Effective inhibition of coronavirus replication by Polygonum cuspidatum

Institute of Chemical Biology, Shenzhen Bay Laboratories, 518132 Shenzhen, Guangdong, China, New Drug R&D Center, North China Pharmaceutical Corporation, 050015 Shijiazhuang, Hebei, China, School of Pharmaceutical Sciences, South-Central University for Nationalities, 430000 Wuhan, Hubei, China, Nanjing Gemni Biotechnology Co., Ltd, 210023 Nanjing, Jiangsu, China, Guangdong Provincial Key Laboratory of Cell Microenviroment and Disease Research, 518055 Shenzhen, Guangdong, China, State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 100191 Beijing, China, Institute for Advanced Study, Shenzhen University, 518052 Shenzhen, Guangdong, China, Department of Biology, Southern University of Science and Technology, 518055 Shenzhen, Guangdong, China, Guangdong Provincial Key Laboratory of Computational Science and Material Design, 518055 Shenzhen, Guangdong, China, School of Nursing, Hebei Medical University, 050000 Shijiazhuang, Hebei, China, Department of Pharmacognosy, Pharmaceutical College, Hebei Medical University, 050017 Shijiazhuang, Hebei, China


Abstract
Background: The coronavirus disease 2019 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 210 million individuals globally and resulted in over 4 million deaths since the first report in December 2019. The early use of traditional Chinese medicine (TCM) for light and ordinary patients, can rapidly improve symptoms, shorten hospitalization days and reduce severe cases transformed from light and normal. Many TCM formulas and products have a wide application in treating infectious and non-infectious diseases. Polygonum cuspidatum Sieb. et Zucc. (P. cuspidatum), is an important Traditional Chinese Medicine with actions of clearing away heat and eliminating dampness, draining the gallbladder to relieve jaundice, removing blood stasis to alleviate pain, resolving phlegm and arrest cough. In the search for anti-SARS-CoV-2, P. cuspidatum was recommended as as a therapeutic drug of COVID-19 pneumonia.In this study, we aimed to identifies P. cuspidatum is the potential broad-spectrum inhibitor for the treatment of coronaviruses infections. Methods: In the present study , we infected human malignant embryonal rhabdomyoma (RD) cells with the OC43 strain of the coronavirus, which represent an alternative model for SARS-CoV-2 and then employed the cell viability assay kit for the antiviral activity. We combined computer aided virtual screening to predicte the binding site and employed Surface plasmon resonance analysis (SPR) to comfirm the interaction between drugs and coronavirus. We employed fluorescence resonance energy transfer technology to identify drug's inhibition in the proteolytic activity of 3CLpro and Plpro. Results: Based on our results, polydatin and resveratrol derived from P. cuspidatum significantly suppressed HCoV-OC43 replication. 50% inhibitory concentration ( Finally, SPR assay confirmed that polydatin and resveratrol had high affinity to SARS-CoV-2, SARS-CoV 3Clpro, MERS-CoV 3Clpro and PLpro protein. Conclusions: we identified the antiviral activity of flavonoids polydatin and resveratrol on RD cells. Polydatin and resveratrol were found to be specific and selective inhibitors for SARS-CoV-2, 3CLpro and PLpro, viral cysteine proteases. In summary, this study identifies P. cuspidatum as the potential broadspectrum inhibitor for the treatment of coronaviruses infections.

Introduction
An unusual pneumonia of unknown origin was reported in December 2019 [1]. Its clinical features are similar to those of severe acute respiratory virus (SARS) [2]. The viral genome isolated from patients clustered into a clade of betacoronaviruses was distinct from that of the severe acute respiratory syndrome coronavirus (SARS-CoV) [3]. Thus, this disease, named the coronavirus disease 2019 (COVID-19) was caused by a novel coronavirus (2019-nCoV), which was later renamed the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). On March 11, 2020, COVID-19 was declared a global pandemic by the World Health Organization (WHO) [4]. In December 2020, several vaccines shown to be highly effective were granted emergency use authorization. While vaccines will prevent disease occurrence, infected individuals still need treatment options, and repurposing drugs circumvents the lengthy and costly process of drug development. Therefore, it is urgent to excavate the efficacy of currently approved drugs for the treatment of COVID-19 [5][6][7].
Traditional Chinese medicine (TCM) is an important party of the world complementary and alternative medicine. TCM offers a wide variety of application of traditional medicines and herbs for treating human diseases. Treating epidemic diseases with TCM and the treatment scheme of integrated Chinese and western medicine have proven their effectiveness in clinical practice in the management of COVID-19 pneumonia [8][9][10]. Effective prescriptions for the treatment of COVID-19 pneumonia include Qingfei Paidu decoction, Pneumonia No.1 Formula, and Shufeng Jiedu capsule. Polygonum cuspidatum Sieb. et Zucc., commonly known as Huzhang in Chinese and Japanese knotweed in English, is a widely used medicinal plant. The roots of Polygonum cuspidatum are used as an important traditional Chinese medicine for centuries, which have the actions of clearing away heat and eliminating dampness, draining the gallbladder to relieve jaundice, removing blood stasis to alleviate pain, resolving phlegm and arrest cough [11]. Of the traditional medicines tested, Polygonum cuspidatum and its active components, resveratrol, were found to inhibit Middle East respiratory syndrome coronavirus (MERS-CoV) in Vero E6 cells [12]. Over 67 compounds have been identified in P. cuspidatum, including quinones, stilbenes, flavonoids, coumarins, and lignans [13]. P. cuspidatum and its active components, resveratrol and emodin, were also found to attenuate influenza viral replication in A549 cells [14]. During the fight against COVID-19 in China, Chinese scientists found some herbs to be symptomatic for COVID-19 through clinical practice and through component screening in the Chinese medicine component library. One of them is P. cuspidatum, in which Polydatin has the strongest inhibitory effect on coronaviruses [15]. Hence, P. cuspidatum was recommended by the academician Zhang boli in the treatment of COVID-19 pneumonia [16].
The first genome sequence of SARS-CoV-2 has been entered into the database by Wu et al. [17] in February 2020. Coronavirus has six open reading frames (ORFs) and the first ORF (ORF1a/b), which comprises approximately 67% of the entire genome, encodes 16 non-structural proteins [18]. The remaining ORFs encode four major structural proteins, which are spike surface glycoprotein (S), small envelope protein (E), matrix protein (M), nucleocapsid protein (N), and accessory proteins.
The polypeptides are released from each polyprotein through extensive proteolytic processing, primarily performed by the virally encoded chymotrypsin-like protease 3CL protein (3CLpro, also called the main protease, Mpro) with additional cleavage performed by the viral papain-like protease Pl protein (Plpro) [19,20]. Owing to their essential roles in viral replication, both the proteases are recognized as attractive targets for the development of anti-SARS-CoV-2 therapeutics [21,22]. While searching for a SARS-CoV-2 3CLpro and PLpro inhibitor from natural sources, we found that polydatin and resveratrol from P. cuspidatum possessed inhibitory activity against both the proteases. Herein, we identified the active ingredients from P. cuspidatum and their mechanism of inhibiting coronavirus.

Materials
Polydatin and resveratrol were purchased from solarbio company (Solarbio Science & Technology Company, Beijing, China).

Viruses and Cell lines
HCoV-OC43(OC43) strain was purchased from American Type Culture Collection (Human Coronavirus OC43(ATCC VR-1558) and propagated using RD cells (ATCC CCL-136). OC43 stock virus was obtained in RD cells in DMEM supplemented with 2% FBS at 72 h postinfection (hpi). Viral titer (PFU/mL) was determined by plaque assay.

OC43 Viral infection inhibition assay
RD cells were infected OC43 at the multiplicity of infection (MOI = 0.01 IU) in the presence of indicated compound diluted in DMEM supplemented with 2% FBS [23]. The compounds and the virus were maintained with the cells during the 48 h incubation at 37 • C. OC43 infection efficiency in RD cell was evaluated by immunofluorescence staining against the Nucleoprotein. In general, RD cells were fixed by 100% methanol at 48 hpi, blocked by 1% BSA/PBS at 37 • C for 1 h, stained with anti-OC43 Np mAb (Sigma, Saint Louis, MO, USA) at 1:1000 at 37 • C for 1 h, and followed by secondary antibody incubation for an-other 1 h. Nuclei were stained with Hoechst 33342 (Thermo Scientific, Waltham, MA, USA) at 1:5000 for 15 min. Images were captured by an Mshot fluorescence microscrope (Olympus, Tokyo, Japan).

Determination of the median cytotoxic concentration (CC 50 )
The median cytotoxic concentration (CC 50 ) of the compounds was determined by the CellTiter-Glo 2 (Promega Corporation, Madison, WI, USA) cell viability assay kit [24]. In general, 293T-hACE2 cells were seeded in 96-well plates with three-fold serial-diluted compounds (from 100 µM to 0.137 µM). After 24 h, the cell viability was detected by CellTiter-Glo 2 reagent by following the manufacture's instructions, and the luminescence was determined by a Spectra MaxiD3 multi-well Luminometer 458 (Molecular Devices, San Jose, CA, USA). The CC 50 value was determined by nonlinear regression analysis [25].

Computer aided virtual screening
The SARS-CoV-2 3CLP (pdb:6lu7) and PLP (pdb:6w9c) program database (PDB) files were downloaded from the PDB website (http://www.rcsb.org/). All heterogeneous atoms and the 3CLP ligand were removed and 6lu7 chain A was selected for subsequent molecular docking. The 3CLP docking grid was maximized for polydatin docking. All heterogeneous atoms and the PLP ligand were removed and 6w9c chain A was selected for subsequent molecular docking. The PLP docking grid was maximized for polydatin docking.
PDB file (6lu7 chain A) and (6w9c chain A) were converted to the PDBQT format as macromolecules before virtual screening. The grid (ligand docking search space) was located as described above. Then, Autodock Vina 1.1.2 [26] was used for the subsequent molecular docking. Protein-ligand interactions were visualized using Pymol version 1.7.4.5 (Schrödinger, New York, NY, USA). The amino acid residues of 3CLP protein close to the hit ligands (≤1 Å) were highlighted as potential interactive residues involved in the protein-ligand interaction.

Surface plasmon resonance (SPR) analysis
To conform the binding affinities of polydatin and resveratrol to SARS-CoV-2, SARS-CoV, MERS-CoV 3CLpro and PLpro, SPR technology based Biacore T200 biosensor was used (Biacore AB, Uppsala, Sweden) [27].   All the screening assays were performed over the unmodified dextran surface and the protein surface. During the 3CLpro and PLpro binder assay, the sample concentrations were set at 100 µM, 50 µM, 25 µM, 12.5 µM, 6.25 µM, 3.125 µM, 1.5625 µM, 0.78125 µM, 0 µM. For sample dilution, the pH and the concentration of both DMSO and buffer substances in samples and running buffer were carefully matched. Each sample assay consisted of a 180s buffer injection and a 300-s dissociation phase and the blank injection was used to check the carryover effects. The signal was adjusted for nonspecific binding of the samples to dextran matrix by subtracting the signal in the reference channel from the signal in the active channel. The experimental data were fitted and analyzed using the BIA evaluation software by steady state analysis.

FRET technology-based IC 50 determination
To further investigate possible inhibitory activities of polydatin and resveratrol against SARS-CoV-2, SARS-CoV, and MERS-CoV 3CLpro, and PLpro, the inhibitory effects of the compounds were tested by the FRET method [28]. For this assay, the internally quenched fluorogenic substrate Dabcyl-KNSTLQSGLRKEE-Edans and Dabcyl-KRLKGGAPIKGE-Edans substrate (synthesized by genescript company) was applied and the compounds were diluted to 100 mM, 20 mM, 10 mM, 2 mM, 1 mM, 200 µM, 100 µM, and 20 µM. A 1.2 µL concentration of the solution of the compounds was taken and thoroughly mixed with 120 µL of 2 µM 3CL protease (final concentration of DMSO is 0.5%) and incubated at 4 • C for 2 h. Thereafter, 100 µL of each concentration sample was taken into a 96-well plate, and then added 100 µL of 20 µM fluorescent substrate solution to start the reaction. At this time, the final concentration of the 3CL or PL protease is 1 µM, the final concentration of the fluorescent substrate is 10 µM, and the final concentrations of the compounds are 500 µM, 100 µM, 50 µM, 10 µM, 5 µM, 1 µM, 0.5 µM, and 0.1 µM, respectively. The enhanced fluorescence emission upon substrate cleavage was monitored at the excitation and emission wave lengths of 340 and 488 nm. The reaction was measured continuously for 1 h. According to the logistic formula, the origin software was used to perform a non-linear fitting to calculate the IC 50 value of compounds in the inhibition of 3CL protease activity. The formula is as follows: In the above formula, A 0 refers to the enzyme activity when 0.5% DMSO is added, A(I) refers to the enzyme activity under different concentrations of compounds, I refers to the concentration of the compound, and p refers to the u factor.

Identification of anti-HCoV-OC43 activity
To verify the inhibitory effect of polydatin and resveratrol on coronavirus at the cellular level in vitro, we measured the antiviral activity of polydatin and resveratrol by indirect immunofluorescence assay (IFA) on RD cells infected with OC43-CoV which is an alternative model for SARS-CoV-2. As shown in Fig. 1A and 1B, polydatin and resveratrol significantly suppressed HCoV-OC43 replication, compared with that of the control (DMSO), and with over 90% inhibitory effect at a concentration of 100 µM, respectively. Cytotoxicity of these drugs to HEK293T cells was measured by CellTiter-Blue assay. As shown in Fig. 2A and 2B, cytotoxicity concentration 50% [CC 50 ] of polydatin and resveratrol was 420.6 µmol/L and 182.4 µmol/L, respectively.

SPR technology-based binder identification
To confirm the interaction between polydatin and resveratrol and SARS-CoV-2, SARS-CoV, MERS-CoV 3CLpro, and PLpro protein, we tested whether polydatin and resveratrol had high affinity to target protein using the SPR assay. The SARS-CoV-2, SARS-CoV, and MERS-CoV 3CLpro and PLpro protein were immobilized separately on a CM5 chip with the tested compounds flowing across their surface. We found that polydatin bound to SARS-CoV-2, SARS-CoV, and MERS-CoV 3CLpro and PLpro protein exhibiting a strong dose-dependent response, with respective KD (equilibrium dissociation constant) val-  Fig. 7A-E). These results suggested that polydatin and resveratrol had a specific affinity   to SARS-CoV-2, SARS-CoV 3CLpro and the PLpro protein, and thus can inhibit virus replication by binding to the active center of 3CLpro and the PLpro protein.

Discussion
Since the COVID-19 pandemic occurred in early 2020, scientists have been trying to find effective treatment methods and remedys. TCM has been proven effective for COVID-19 treatment [29][30][31]. P. cuspidatum, is a traditional Chinese medicine with actions of clearing away heat and eliminating dampness, draining the gallbladder to relieve jaundice, removing blood stasis to alleviate pain, resolving phlegm and arrest cough [32]. Polygonum cuspidatum and its active components were found to inhibit Mid-dle East respiratory syndrome coronavirus (MERS-CoV) in Vero E6 cells [12] and attenuate influenza viral replication in A549 cells [14].
In this study, we focused on the SARS-CoV-2 3CL and PLP protein as target sites for the identification of possible Food and Drug Administration-approved drugs for repositioning in COVID-19 treatment.
As mentioned above, 3CLpro and PLpro of SARS-CoV-2 are good targets to design inhibitory chemicals since some flavonoids inhibit the proteolytic activity of SARS-CoV 3CLpro [33,34]. The similar sequence between them [35,36] together with their conserved active site suggests that similar flavonoids may work for SARS-CoV-2 3CLpro and PLpro. We found that polydatin and resveratrol revealed the prominent inhibitory activity against SARS-CoV-2 and MERS-CoV 3CLpro and PLpro. The measured IC 50 values against SARS-CoV-2 3CLpro were 18.66 and 29.81 µM, respectively. Polydatin and resveratrol also revealed the prominent inhibitory activity against OC43-CoV at the cellular level. The assay and virtual screening results indicate an important conclusion of P. cuspidatum as a broad-spectrum inhibitor of coronaviruses. Since the epithelial cells of the upper airways are the primary target site of SARS-CoV-2 infection. Nasal spray will be a suitable administration route of P. cuspidatum. Clinical trials and in vitro experiments supporting these findings would be of great importance towards overcoming COVID-19.

Conclusions
The COVID-19 pandemic wreaked havoc on human health from the beginning of 2020. In the search for anti-SARS-CoV-2 therapeutics, we identified the antiviral activity of flavonoids polydatin and resveratrol from P. cuspidatum on RD cells infected with the OC43 strain of the coronavirus. In order to elucidate the mechanism of polydatin and resveratrol on inhibition of virus replication, we combined virtual screening with experimental studies. Polydatin and resveratrol were found to be specific and selective inhibitors for SARS-CoV-2, 3CLpro and PLpro, viral cysteine proteases. In summary, this study identifies P. cuspidatum as the potential broad-spectrum inhibitor for the treatment of coronaviruses infections.

Author contributions
HX and NW wrote the manuscript, HX performed OC43 Viral infection inhibition assay and determination of the median cytotoxic concentration and computer aided virtual screening, JL and SS performed Surface plasmon resonance (SPR) analysis and FRET technology-based IC 50 determination and provided comments. ZX, XC, BH, GS, DZ provided the comments and checked the manuscript. MS offered technical support for the analysis of surface plasmon resonance. NW, GW, RH designed study and offered professional advice. All authors read and approved the final manuscript.

Ethics approval and consent to participate
Not applicable.

Acknowledgment
Thanks to all the peer reviewers for their opinions and suggestions. We warmly appreciate professor Huan Yan (Wuhan University) for gifting OC43.