key: cord-1024834-0j53vg0k
authors: Bahadur Gurung, Arun; Ajmal Ali, Mohammad; Lee, Joongku; Abul Farah, Mohammad; Mashay Al-Anazi, Khalid; Al-Hemaid, Fahad
title: Identification of SARS-CoV-2 inhibitors from extracts of Houttuynia cordata Thunb
date: 2021-09-06
journal: Saudi J Biol Sci
DOI: 10.1016/j.sjbs.2021.08.100
sha: 610cd224d4a6bfc457ab42302559e7b73bd7b851
doc_id: 1024834
cord_uid: 0j53vg0k

Houttuynia cordata Thunb., a perennial herb belonging to the Saururaceae family is a well-known ingredient of Traditional Chinese medicine (TCM) with several therapeutic properties. During the severe acute respiratory syndrome (SARS) outbreak in China, it was one of the approved ingredients in SARS preventative formulations and therefore, the plant may contain novel bioactive chemicals that can be used to suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a virus for which there are currently no effective drugs available. Like all RNA viruses, SARS-CoV-2 encode RNA-dependent RNA polymerase (RdRp) enzyme which aids viral gene transcription and replication. The present study is aimed at understanding the potential of bioactive compounds from H. cordata as inhibitors of the SARS-CoV-2 RdRp enzyme. We investigated the drug-likeness of the plant's active constituents, such as alkaloids, polyphenols, and flavonoids, as well as their binding affinity for the RdRp enzyme. Molecular docking experiments show that compounds 3 (1,2,3,4,5-pentamethoxy-dibenzo-quinolin-7-one), 14 (7-oxodehydroasimilobine), and 21 (1,2-dimethoxy-3-hydroxy-5-oxonoraporphine) have a high affinity for the drug target and that the complexes are maintained by hydrogen bonds with residues like Arg553, Cys622 and Asp623, as well as hydrophobic interactions with other residues. The lead compounds' complexes with the target enzyme remained stable throughout the molecular dynamics simulation. Analysis of molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) and molecular mechanics generalized Born surface area (MM-GBSA) revealed the key residues contributing considerably to binding free energy. Thus, the findings reveal the potential of H. cordata bioactive compounds as anti-SARS-CoV-2 drug candidate molecules against the target enzyme.

Title: Identification of SARS-CoV-2 inhibitors from extracts of Houttuynia cordata Thunb.

Houttuynia cordata Thunb., a perennial herb belonging to the Saururaceae family is a wellknown ingredient of Traditional Chinese medicine (TCM) with several therapeutic properties.

During the severe acute respiratory syndrome (SARS) outbreak in China, it was one of the approved ingredients in SARS preventative formulations and therefore, the plant may contain novel bioactive chemicals that can be used to suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a virus for which there are currently no effective drugs available. Like all RNA viruses, SARS-CoV-2 encode RNA-dependent RNA polymerase (RdRp) enzyme which aids viral gene transcription and replication. The present study is aimed at understanding the potential of bioactive compounds from H. cordata as inhibitors of the SARS-CoV-2 RdRp enzyme. We investigated the drug-likeness of the plant's active constituents, such as alkaloids, polyphenols, and flavonoids, as well as their binding affinity for the RdRp enzyme. Molecular docking experiments show that compounds 3

(1,2,3,4,5-pentamethoxy-dibenzo-quinolin-7-one), 14 (7-oxodehydroasimilobine), and 21

(1,2-dimethoxy-3-hydroxy-5-oxonoraporphine) have a high affinity for the drug target and that the complexes are maintained by hydrogen bonds with residues like Arg553, Cys622 and Asp623, as well as hydrophobic interactions with other residues. The lead compounds' complexes with the target enzyme remained stable throughout the molecular dynamics simulation. Analysis of molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and molecular mechanics generalized Born surface area (MM-GBSA) revealed the key residues contributing considerably to binding free energy. Thus, the findings reveal the potential of H. cordata bioactive compounds as anti-SARS-CoV-2 drug candidate molecules against the target enzyme.

Keywords: Coronaviruses, coronaviral RdRp, Houttuynia cordata, bioactive compounds, natural products, COVID-19, SARS-CoV-2, virtual screening, molecular docking, molecular dynamics simulation Manuscript (WITHOUT Author Information) Click here to access/download;Manuscript (WITHOUT Author Information);3. manuscript_unmarked.doc Click here to view linked References

Houttuynia cordata Thunb. is a flowering and perennial herb native to China, Japan, Korea, and Southeast Asia. It is the single species in the genus Houttuynia, which belongs to the Saururaceae family. It thrives in wet, shaded hillside, roadside, and field ridges between 300 and 2600 meters in elevation (Jiangang et al., 2013) . H. cordata has a slender stem and heartshaped leaves and bears greenish-yellow flowers. It grows up to an average height of 15-50 cm. When rubbed, it has a fishy odour and a somewhat astringent flavour. When the stalk and leaves have matured, it is generally harvested in the summer or autumn (Yang and Jiang, 2009 ). In Southeast Asia's indigenous medicine systems, H. cordata is a well-known traditional medicinal ingredient (Jiangang et al., 2013) . It relieves fever, resolves toxins, reduces edema, drains pus, and promotes urination (Zheng et al., 1998) . It was one of the components in SARS preventive formulations authorized by the Chinese Ministry of Health during the epidemic of Severe Acute Respiratory Syndrome (SARS) (Lau et al., 2008) . H. cordata has been utilized in China as an edible vegetable and an effective traditional Chinese medicine (TCM) since ancient times (Yang and Jiang, 2009 ). It has antileukemic (Kwon et al., 2003) , antimutagenic , anti-inflammatory (Chiang et al., 2003) , and antianaphylaxis (Li et al., 2005) properties, as well as the potential to boost immunologic function. Amino acids, vitamins, and trace elements such as potassium, zinc, iron, copper, and manganese are the nutrients present in H. cordata. The active components in the plant include volatile oils, organic acids, flavonoids, alkaloids, polyphenols, water-soluble polysaccharides etc (Yang and Jiang, 2009 ).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and pathogenic coronavirus that first appeared in late 2019 and has since caused a pandemic of an acute respiratory disease known as coronavirus disease 2019 (COVID-19), which poses a threat to human health and public safety (Hu et al., 2020) . SARS-CoV-2 is a new betacoronavirus with a genomic sequence that is 79 % similar to severe acute respiratory syndrome coronavirus (SARS-CoV) and 50 % similar to Middle East respiratory syndrome coronavirus (MERS-CoV) (Lu et al., 2020) . SARS-CoV-2 is a positive-strand RNA virus with a genome of around 30 kb which encode 14 open reading frames (ORFs) (Jiang et al., 2021) . All RNA viruses encode RNA-dependent RNA polymerases (RdRps) enzyme which aids viral gene transcription and replication in collaboration with other viral and host components (Gorbalenya et al., 2002) . The RdRps are multi-domain proteins that catalyze the formation of phosphodiester linkages between ribonucleotides in the presence of a divalent metal ion using an RNA template (Jia and Gong, 2019) . SARS-CoV-2 RdRp (also known as nsp12) is an important element of the replication/transcription machinery (Pachetti et al., 2020) . Nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain, interface domain, and C-terminal RdRp domain are all found in the nsp12 subunit (Gao et al., 2020) .

The RdRp domain, which is present in all single-subunit polymerases, is shaped like a right hand, with fingers, palm, and thumb subdomains (Kirchdoerfer and Ward, 2019) . RdRp is one of the most important targets for antiviral medication research, as it is found in a wide range of viruses (Pachetti et al., 2020) . Favipiravir (Furuta et al., 2013) , Galidesivir (Lim et al., 2017) , Remdesivir (Agostini et al., 2018) , and Ribavirin (Morgenstern et al., 2005) are several RdRp inhibitors that have been proposed to target SARS-CoV-2.

The absence of effective treatments for human coronaviral infections (Jean et al., 2020) , as well as the high fatality rates associated with the novel coronavirus (2019-nCoV) (Piroth et al., 2021) , has prompted the development of new vaccines. In this study, we looked at the possibilities of utilizing H. cordata bioactive molecules to halt SARS-CoV-2 replication. We screened out drug-like molecules from H. cordata using in silico toxicity filters and utilized molecular docking and molecular dynamics to describe the binding interaction of the chosen bioactive compounds with the target enzyme (SARS-CoV-2 RdRp).

The information on the various bioactive compounds of H. cordata was obtained from a literature search (Jiangang et al., 2013; Ma et al., 2017) . A total of 49 molecules consisting of 22 alkaloids, 11 flavonoids and 16 polyphenols were chosen for the study. The 3D structures of the molecules were retrieved from the PubChem database (Kim et al., 2016) and the molecules whose three-dimensional structures were not available in the chemical databases were sketched using ACD/ChemSketch (Freeware) 2019.1.2 software and were processed into 3D structures using Open Babel version 2.4.1 software (O'Boyle et al., 2011) and further energy-optimized using Merck molecular force field (MMFF94) (Halgren, 1996) following our previously described protocol (Gurung et al., 2016) . The molecules were prepared for docking using AutoDock Toos-1.5.6 by the addition of Gasteiger charges and hydrogen atoms and torsions for each molecule were optimally defined.

The bioactive molecules were screened based on various drug-like filters such as Lipinski's rule of five parameters (Lipinski, 2004) : molecular weight (MW) (<=500), hydrogen bond acceptor (HBA) (<=10), hydrogen bond donor (HBD) (<=5), partition coefficient between noctanol and water (clogP) (<=5) and in silico toxicity filters such as mutagenicity, irritancy, tumourigenicity, reproductive health etc. DataWarrior program version 5.0 software (Sander et al., 2015) was used to analyze the physico-chemical characteristics of the selected compounds, such as drug-like properties and toxicity.

The three-dimensional cryo-electron microscopy structure of the enzyme target-SARS-CoV-2 RdRp (PDB ID: 7BV2) at a resolution of 2.50 Å, was retrieved from Protein Data Bank (http://www.rcsb.org/). This crystal structure contains a ternary complex of RdRp enzyme (nsp12) with cofactors nsp7 and nsp8 bound to the template-primer RNA and triphosphate form of remdesivir (Yin et al., 2020). The target enzyme (nsp12) was prepared by deleting the cofactors and removing the heteroatoms including ions, co-crystallized ligands and water molecules. Further, an optimum number of polar hydrogen atoms and Kolmann charges were added to the target enzyme using AutoDock Toos-1.5.6.

The binding affinity of each molecule along with the control inhibitor was evaluated against the enzyme target using a molecular docking approach. The binding sites for the compounds were defined by choosing a grid box of dimensions of 60×60×60 Å 3 with a grid spacing value of 0.375 Å centred at x:92.5053, y:93.2594, z:103.4061 around the bound co-crystallized ligand. AutoDock 4.2 (Morris et al., 2009 ) was used for performing molecular docking study using Lamarckian genetic algorithm with fifty independent docking runs for each molecule including the cocrystal ligand (triphosphate form of remdesivir).

The binding poses for each molecule was considered based on the lowest binding energy score. LigPlot+ tool version v.1.4.5 was used to analyse the molecular interactions (hydrogen bonds and hydrophobic interactions) between the target enzyme and compounds (Laskowski and Swindells, 2011) .

The AMBER16 software, which is accessible on ligand and receptor molecular dynamics (LARMD) (http://chemyang.ccnu.edu.cn/ccb/server/LARMD/), was used to simulate the protein-ligand complexes for a 4-ns MD simulation in an explicit water model (Yang et al., 2020) . The following equation (1) was used to determine the binding free energy (Δ )

where Δ is the binding energy, Δ is the solvation entropy, and Δ is the conformational entropy. The entropy was estimated using the MM/PB (GB) SA technique (Hou et al., 2011) , and the enthalpy was computed using an empirical approach (Hao et al., 2009; Pan et al., 2008) .

A total of 49 major bioactive molecules as shown in The binding affinities of these drug-like bioactive molecules were evaluated against the target enzyme-SARS-CoV-2 RdRp using molecular docking. The docking scores of the molecules were compared with the control inhibitor Remdesivir which is bound as a co-crystal structure.

The top three lead molecules identified for SARS-CoV-2 RdRp were 7oxodehydroasimilobine (14), 1,2,3,4,5-pentamethoxy-dibenzo-quinolin-7-one (3) and 1,2dimethoxy-3-hydroxy-5-oxonoraporphine (21) with binding energies of -6.38 kcal/mol, -6.24 kcal/mol and -6.15 kcal/mol respectively and their corresponding inhibition constants were 21.14 µM, 26.88 µM and 31.31 µM ( Table 2) (Figure 3c) . Rg can be explained as the root mean square distance from each atom of the system to its centre of mass (Lobanov et al., 2008) . The Rg values for protein-ligand complexes: RdRp_14, RdRp_3 and RdRp_21 show stable fluctuation between 28.8 to 29.2 Å, 28.6 to 29.2 Å and 28.7 to 29.1 respectively (Figure 4) .

With a folding free energy barrier, the Q (fraction of native contacts) represents conformational dynamics and transition states of a protein (Best et al., 2013) . Table 3 ). In all the three protein-ligand complexes except RdRp_3, the major contribution to the binding energy is by the van der Waals energy component. The top ten residues contributing towards the binding interaction between RdRp and 14 include Asp452, Tyr455, Ser549, Lys551, Arg553, Pro620, Lys621, Cys622, Asp623 and Arg624 (Figure 7a) . The residues such as Tyr455, Lys551, Arg553, Asp618, Tyr619, Lys621, Asp623, Asp760, Asp761 and Lys798 contribute significantly to the total binding energy between RdRp and 3 (Figure 7b) . Similarly, the top ten residues contributing towards the binding interaction between RdRp and 21 include Asp452, Tyr455, Arg553, Thr556, Val557, Asp623, Arg624, Ser681, Ser682 and Thr687 (Figure 7c) .

Medicinal plants have long been recognized as a source of therapeutics, and they continue to be a valuable resource for discovering new drug candidates (Atanasov et al., 2015) . Many of these plants have been used in traditional medicine to treat diseases that are viral in origin (Ben-Shabat et al., 2020) . Besides gaining a better understanding of pathological processes, the pharmaceutical industry has been concerned about the source of molecules. Natural medicines are gaining popularity due to several advantages, including lower costs, acceptability due to a long history of usage, better patient tolerance, and fewer or no adverse effects (Akram et al., 2018) . In the present study, we explored the potential of three major classes of phytochemicals-alkaloids, flavonoids and polyphenols from H. cordata as inhibitors of the SARS-CoV-2 RdRp enzyme. H. cordata (Saururaceae) is a traditional Chinese medicine (TCM) that has been used for hundreds of years to treat pulmonary-related problems such as abscesses, phlegm, cough, and dyspnea and is effective in the treatment of pneumonia, infectious disease, and other respiratory disorders (Lau et al., 2008) . Besides, the plant has anti-inflammatory (Park et al., 2005) , anti-allergic (Kim et al., 2007) , virucidal (Chiang et al., 2003) , anti-oxidative (Ng et al., 2007) , and anti-cancer properties (Kim et al., 2001) . RdRp, one of the most important drug targets found in several viruses is a major component of the SARS-CoV-2 replication/transcription machinery (Pachetti et al., 2020) . In our present studies, compounds 14 (7-oxodehydroasimilobine), 3 (1,2,3,4,5-pentamethoxydibenzo-quinolin-7-one) and 21 (1,2-dimethoxy-3-hydroxy-5-oxonoraporphine) were found to be the most potent bioactive molecules interacting with the enzyme target with a binding affinity higher than the control (remdesivir). The complexes of these lead molecules with the target enzyme remained stable throughout the simulation time in terms of the root mean square deviation (RMSD), radius of gyration (Rg), and percentage of native contacts (Q) plots. Remdesivir, an adenosine analogue first designed for hepatitis C and later investigated for Ebola is a competitive inhibitor of RdRp enzyme (Triggle et al., 2021) . All the three bestdocked molecules of H. cordata in our studies belong to the alkaloids class. Alkaloids are a class of natural compounds produced from plants that have potent antiviral properties and therefore, represent potential candidates for finding effective COVID-19 therapies (Majnooni et al., 2021) . Compound 14 had substantial protein tyrosine phosphatase 1B (PTP1B) inhibitory action with an IC50 value of 2.672 µM while 3 (10 µM) had modest hepatoprotective efficacy against D-galactosamine-induced WB-F344 cell injury (Ma et al., 2017) . PTP1B is a validated therapeutic target for type 2 diabetes since it acts as a negative regulator of insulin signalling pathways (Shrestha et al., 2019) . Previous studies investigating the immunological and antiviral aspects of SARS-preventive mechanisms of H. cordata found that the HC water extract stimulates significant proliferation of mouse splenic lymphocytes, increased the proportion of CD4+ and CD8+ T cells, and a significant increase in the secretion of IL-2 and IL-10, and exhibited significant antiviral properties by inhibiting SARS-CoV RdRp and 3C-like protease (3CL pro ) enzymes (Lau et al., 2008) . Further, the anti-SARS-CoV-2 potential of H. cordata was recently demonstrated by Das et al. (2021) whose studies suggested 6-Hydroxyondansetron and Quercitrin as a new therapeutic drug against COVID-19. Both these compounds showed good binding with three SARS-CoV-2 protein receptors such as main protease (M pro ), papain-like protease (PL pro ) and ADP-ribose phosphatase (ADRP).

The binding of drug-like bioactive compounds of H. cordata to the RdRp, an enzyme involved in the replication and transcription of SARS-CoV-2, was investigated using molecular modelling techniques such as molecular docking and dynamics simulation.

Compounds 14 (7-oxodehydroasimilobine), 3 (1,2,3,4,5-pentamethoxy-dibenzo-quinolin-7one) and 21 (1,2-dimethoxy-3-hydroxy-5-oxonoraporphine) were found to be best docked to the target enzyme and formed stable protein-ligand complexes throughout the simulation time. These compounds may be developed into promising drug candidates for SARS-CoV-2

infections.

The authors report no conflicts of interest in this work.

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The authors report no conflicts of interest in this work.

The authors would like to extend their sincere appreciation to the Researchers Supporting Project number (RSP-2021/154)