as the reported procedure [21] . Firstly, two incubation mixture groups (group A and group B) were prepared for use. 

The covalent docking was carried out through the covalent docking module of MOE (Molecular Operating Environment 2019.01, Chemical Computing Group Inc., Montreal, Canada). Firstly, the crystal structure of SARS-CoV-2 3CL pro (PDB Code: 6XHU, [22] ) was download for preliminary treatment by using the QuickPrep module, including adding hydrogens and partial charges, optimizing the hydrogen bond network and minimizing energies. Then, the orthoquinone form of myricetin (QFM) was constructed and minimized energy in the Builder panel. Next, the covalent reaction formula for QFM and cysteine residuals was imported to MarvinSketch for covalent docking module. Finally, with the help of GBVI/WSA dG, these generated conformations of the rigid receptor were refined and estimated the binding scores [23] . The pose with the lowest S score was selected as the initial J o u r n a l P r e -p r o o f Journal Pre-proof conformation of QFM-3CL pro complexes.

To perform the molecular dynamic (MD) simulations for QFM-3CL pro complexes, it was necessary to get the force field of non-standard amino acid (Cys-QFM), which was generated by AmberTools20 (AMBER 2020, University of California, San Francisco, USA). The detailed procedures were as follows. At first, AM1-BCC charges of the Cys-QFM complex were calculated [24] . Then, atoms, bonds, angles, and dihedral parameters of Cys-QFM complex were established via atom types of amber99sb-ildn force field and checked by parmchk2 [25] . Next, Coordinate and topology files of Cys-QFM complex were created by leap program, and then translated to the GROMACS topology file via ACYPE [26, 27] .

Finally, manually written residue topology parameter file (rtp file) as per the GROMACS topology file and manually written hydrogen database file (hdb file) were loaded into the libraries files of amber99sb-ildn force field.

Systems of QFM-3CL pro complexes was established for simulations. Prior to MD simulations, an energy minimization of 50,000 steps steepest descent was performed. To neutralize the charges of the protein-solvent system, SARS-CoV-2 3CL pro and QFM-3CL pro complexes were separately solvated with the TIP3P water model including sodium ions. Then, the system was equilibrated, including 100 ps for NVT heating to 310 K, and 100 ps for NPT. Finally, the system was subjected to 50 ns MD at 310 K (V-rescale thermostat) under a pressure of 1 bar (Parrinello-Rahman barostat). To analyze the interactions of QFM-3CL pro complexes, we clustered the equilibrium conformations [28] , and the largest center structure was selected to study the interactions by creating its stereoscopic picture via Discovery 

Firstly, the inhibitory potentials of 105 herbal extracts (100 μg/mL, final concentration) on SARS-CoV-2 3CL pro were assayed by using Dabcyl-KNSTLQSGLRKE-Edans as the fluorescent substrate. From the preliminary screening, AGE exhibited the most potent SARS-CoV-2 3CL pro inhibition activity (Fig.1) . The residual activity of SARS-CoV-2 3CL pro in the presence of AGE (100 μg/mL, final concentration) was 0.26 %. As depicted in Fig.2 when AGE was pre-incubated with SARS-CoV-2 3CL pro at various preincubation times.

These findings demonstrate that AGE strongly inhibits SARS-CoV-2 3CL pro in dose-and time-dependent manners, implying that some natural constituents in AGE may covalently bind with SARS-CoV-2 3CL pro .

J o u r n a l P r e -p r o o f

Then, a practical strategy via integrating bioactivity-guided fractionation and purification, as well as inhibition assay, was used to identify the key constituents in AGE. From the results exhibited in Fig.3 , within ten fractions, F5, F6 and F7 possessed strong inhibitory properties against SARS-CoV-2 3CL pro . After then, five major peaks of the bioactive fractions (10-17.5 min) were isolated, while their structures and effects of inhibiting SARS-CoV-2-3CL pro were characterized. The MS 1 and MS 2 spectra of these natural compounds were shown in Table 1 , SARS-CoV-2 3CL pro were also plotted by using increasing concentrations of each flavonoid (Fig.5, Fig.S12 ). As listed in Table 2 (Table 2, Fig.5) . These findings suggest that dihydromyricetin, iso-dihydromyricetin, myricitrin and myricetin are the key bioactive constituents in AGE that can dose-and time-dependently inhibit SARS-CoV-2 3CL pro .

Next, the covalent binding sites of dihydromyricetin, iso-dihydromyricetin and myricetin on SARS-CoV-2 3CL pro were identified by using mass spectrometry. From the view of chemical structures of these naturally occurring flavonoids, all these compounds bear a catecholic group at the B-ring, which can be easily oxidized to form orthoquinones that can covalently bind on the biothiols or the cysteines in target proteins (Fig.6 ) [28, 29] . In this case, the generated MS/MS spectra were analyzed by searching covalent modifications on cysteines of SARS-CoV-2 3CL pro , with the molecular mass increments of 316.24 Da (myricetin) and 318.25 Da (dihydromyricetin or iso-dihydromyricetin). Table 3 , following co-incubation of SARS-CoV-2 3CL pro with each tested flavonoid, several cysteine residues in the peptides of SARS-CoV-2 3CL pro could be modified by the orthoquinone forms of these three flavonoids. From Fig.7, Fig.S14, Fig.S15 , all tested flavonoids (myricetin, dihydromyricetin and iso-dihydromyricetin) could covalently modify Cys300, a key residue located at domain III (residuals 198-303) of SARS-CoV-2 3CL pro (Fig.S13) . Several reports state that domain III (especially 290 E-V 303 ) functions as a crucial J o u r n a l P r e -p r o o f part to maintain the dimer conformation of active 3CL pro , mutation or modification of the key residuals (such as Gln290, Arg298 and Gln299) would result in the instability or inactivation of this key enzyme [31] [32] [33] . Thus, it was easily conceivable that the surrounding micro-environment or the pivotal interactions for the formation of the dimer of active SARS-CoV-2 3CL pro might be changed or destroyed via modification of Cys300 by these naturally occurring flavonoids.

In addition to Cys300, myricetin can also covalently bind on Cys44, which is near the catalytic site of SARS-CoV-2 3CL pro (Fig.7, Fig.S13 ). Recently study has found that Cys44 is a hyper-reactive cysteine with higher nucleophilicity than Cys145, which is recognized as a promising binding site for designing and developing covalent inhibitors of this key enzyme [34] . The covalent binding of myricetin on Cys44 might block 3CL pro -catalyzed peptide-cleavage reactions. These findings suggest that myricetin and its analogous in AGE can covalently bind on some key cysteines of SARS-CoV-2 3CL pro , while myricetin can concurrently modify both Cys300 and Cys44. Meanwhile, these findings can partially explain the potent inhibition efficacy of myricetin, in comparison with its analogous (such as dihydromyricetin or iso-dihydromyricetin).

The inactivation kinetics for three flavonoids were further investigated to evaluate the inactivation potency of these naturally occurring SARS-CoV-2 3CL pro inhibitors. To this end, the inactivation kinetic curves were plotted by using various inhibitor concentrations with increasing pre-incubation times. As shown in Fig.S16 and Fig.8 

Finally, covalent docking simulations and molecular dynamics simulations were carefully conducted for QFM that covalently bound in site 1 (near Cys300) and site 2 (near Cys44) of SARS-CoV-2 3CL pro to explore the key interactions between this agent and the target enzyme.

As shown in Fig.9B and Fig.S17 , when QFM covalently bound to the sulfur atom of Cys300, this agent mainly interacted with the surrounding amino acid residuals (including Val296, Val297, Gly2 and Ile213) by hydrogen bonding. As for site 2 (near Cys44), it was observed from Fig.9D and [35] . Over the past one year, a variety of SARS-CoV-2 3CL pro inhibitors have been found, but only several compounds are identified as the covalent inhibitors of this vital enzyme via forming chemically stable and irreversible bonds [13] . Given that the covalent inhibitors always display good inhibition potency in living systems, the covalent inhibitors of 3CL pro are considered as a good choice to block the SARS-CoV-2 multiplication via inactivating the proteolytic activity of 3CL pro . Thus, it is urgent and highly desirable to find more efficacious SARS-CoV-2 3CL pro covalent inhibitors with improved safety profiles, which may offer the promising lead compounds for developing novel anti-COVID-19 agents.

In these cases, a high-throughput screening campaign was conducted for discovering effective SARS-CoV-2 3CL pro covalent inhibitors from herbal products. Among all tested herbal products, AGE demonstrated the most potent SARS-CoV-2 3CL pro inhibition activity, while this herbal extract inhibited this key enzyme in time-and dose-dependent manners.

This finding intrigued us to reveal the key bioactive constituents in AGE that could covalently bind to SARS-CoV-2 3CL pro . In Southeast China, Ampelopsis grossedentata is a flavonoid-rich (w/w > 40%) medicinal herb, whose dried leaves and stems are popularly used as healthy tea to prevent chronic disorders by reducing hypertension, regulating plasma lipids J o u r n a l P r e -p r o o f Journal Pre-proof and blood glucose [36] [37] [38] [39] . Herein, we identified that three abundant flavonoids (dihydromyricetin, iso-dihydromyricetin and myricetin) in AGE could strongly inhibit SARS-CoV-2 3CL pro by covalently binding with two key cysteines on this target enzyme.

Previous reports have reported that the flavonoids (myricetin and dihydromyricetin) in AGE exhibit multiple beneficial effects including anti-inflammatory, anti-coagulative, as well as pulmonary fibrosis inhibition activities [34, [40] [41] [42] . Thus, Ampelopsis grossedentata could be used as a healthy plant-based supplement for treating COVID-19, which might relieve the COVID-19-related symptoms in both the respiratory tract system and alimentary system.

Scutellaria baicalensis [43] , Glycyrrhiza uralensis [44] and Ephedra sinica [45] ) that contain other anti-SARS-CoV-2 phytochemicals with diverse inhibition mechanisms or various binding targets, in which the combination use may bring additive or synergistic antiviral effects for the treatment of COVID-19.

Notably, three newly identified SARS-CoV-2 3CL pro covalent inhibitors (dihydromyricetin, iso-dihydromyricetin and myricetin) in AGE were also found with the inhibition against SARS-CoV helicase [46] and SARS-CoV3CL pro [47] , as well as high affinities with the ACE2 receptor [40] , suggesting that these agents hold sufficient potentials to develop as the broad-spectrum anti-coronavirus agents via targeting multiple key druggable targets.

However, the poor cell-permeability and poor metabolic stability of these natural flavonoids strongly hampered the wide applications of these flavonoids in clinical settings [42] .

Therefore, it is necessary to used more practical approaches (such as structural optimizations or drug delivery technologies) to develop more efficacious agents for combating J o u r n a l P r e -p r o o f pandemic. Considering that the naturally occurring flavonoids could be extensively metabolized by UDP-glucuronosyltransferases (UGTs) or other conjugative enzymes in humans [48] , AGE could be co-administrated with other herbal medicines containing strong inhibitors against human UGTs, such as Fructus Psoraleae [49] , or UGTs inhibitors like amentoflavone [50] and licochalcone A [51] , which might improve the in vivo therapeutic effects of AGE against COVID-19. Furthermore, this study also offered several leading compounds and a key warhead for designing and developing more efficacious SARS-CoV-2 3CL pro covalent inhibitors. Among all tested flavonoids isolated from AGE, myricetin was identified as the most potent SARS-CoV-2 3CL pro covalent inhibitor, owing to this agent could concurrently label both Cys300 and Cys44 of this key enzyme. Our findings suggested that the ortho-trihydroxyl group in the B ring of these flavonoids was a crucial pharmacophore for covalently binding on 3CL pro , as well as SARS-CoV-2-3CL pro inhibitory activities. Meanwhile, the plane structures without glycosides were propitious to the process of covalent reactions. In future, this key warhead will be conducive to design a new generation of efficacious anti-COVID-19 medications, while these pyrogallol-containing compounds can be used as practical probes or tools to identify the covalent inhibitors for cysteine proteases. More importantly, compared with the cysteine residuals in SARS-CoV-2 3CL pro (such as Cys44 and Cys145), Cys300 is suggested as a more desired ligand-binding site for developing covalent inhibitors of SARS-CoV-2 3CL pro , owing to its unique location at the dimeric surface of SARS-CoV-2 3CL pro that is more liable to be covalently modified by 

In summary, this study reported that both AGE and the major constituents or the flavonoid-rich fractions of this herbal extract could strongly inhibit SARS-CoV-2 3CL pro in 

No competing interests. J o u r n a l P r e -p r o o f Journal Pre-proof J o u r n a l P r e -p r o o f

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We thank professor Guo-Qiang Lin and Dr. Ding-Ding Gao for the technical supports.