Targeting SARS-CoV-2 main protease 3CL pro and human ACE2 with Paeonia Phytochemicals by in silico and in vitro Studies in Terms of Possible COVID- 19 Therapeutics

doi:10.21203/rs.3.rs-3666689/v1

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Targeting SARS-CoV-2 main protease 3CL pro and human ACE2 with Paeonia Phytochemicals by in silico and in vitro Studies in Terms of Possible COVID- 19 Therapeutics

https://doi.org/10.21203/rs.3.rs-3666689/v1

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As important medicinal herbs, Paeonia species have been used in ancient medicine. Although its therapeutic potential is well known, the potential efficacy of Paeonia phytochemicals against the emerging coronavirus (SARS-CoV-2) has yet to be tested. This study selected seventy-six Paeonia compounds to determine their potential druggable impact on SARS-CoV-2 main protease (3CL^pro) and human ACE2 proteins. Structure-based virtual screening (SBVS) approach was performed by PyRx molecular docking software, including the Open Babel v2.4 and AutoDock 4.2.6 tools. The lowest affinity score and desired hydrogen bonding interactions were selected, and SwissADME was used to predict drug-likeness and pharmacokinetics properties. In addition, the potential cytotoxic effect of five Paeonia root extracts was tested in cancer (HCT116 and HeLa) and fibroblast (HFF) cell lines. The results showed that nine Paeonia ligands (catechin, apigenin, palbinone, kaempferol, paeoniflorigenone, eriodictyol, paeonilactone C, cassythicine, and 3-O-methylquercetin) were able to interact with SARS-CoV-2 at high affinity (from − 7.5 to -9.0 kJ/mol), as possible SARS-CoV-2 inhibitors. Molecular dynamics simulation (MDS) analysis revealed that five of these phytochemicals -cathecin, apigenin, palbinone, paeoniflorigenone, and eriadictyol- have the potential to act as effective compounds. In addition, the plant extracts at low concentrations is not cytotoxic for selected cell lines. Overall, this study points to the inhibitory potential of Paeonia phytochemicals as novel therapeutics against SARS-CoV-2. Their druggable potential can be tested in vivo in further studies.

Paeonia

novel therapeutics

ligand-based virtual screening

SARS-CoV-2

Molecular dynamic simulations

Paeonia species have long been recognized for their ethnomedicinal uses garnering significant attention from the scientific community. Twelve taxa of Paeonia have been discovered in Turkiye, the world's most important genetic resource center for this genus (Cetiz et al. 2023). Their therapeutic properties, such as antimicrobial, anti-inflammatory, anticancer, antioxidant, antiviral, antifungal, antityrosinase, neuroprotective, and hepatoprotective effects, have been shown elsewhere (Kim et al. 2009; Lee et al. 2012; Li et al. 2008; Wang et al. 2012; Yang et al. 2011; Lee et al. 2008; Picerno et al. 2011; Zhang et al. 2018; Magid et al. 2017). Among the various parts of Paeonia, the root is particularly interesting to phytochemists for its medicinal uses. A recent review highlighted their traditional uses, pharmacological activities, and clinical applications (Li et al. 2021). Although their therapeutic potential is well known, the potential efficacy of Paeonia phytochemicals against the emerging coronavirus (SARS-CoV-2) has yet to be tested.

The SARS-CoV-2 is the strain of coronavirus that causes the disease COVID-19 and was first reported in Wuhan, China, in December 2019 (Zhu et al. 2020). The disease affects multi organs (Rabaan et al. 2023a) and caused about seven million death in the last three years (http://worldometer.com). The disease affects individuals of all age groups, including children (Rabaan et al. 2023b). The COVID-19 pandemic is persistent and shows no signs of extinction in the near future due to the virus's rapid mutation rate generating new variants (Amoutzias et al. 2022). The absence of specific biotherapeutics requires new strategies to prevent the spread and prophylaxis of the novel virus and its variants.

Coronaviruses possess the largest genomes among any RNA viruses. As a novel beta coronavirus, SARS-CoV-2 has 79% genomic sequence identity with SARS-CoV and 50% with MERS-CoV (Hu et al. 2021). Its genomic organization is similar to that of other beta coronaviruses. Six functional open reading frames (ORFs) are arranged in order from 5' to 3': two large non-structural polypeptides (ORF1a/ORF1b), spike (S), envelope (E), membrane (M), and nucleocapsid (N). Additionally, seven putative ORFs encoding accessory proteins are interspersed between the structural genes (Michel et al. 2020). These proteins are responsible for replicating the virus in human host cells. The spike (S) protein of SARS-CoV-2 binds to receptor proteins on the host cell’s surface, known as angiotensin-converting enzyme-2 (ACE2). The S protein has a receptor binding domain (RBD) for binding to human host cells. The RBD of the spike glycoprotein interacts with the ACE2 receptor in the host cell's protease domain (PD), causing viral infection (Belouzard et al. 2012). Together with, ORF1a encodes pp1a polypeptide, which contains, among other proteins, two viral proteases, namely papain-like protease (PL^pro) and 3C-like protease (3CL^pro), also known as the main protease (M^pro). These proteases further cleave polypeptides pp1a and pp1ab (encoded by ORF1b) into 16 functional non-structural proteins (nsps). These nsps play essential roles in the activation of the viral replication process. Some of these nsps are single-stranded RNA binding protein (nsp9), growth factor-like protein (nsp10), viral RNA-dependent RNA polymerase (nsp12), RNA helicase (nsp13), exo-ribonuclease (nsp14), endoribonuclease (nsp15) and 2´-O-ribose methyl-transferase (nsp16) (Rehman et al. 2021).

During the previous outbreak of the SARS-CoV crisis, several research groups conducted in vitro analysis and randomized trials of the use of natural medicinal herbs to control the side effects of infection and confirmed their effectiveness (Bormann et al. 2021; Jan et al. 2021; Nie et al. 2021). Besides, there are various synthetic drugs, treatments, and vaccines to break the SARS-CoV-2 viral chain. Preventive measures and incorporating potential antiviral herbal agents into a healthy diet are still recommended. With the advent of computational intelligence, computational biologists and researchers have deployed artificial intelligence and machine learning algorithms to track, monitor, and diagnose SARS-CoV-2 infections worldwide (Mottaqi et al. 2020). By utilizing these methods, researchers have identified specific plant-derived compounds that show promising interactions with the Spike glycoprotein, suggesting their potential as therapeutic agents in combating SARS-CoV-2 (Singh et al. 2021; Kulkarni et al. 2020; Rolta et al. 2021).

In this study, we aimed to find the antiviral therapeutic potential of Paeonia species against the SARS-CoV-2 virus. 76 phytochemicals known as Paeonia-derivate substances in the NPASS database were screened. Their potential interaction with 3CL^pro and ACE2 has been in silico tested by modeling and molecular docking analyses. The results represented the therapeutic potential of Paeonia species against SARS-CoV-2. The polyphenol content and antioxidant activities of methanolic root extracts from twelve taxa of the Paeonia genus have been comparatively analyzed by our group (Cetiz et al. 2023). Among these taxa, the top five plants exhibiting higher antioxidant activity were selected for further in vitro investigation. Besides, the goal was to determine the cell viability and identify non-toxic concentrations of the selected extracts. This step is crucial for evaluating the potential biomedical applications of these plant extracts. Researchers can gain insights into these Paeonia extracts' safety and appropriate dosage ranges for potential therapeutic use by assessing cell viability and establishing non-toxic concentrations.

2.1 Paeonia samples

Twelve taxa of Paeonia genus (P. mascula subsp. mascula (1), P. kesrouanensis (2); P. daurica (3), P. turcica (4); P. mascula subsp. orientalis (5), P. peregrina (6), P. mascula subsp. bodurii (7), P. x kayae (8), P. mascula subsp. arasicola (9), P. arietina (10), P. wittmanniana (11), P. tenuifolia (12)), which is collected from different provinces in Turkiye (Rize, Samsun, Yozgat, Gümüşhane, Mersin, Antalya, Konya Afyonkarahisar, Çanakkale, Edirne and Bursa), were used in this study. The powdered root (10 g) was added to 50 mL of 100% methanol at 37°C and shaken on a shaker at a controlled speed for 2.5 h (Heidolph Unimax 1010, Germany), and ultrasonic (Elma Clean Box, Elma) extraction have been applied at 25°C for one hour, then centrifuged at 4,000 rpm for 20 min at 4°C. The supernatants were collected and evaporated (EZ-2 Evaporator, GeneVac).

2.2 Collection and preparation of protein structures

Three-dimensional .pdb structures of the SARS-CoV-2 related proteins (ACE2: 6LZG and 7WPA), and a highly conserved protease for SARS-CoV-2 replication (3CL protease: 6LU7 and 6M2N) of SARS-CoV-2 were obtained from RCSB-PDB database (Berman, 2000) (https://www.rcsb.org/). All water molecules and other heteroatoms (HETATMs) in crystal structures were removed, missing residues were repaired, and polar hydrogens and Kollman charges were added. Finally, the .pdb files were converted into .pdbqt file format using AutoDock Tools (Morris et al., 2009).

2.3 Selection of phytochemicals and ligand preparation

A total of 76 phytochemicals that are known Paeonia-derivate substances in the NPASS database (Zeng et al., 2018) (https://bidd.group/NPASS/) were collected, and 3D-sdf files were downloaded according to their CID code from the Pubchem database (S. Kim et al., 2023) (http://pubchem.ncbi.nlm.nih.gov/). The files were minimized and converted into .pdbqt file using OpenBabel v3.1.1 software (O’Boyle et al., 2011).

2.4 Molecular docking

After the compounds and enzymes were prepared, docking analysis was carried out using PyRx (Dallakyan & Olson, 2015) (https://pyrx.sourceforge.io/). The potential binding pockets and subpockets in protein structures were detected by DockSiteScorer (Volkamer et al., 2012) (https://proteins.plus/#dogsite). Docking grid parameters were set according to their co-crystallized ligands and known active site residues. After docking, the lowest binding energy (kJ/mol) for 3CLpro and ACE2 was selected and visualized in PyMOL (The PyMOL Molecular Graphics System, Version 2.5, Schrödinger, LLC) (https://pymol.org/2/) to identify interacting residues with H-bond.

2.5 Molecular dynamics simulation

Molecular dynamics simulation (MDS) was implemented to validate the docking study and evaluate the change in protein conformation. Gromacs v2023.1 (Abraham et al., 2015) (https://www.gromacs.org/) was used to obtain more reliable data on binding affinity in the system of CHARMM36 force field (ff) (Vanommeslaeghe et al., 2009). The water molecules (spc) were added, and the complex was solvated in 150 mM NaCl salt at 310 K temperature. A triclinic simulation cell was created to simulate the protein, which was 1.0 Å bigger than the protein. The Particle Mesh Ewald method was applied to calculate long-range electrostatic interaction, and the modified Berendsen thermostat (V-rescale) was used to control the temperature of the simulation system. The MD simulation time was 10 ns at 310 K temperature. The simulation result was incorporated with the default script of gmx rms to obtain root mean square deviation (RMSD), and gmx rmsf to obtain root mean square fluctuation (RMSF). Hydrogen bonds (H-bonds) were analyzed by molecular visualization program, VMD. The Molecular Mechanics Generalized Born method was applied to calculate the binding free energy. gmx_MMPBSA (Valdés-Tresanco et al. 2021) is a tool based on AMBER's MM/PB(GB)SA calculation aiming to perform end-state free energy calculations with GROMACS files. The last three ns were considered for MM/GBSA calculation.

ΔG (bind) = ΔG (complex) – [ΔG (receptor) + ΔG (ligand)]

2.6 Cell culture and assays

Cytotoxic effects of five plant extracts (Sample 4, 7, 14, 16, and 18) were assessed in three cell lines: HCT116 (human colorectal carcinoma), HeLa (human cervical carcinoma), and HFF (human foreskin fibroblast). The cells were maintained in humidified conditions and incubated at 37 ˚C with 5% CO₂. The cells were cultured in DMEM culture media, supplemented with 10% heat-inactivated fetal bovine serum, 1% Pen Strep, and 1% MEM-NEAA. All cell culture reagents were purchased from Thermo Fisher Scientific.

For the MTT cell viability assay, 20,000 cells/well were plated in 96-well plates. Cells were then treated with the different plant extract samples at 5, 20, 45, and 80 µg/ml for 48 h. After that, MTT solution was added to each well and incubated at 37 ˚C for 3 h, followed by solubilization with 0.04 N HCl isopropyl alcohol. Absorbance was measured at 570 nm using the SYNERGY-Neo2 BioTek plate reader. The results were analyzed using the following equation:

Cell Viability (%) = (absorbance of treated well/ absorbance of the no-treatment negative control) x 100.

2.7 Statistical analysis

The MTT cell viability assay was performed in four independent biological repeats (n = 4). Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparison test. Statistical analysis was performed using Prism 9.5.1. software (GraphPad, La Jolla, CA). Error bars ± standard error of the mean (SEM).

3.1 Molecular docking studies and SwissADME calculation

Three-dimensional structures of 3CL^pro (6LU7, 6M2N) and ACE2 (6LZG, 7WPA) were retrieved from PDB database. Structurally, 3CL^pro contains 306, ACE2 contains 596 (6LZG), and 805 (7WPA) residues. Figure S1 depicts the DoGSiteScorer analysis results to assess potential druggable pockets and subpockets in the protein structure. Results of volume Å³, surface Å², and drug score were considered to select suitable binding pockets. In addition, 3CL^pro structures include inhibitors in the active site located at the cleft region between domain I and II. The predicted binding sites were considered for Autogrid preparation.

In the next step, the chemical structure of selected compounds (in total 76; Table S1) was modified by using OpenBabel3.1.1 to prepare molecular docking. After the molecular docking using AutoDock tool of PyRx, favorable protein structures were selected according to their hydrogen bond interactions between ligands and active residues of proteins. List of the natural products having favorable docking scores were listed in Table S2. The results showed that nine Paeonia ligands (catechin, apigenin, palbinone, kaempferol, paeoniflorigenone, eriodictyol, paeonilactone C, cassythicine, and 3-O-Methylquercetin) were able to interact with SARS-CoV-2 at high affinity (from − 7.5 to -9.0 kJ/mol). Table 1 shows the favorable binding energies of these compounds with 3CLpro (6LU7, and 6M2N) and ACE2 (6LZG, 7WPA) of the SARS-CoV-2.

Table 1

List of the natural products with their docking affinity scores (kJ/mol).
	ACE2		3CLpro
Name	6LZG	7WPA	6LU7	6M2N
(-)-Catechin	-9	-8	-7.3	-7.5
Apigenin	-8.8	-8.2	-7.5	-7.6
Palbinone	-8.5	-8.9	-7.5	-7.8
Kaempferol	-8.3	-8.2	-7.4	-7.4
Paeoniflorigenone	-8.1	-8	-7.4	-7.8
Eriodictyol	-7.9	-8	-7.1	-7.5
Paeonilactone C	-7.7	-7.7	-7.1	-7.3
Cassythicine	-7.5	-8.3	-7.7	-7.8
3-O-Methylquercetin	-7.5	-8.3	-6.7	-7.8

3.2 Molecular dynamics (MD) simulation and free energy calculation

The RMSD of protein complexes (lig-fit-prot) during the period of simulation (10 ns) are presented in Fig. 1. The protein and ligand bound ACE2 and 3CLpro exhibit initial rise and then stable nature. However, the RMSD descriptor of complexes such as Kaempferol (L04), Paeonilactone C (L07), Cassthicine (L08), and 3-O-Methylquercetin (L09) showed exceeded 1.0 nm. Unfavorable fluctuation values may be seen in the RMSD profile for longer simulation. Therefore, ligands that have lower values of RMSD profile, such as (-)-Cathecin (L01), Apigenin (L02), Palbinone (L03), Paeoniflorigenone (L05), and Eriadictyol (L06) were selected for further analysis. In Fig. 1, when the RMSD graph Ligands fit to 6LU7 considered, (-)-Cathecin and Eriadictyol showed a more stable nature, and ligand-complex were found to be less than 0.5 nm value. In the RMSD graph Ligands fit to 6LZG, the complexes (-)-Cathecin, Palbinone, and Eriadictyol binding with ACE2 showed a stable profile and their RMSD values were found to be less than 0.4 nm. In the RMSD graph Ligands fit to 6M2N, RMSD values of (-)-Cathecin (L01), Apigenin (L02), Palbinone (L03), and Eriadictyol (L06) were found to be less than 0.4 nm except for Paeoniflorigenone (L05).

Figures 2 and 3 showed that polar and hydrophobic amino acid residues in the 2-D display of binding interactions after 10 ns MD simulation. The 3-D surface view structure with ligand binding sphere view shows the ligands in the binding site of ACE2 and 3CLpro proteins. Binding site surfaces were displayed according to residue charges by specific coloring. Seven amino acid residues of proteases (6LU7, 6M2N), such as Thr26, Hsd41, Cys44, Hsd163, Hsd164, Glu166, and Asp187, were identified to have H-bond interactions with the ligands. On the other hand, five amino acid residues of ACE2 (6LZG), such as Ile291, Asp382, Asn394, Hsd540, and Lys562 formed H-bond interactions with the ligands.

In Fig. 2, the SwissADME results allow acknowledgment of pharmacokinetic properties, druglike nature, and medicinal chemistry friendliness of one or multiple small molecules to support drug discovery. The colored zone indicates the range of physicochemical properties within which compounds are more likely to have favorable oral bioavailability (LIPO (Lipophility): -0.7 < XLOG3 < + 5.0, SIZE: 150g/mol < MV < 500g/mol, POLAR (Polarity): 20 Â² < TPSA < 130 Â², INSOLU (Insolubility): -6 < Log S (ESOL) < 0, INSATU (Insaturation): 0.25 < Fraction Csp3 < 1, FLEX (Flexibility): 0 < Num. rotatable bonds < 9). All compounds were in desired range and no violation for Lipinski filter (≤ 500 Da, LogP value ≤ 5, number of H-bond donor ≤ 5, and H-bond acceptor is ≤ 10).

The gmx-MMPBSA is a free energy calculation tool based on AMBER's MM/PB(GB)SA calculation aiming to perform end-state free energy calculations with GROMACS files. In the study, the Molecular Mechanics Generalized Born model method was applied to calculate the binding free energy, and the last 3 ns of a 10 ns simulation were considered for MM/GBSA calculation. Total free energy (ΔTOTAL) (kJ/mol) mean values were showed in Fig. 4. ΔTOTAL accounts for the interaction energy between the ligand and protein, the solvation effect, and other factors contributing to the free energy change. According to Fig. 4, all the selected compounds exhibited a total free energy below − 20 kJ/mol, indicating that these complexes possess reasonable interaction energies.

3.4 Cytotoxic effects of root extracts on different cell lines

To assess the cytotoxic effects of five plant extracts, we performed the MTT assay on HCT116, HeLa, and HFF cell lines (Fig. 5). While HCT116 and HeLa cell lines were generated from a cancerous origin, the HFF cell line was generated from a non-cancerous origin. However, all plant extract samples showed a cytotoxic effect in all three cell lines at higher concentrations. However, the cytotoxic effect was observed in a dose-dependent manner. The lowest dose (5 µg/ml) resulted in insignificant changes in cell viability compared to the no-treatment control. Therefore, the use of these plant extracts at low concentrations is potentially not cytotoxic. Upon closer inspection, sample 4 resulted in the highest toxicity in all three cell lines, even at lower concentrations than the other plant extracts. On the other hand, sample 16 seemed to have the lowest effect. Therefore, the ultimate purpose of using these plant extracts must be considered when choosing which sample and what concentration to use.

This study aimed to determine possible interactions of phytochemicals found in Paeonia species with human ACE2 protein and the highly conserved SARS-CoV-2 3CL protease (3CL^pro). Seventy-six compounds were retrieved from known Paeonia content in the NPASS database. These compounds were then subjected to molecular docking with the structures of ACE2 from the first SARS-CoV-2 variant (6LZG) and ACE2 from the Omicron variant (7WPA), along with the structures of 3CL^pro containing inhibitors (6LU7 and 6M2N). The results in Table 1 revealed that nine out of the 76 Paeonia compounds (catechin, apigenin, palbinone, kaempferol, paeoniflorigenone, eriodictyol, paeonilactone C, cassythicine, and 3-O-Methylquercetin) exhibited a high affinity for interacting with ACE2 and 3CL^pro structures, with binding energies ranging from − 7.5 to -9.0 kJ/mol.

Subsequently, the nine compounds with favorable affinity values underwent 10ns Gromacs MD simulations. Based on the lig-fit-prot RMSD graphs obtained in Fig. 1, (-)-catechin (L01), apigenin (L02), palbinone (L03), paeoniflorigenone (L05), and eriodictyol (L06) showed lower deviation values, indicating stable interactions. Conversely, compounds with deviations higher than 1.0 nm, namely kaempferol, paeonilactone C, and 3-O-Methylquercetin were excluded from further analysis. Next, the protein-ligand complexes were evaluated using the MM/GBSA method to calculate the ΔTOTAL free energy, which demonstrated a reasonable total binding free energy between the ligands - ACE2 and − 3CL^pro proteins. Examining the results in Fig. 4, all compounds had a total binding free energy < -20 kJ/mol. Upon evaluating all the findings, it is suggested that (-)-catechin (L01), apigenin (L02), palbinone (L03), paeoniflorigenone (L05), and eriodictyol (L06) compounds derived from Paeonia species may have potential interactions with the known SARS-CoV-2 target proteins, ACE2 and 3CL^pro. In Fig. 2, seven amino acid residues of 3CL^pro (6LU7, 6M2N), including Thr26, His41, Cys44, His163, His164, Glu166, and Asp187, were identified to have hydrogen-bond interactions with the ligands after 10 ns MDS. Additionally in Fig. 3, five amino acid residues of ACE2 (6LZG), namely Ile291, Asp382, Asn394, His540, and Lys562, formed hydrogen-bond interactions with the ligands after 10 ns MDS. Based on the findings of this study, it is conceivable that these five compounds from Paeonia extract ((-)-catechin (L01), apigenin (L02), palbinone (L03), paeoniflorigenone (L05), and eriodictyol (L06) compounds) could be potentially utilized in the treatment of SARS-CoV-2, and further investigations can be conducted to explore their application in therapeutic approaches. The following sections discuss their druggable potential in various diseases.

4.1 Catechins

Catechins are a group of flavonoids that are present in a wide variety of plant-based foods, especially tea, chocolate, and fruits like berries and apples (Zanwar et al 2014). According to the research, catechins may have anti-cancer, anti-diabetic, antioxidant, and anti-inflammatory properties that are helpful to human health (Higdon and Frei 2003). Additionally, they may enhance cognitive performance, lower the risk of neurodegenerative disorders, and have cardioprotective properties (Khan and Mukhtar 2013). The health advantages of catechins have been explained by a number of different mechanisms. For instance, they might function as free radical scavengers, guarding against oxidative stress and DNA damage in the cells (Lambert and Yang 2003). They have been demonstrated to limit the development and spread of cancer cells in vitro and animal models, and they may also affect signaling pathways involved in cell proliferation and death (Lambert and Yang 2003). Additionally, catechins have been demonstrated to increase lipid metabolism and lower blood pressure, which are good for cardiovascular health (Kim et al. 2007). They also may have neuroprotective properties that could lower the likelihood of cognitive decline and neurodegenerative illnesses like Alzheimer's and Parkinson's (Pervin et al. 2018). According to a number of in-silico studies regarding its antiviral effect, catechins may be able to obstruct the SARS-CoV-2 viral entrance process by binding to the spike protein and it can inhibit the activity of the SARS-CoV-2 main protease M^pro enzyme. Ghosh et al. (2021) examined the possible binding of eight natural substances, including catechins, to the SARS-CoV-2 M^pro enzyme using molecular docking models. The research discovered that catechins have a high affinity for the enzyme and might be able to reduce its activity and represent viable COVID-19 therapy options (Ghosh et al. 2021). Another study, also published in 2021, looked into the possibility of catechins interacting with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein using molecular docking and molecular dynamics simulations. The study discovered that catechins might attach to the RBD of the spike protein and interact hydrophobically with the amino acid residues to produce hydrogen bonds. The interaction between the spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptor, which is necessary for viral entrance into host cells, may be affected by catechins, the researchers hypothesized (Jena et al. 2021).

4.2 Apigenin

Numerous plants contain apigenin, such as parsley, chamomile, celery, and oranges. It has several pharmacological activities that have been identified, including anti-inflammatory, antioxidant, and anticancer actions (Salehi et al. 2019). It might help treat neurological conditions, including Parkinson's and Alzheimer's disease (Salehi et al. 2019). Additionally, apigenin has been shown to have antiviral effects against several viruses, including the influenza virus (Sithisarna et al. 2013) and hepatitis C virus (Manvar et al. 2012), and it might be useful in the management of viral diseases, such as HIV (Critchfield et al. 1996). Research on apigenin's potential effects on COVID-19 is still in its early phases, and little is known about this subject. The main protease (M^pro) of SARS-CoV-2 can be inhibited by apigenin, suggesting that it may stop viral replication and lessen the severity of COVID-19 (Matondo et al. 2021). According to another in silico study, Apigenin and Narcissin are possible Covid-19 viral inhibitors because they directly bind to RNA polymerase and important viral proteins such as the primary protease and the spike-RBD (Owona et al. 2021).

4.3 Palbinone

Palbinone was originally discovered in Paeonia albiflora in 1993 and then in Paeonia delavayi in 2005 (Kadota et al. 1993; Smolecule 2023). It has been investigated for potential therapeutic activities, including antifungal, antibacterial, anti-inflammatory, and antioxidant (Tuan et al. 2009). There is no study addressed its potential antiviral effect on viruses such as SARS-CoV-2. Studies on Palbinone's possible anti-inflammatory effects revealed that it inhibits the activity of the Janus kinase (JAK)1, 3a-hydroxysteroid dehydrogenase, human monocyte interleukin-1b, and 3a-hydroxy dehydrogenase. (Kadota et al. 1993; 1995; Xiao et al. 2022).

4.4 Kaempferol

Tea, broccoli, kale, and berries are just a few examples of the many plant sources that contain kaempferol (Kashyap et al. 2017). It has been researched for potential health advantages, including its anti-inflammatory, anticancer, and antioxidant effects (Kashyap et al. 2017). Its antiviral properties are also attracting more attention. The antiviral properties of kaempferol have been studied in many investigations. For instance, a study published in 2021 investigated its activity against a variety of viruses, including poliovirus type 1, HSV-1 and 2, respiratory syncytial virus (RSV), Coxsackie B virus type 1, HCV, Canine distemper virus, SARS-CoV, and HIV-1 viruses), human rhinovirus (HRV), Enterovirus A71 (EV-A71) and severe fever with thrombocytopenia syndrome virus (SFTS) and found that kaempferol had a board antiviral effects (Mouffouk et al. 2021). Other studies that assessed kaempferol's activity against SARS-CoV-2 discovered that it may be an effective antiviral agent against the virus's enzymes and proteins, including chymotrypsin-like protease (3CLpro), papain-like protease (PLpro), spike protein (S protein), and RNA-dependent RNA polymerase (RdRp), as well as their capacity to interact with the ACE2 receptor (Mouffouk et al. 2021; Pan et al. 2020; Khan et al. 2021).

4.5 Paeoniflorigenone

Paeoniflorigenone (PF) has demonstrated positive biological actions, such as anti-oxidative (Kim et al. 2009), anti-cancer (Huang et al. 2017; Park et al. 2022), anti-inflammatory (Li et al. 2022), anti-viral and bacterial properties (Demir et al. 2019). PF prevents numerous cancer cells, including head and neck cancer, from proliferating, according to in vitro research (Park et al. 2022). Regarding its antiviral activity, it has been noted to exhibit antiviral activity against the hepatitis B virus (HBV) (Wu et al. 2020).

4.6 Eriodictyol

Eriodictyol is a flavonoid that belongs to the flavanones subfamily and can be found in several medically important plant species like Paeonia. It is among the currently available compounds that were shown to have anti-inflammatory and antiviral properties and thus can be considered a therapeutic agent against COVID-19 (Ayipo et al. 2021; Deshpande et al. 2020). Many researchers tested the inhibitory effects of eriodictyol on SARS-CoV-2 infection based on molecular docking studies. For instance, Lin et al. (2021) studied the Yinqiao powder potential for treating COVID-19, in which eriodictyol is one of its active ingredients. Eriodictyol had a good binding score with 3CLpro, which could inhibit SARS-CoV-2 entry. This was also supported by (Glinsky, 2020), who found that eriodictyol, along with other flavonoids, can be developed as a therapeutic agent for the treatment of COVID-19, based on docking screening and gene expression profile analyses. However, these inhibitory effects must be directly verified in animal experiments and clinical trials (Lin et al. 2021). Others performed a molecular docking study of the spike protein with eriodictyol and it had a good binding energy of -7.9 KJ/mol within the active site of the SARS-CoV-2 ACE2 receptor, suggesting an effective docking ligand. They postulated that eriodictyol can shorten the virus’s lifecycle thanks to its positive drug score, anti-inflammatory, and antioxidant properties (Deshpande et al. 2020). (Rudrapal et al. 2022) also performed in silico screening and molecular docking study against the main protease (M^pro) and papin-like protease enzymes. Among the eleven tested compounds, eriodictyol exhibited a remarkable binding affinity with the proteases with a docking score of -9.027 and binding energy of -212.66 KJ/mol, indicating a strong protease inhibition. Furthermore, (Zhao et al. 2022) evaluated the phytochemicals of Eurya chinensis leaves for their antiviral activity in vitro. Cytopathic effect reduction and antibody-based EC₅₀ assays revealed that the phytochemicals, including the active compound eriodictyol, had antiviral effects against HCoV-OC43. Eriodictyol could reduce the severity of COVID-19 by inhibiting mast cell activation mediated by the inflammatory response of the infection (Theoharides & Kempuraj, 2023). However, further in vitro and in vivo studies must employ the effect of eriodictyol as a potent therapeutic target for COVID-19.

4.7 Paeonilactone C

Paeonilactone C is a monoterpene that can reduce the toxicity of reactive oxygen species (ROS) and therefore an effective antioxidant (Parker et al. 2016). Most published studies discussed and tested the antioxidant properties of paeonilactone C, while there is a lack of data regarding the antiviral effects against SARS-CoV-2. In an effort to test its potency as a neuroprotective compound against oxidative stress in vitro, a study found that paeonilactone C, isolated from Paeonia lactoflora roots, successfully protected primary cortical cell cultures of rats against cytotoxicity induced by H₂O₂, thereby demonstrating a neuroprotective activity determined by MTT assay within the concentration range 0.1–10 µM. They hypothesized that benzoyl moiety contained in the paeonilactone C structure contributes to this neuroprotection activity (Kim et al. 2009). In addition, it was found that paeonilactone C suppresses muscle twitching of frog sciatic nerve-sartorius muscles (Zhao et al. 2016). Further investigations are needed to evaluate the antiviral and anti-inflammatory effects of paeonilactone C (Kim et al. 2009).

4.8 Cassythicine

Cassythicine, a natural product belonging to aporphines, is a quinoline alkaloid that can be found in different medical plants. Several studies proved the inhibitory potential of Cassythicine against COVID-19 as well as other diseases. (Cavallaro et al. 2020) suggested that cassythicine can be used as a competitive therapy against the current therapeutic drug galanthamine for the treatment of Alzheimer’s disease. Molecular modeling studies were performed to illustrate the interactions between the active Cassythicine and acetylcholinesterase and butylcholinesterase enzymes. Their results revealed dual in vitro inhibitory activity against acetylcholinesterase and butylcholinesterase, showing a better inhibitory action compared with galanthamine (Cavallaro et al. 2020). Potent antiviral phytomedicines, including Cassythicine, were also tested against SARS-CoV-2 by targeting the active pocket of its main protease (M^pro). Using virtual screening, cassythicine extracted from the Lauraceae family achieved a stable M^pro-cassythicine docking complex with a docking score of -8.6 KJ/mol. Furthermore, (Barbosa et al. 2021) showed the potential of cassythicine extracted from Octea puberula for having anti-protozoa activity, as evaluated by in vitro studies. Cassythicine should be considered for future in vitro and in vivo studies due to its availability, low toxicity, and cost-effectiveness (Bora et al. 2022).

4.9 3-o-methylquercetin

3-o-methylquercetin is a flavonoid having strong antioxidant and antiviral properties. (Alshawaf et al. 2022) targeted the neuropilin-1 (NRP1) entry point of SARS-CoV-2 to test a 200 ns molecular dynamics (MD) simulation with natural extracts to find a potential antiviral compound against SARS-CoV-2. 3-o-methylquercetin formed a stable complex with NRP1-b1 with tight affinity and a binding energy of -25.52 ± 0.04 KJ/mol and a docking score of -5.91 KJ/mol. Their results indicate the ability of this complex to disrupt the NRP1-b1-S protein interaction (Alshawaf et al. 2022). In addition, 3-o-methylquercetin was tested against the influenza A virus and Mycobacterium tuberculosis in vitro and showed effective antiviral and antituberculosis properties (Lall et al. 2006). A study also showed that 3-o-methylquercetin attenuated the formation of ROS induced by H₂O₂ in liver and lung cells (Kumar et al. 2016). These results make 3-o-methylquercetin an attractive therapeutic candidate for SARS-CoV-2 infection, however, more functional in vivo and in vitro tests are needed (Alshawaf et al. 2022).

Paeonia has garnered significant attention as an important medicinal herb resource, supported by numerous phytochemical and pharmacological reports. Traditional uses of Paeonia, such as treating inflammation, cardiovascular diseases, bacterial and viral DNA polymerases inhibition and neuroprotection, have been partially validated through modern pharmacological studies. This study revealed the potential efficacy of Paeonia phytochemicals against the emerging coronavirus (SARS-CoV-2). Among seventy-six compounds, cathecin, apigenin, palbinone, paeoniflorigenone, and eriadictyol were found to have potential druggable impact on SARS-CoV-2 main protease (3CL^pro) and human ACE2 proteins. In addition, the plant extracts at low concentrations are not cytotoxic for selected cell lines. Overall, this study points to the inhibitory potential of Paeonia phytochemicals as novel therapeutics against SARS-CoV-2. It is important to note that further exploration and investigation of the remaining compounds isolated from Paeonia plants are needed. By conducting more comprehensive studies on a wider range of compounds, researchers can unravel the full potential and therapeutic properties of Paeonia as a valuable medicinal herb. Toxicology studies are also necessary to assess the safety profiles and potential side effects of Paeonia extracts and their isolated compounds. By incorporating these aspects into future studies, a more comprehensive understanding of Paeonia's medicinal potential can be achieved, leading to the development of safe and effective treatments derived from these valuable plant resources.

Acknowledgements

The authors would like to acknowledge Ms. Dorothy Joy H. Huelar for her assistance in the in vitro studies.

Disclosure statement

Authors declare no conflict of interest.

Ethical Approval

Not applicable

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Availability of data and materials

Not applicable

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No competing interests reported.

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Targeting SARS-CoV-2 main protease 3CL pro and human ACE2 with Paeonia Phytochemicals by in silico and in vitro Studies in Terms of Possible COVID- 19 Therapeutics

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Abstract

Figures

1 Introduction

2 Materials and Methods

2.1 Paeonia samples

2.2 Collection and preparation of protein structures

2.3 Selection of phytochemicals and ligand preparation

2.4 Molecular docking

2.5 Molecular dynamics simulation

2.6 Cell culture and assays

2.7 Statistical analysis

3 RESULTS

3.1 Molecular docking studies and SwissADME calculation

3.2 Molecular dynamics (MD) simulation and free energy calculation

3.4 Cytotoxic effects of root extracts on different cell lines

4 DISCUSSION

4.1 Catechins

4.2 Apigenin

4.3 Palbinone

4.4 Kaempferol

4.5 Paeoniflorigenone

4.6 Eriodictyol

4.7 Paeonilactone C

4.8 Cassythicine

4.9 3-o-methylquercetin

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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