Virtual Screening Approach in Identification of Bioactive Lignans from Medicinal Plants as Potential Lead Compounds against Main Protease of SARS-CoV-2 COVID-19

Introduction

The 2019 novel coronavirus (2019-nCoV) is the severe acute respiratory syndrome coronavirus 2 (SARSCoV-2). COVID-19 originated in Wuhan City of Hubei Province of China.¹ Coronaviruses are an etiologic agent of severe infections in both humans and animals, which may cause infectious disorders within the respiratory tract.^2,3 The symptoms of this disease are usually cough, fever, sore throat, breathlessness, fatigue, malaise, etc. Although the disease is mild in people, in a few cases, it may lead to pneumonia, acute respiratory distress syndrome and multi-organ dysfunction.^4,5 Hence, preventive measures and treatment are currently needed. “Since knowledge about this virus is rapidly evolving, readers are urged to update themselves regularly”.⁶

The investigations regarding the treatment of COVID-19 are lacking. Liu et al. have successfully crystallized this virus’s main protease (Mpro), which is accessible to the public in the Protein Data Bank (PDB). This protease is a potential target for the inhibition of Corona virus (CoV) replication.^7,8

Lignans are an important class of phytochemicals obtained from various plant species and show structural similarity.⁹ They possessed a core scaffold formed by two or more phenylpropanoid units. Lignans have many biological and antiviral activities reported in recent years.¹⁰ They are widely distributed in the plant kingdom and are present in plant stems, leaves, flowers, fruits, rhizomes, roots, fruits, and seeds.¹¹ Plants belonging to the Lauraceae family and Machilus, Ocotea, and Nectandra genera are rich sources of lignans.¹² Berberidaceae, Orchidaceae, and Schisandraceae families also showed the presence of many constituents of lignans and neolignans.¹³

Niranthin is the first lignan isolated from Phyllanthus niruri Linn. of the family Euphorbiaceae. It shows liver protection and anti-hepatitis B virus activity.¹⁴ Diphyllin is a natural constituent of plants with naphthalene and one hydroxyl lignans.^15,16 It is an excellent vacuolar ATPase inhibitor in virus cells.¹⁷ Phillygenin is the main constituent of Fructus Forsythiae. It can suppress high glucose-induced lipid accumulation and has antiviral, antibacterial, and antioxidant properties.^18,19 Bicyclol is a biphenyl analog of the active component schizandrin C from Fructus Schiznadrae²⁰ with in-vitro and in vivo antiviral activities. Clinical studies showed that it inhibits virus replication in patients infected with hepatitis viruses.²¹

A literature search revealed that selected lignans (niranthin, diphyllin, phillygenin and bicyclol) have a potential antiviral effect against different viruses and may be effective against molecular targets of COVID-19. Hence, investigating the selected lignans against molecular targets of COVID-19 is necessary using molecular docking techniques. No studies have been reported on the molecular docking of selected lignans against the selected targets of COVID-19. Hence, the present study aimed to investigate the bioactive lignans against the molecular target of a new strain of coronavirus.

Materials and methods

Standard drug

Nelfinavir was used as the standard for comparison.

Lignans/ ligands

Niranthin, diphyllin, phillygenin and bicyclol were used as ligands.

Softwares

PyRx 0.8, Biovia Discovery Studio 2019, Molsoft, Marvin Sketch, and Pre ADME server were used for the analysis.

Ligand preparation

The three-dimensional (3D) structures of the five ligand molecules were retrieved from PubChem (https:// pubchem.ncbi.nlm.nih.gov/) in structural data format (SDF) and converted to PDB format using Discovery Studio 2019. The energy form of all ligand molecules was minimized by using Marvin Sketch mmff94 force field and used for docking.²²

Protein Preparation

3D crystallographic structure of COVID-19 3clpro/ Mpro (PDB ID: 6LU7) was retrieved from PDB (www. rcsb.org). Discovery Studio 2019 was used to remove the water molecules and heteroatoms associated with protein molecules to avoid docking interference and saved in the PDB format. The 6LU7 protein contains two chains- A and C forming a homodimer. Chain A contains SARS-CoV-2 Mpro enzyme; hence, Chain A was selected for macromolecule preparation. The native ligand for 6LU7 is n-[(5-methylisoxazol-3yl) carbonyl]alanyl-l-valyln~1~-((1r,2z)-4-(benzyloxy)-4- oxo-1-{[(3r)-2-oxopyrrolidin 3yl]methyl}but-2-enyl)- lleucinamide.

Drug likeness score of selected bioactive lignans

Drug likeness properties of bioactive molecules were predicted via an online server, Molsoft (http://molsoft. com/mprop/). This was based on molecular weight, logP value, the total number of hydrogen bond donors, and the total number of hydrogen bond acceptors.²³

Pharmacokinetic and toxicological predictions

Pharmacokinetic properties such as absorption, distribution, metabolism, and excretion (ADME) and toxicity studies of ligand molecules are essential in the drug development process. All the possible pharmacokinetic parameters— ADME and toxicity of selected lignans were predicted by an online PreADMET server (http://preadmet.bmdrc.org). PreADMET calculates pharmacokinetic and toxicological parameters such as blood-brain barrier penetration, cellular permeability of CaCO₂ in-vitro, human intestinal absorption, skin permeability, plasma protein binding, mutagenicity, and carcinogenicity.²⁴ The drug molecule having good ADME and minimum toxicity was considered the best drug for the target.

Ligand protein docking

The molecular docking was performed using PyRx 0.8. After the completion of docking, auto dock preferences were obtained for both ligand and target in PDBQT format.The ligand poses with the lowest binding energy were selected for the interaction visualization. Finally, protein and ligand interactions were analyzed under Biovia Discovery Studio 2019.²⁵

Results

Selected ligands were calculated for their drug-likeness properties, and the bioactive Lignans have been previously selected based on adherence to Lipinski’s rule of five. The drug scanning results were calculated, and data is presented in Table 1. The results of pharmacokinetic parameters such as BBB penetration, in vitro Caco-2 permeability, human intestinal absorption, skin permeability, plasma protein binding etc and Toxicological parameters like mutagenicity and carcinogenicity of selected ligands are presented in Table 2. The drug candidates were docked against 6LU7 using PyRx 0.8. The binding energies of the compounds based on their rank are presented in Table 3.

The amino acids in protein play a vital role in the interaction with ligand molecules and form different types of bonds such as hydrogen bonds, electrostatic pi bonds, hydrophobic bonds, etc. The structure of ligand molecules and their 2D interaction with different amino acids on target 6LU7 are presented in Table 4.

Docking analysis visualization of protein 6LU7 and bioactive lignans (nelfinavir, niranthin, diphyllin, phillygenin, and bicyclol) were performed, and their 3D interactions are presented in Figure 1.

Discussion

The present study focused on the main proteases in CoVs PDB ID 6LU7 as potential target proteins for COVID-19 treatment. 6LU7 is the Mpro in COVID-19 and is accessible to the public in PDB. The main protease structure in COVID-19 is a great source to search for drugs of natural and synthetic origin.^{27, 28}

According to Lipinski’s rule of five, if the compound has >500g/mol molecular weight, >10 hydrogen bond acceptors, >5 hydrogen bond donors, and >5 log P values, it shows poor bioavailability and absorption. In the selected lignans in the present study, phillygenin showed the highest drug likeness score (0.1). The compounds niranthin, diphyllin, and bicyclol did not violate Lipinski’s rule of five (Table 1). Nelfinavir and all four compounds showed 90% to 100% human intestinal absorption, CYP2C9 and CYP3A4 inhibition and 89% to 100% plasma protein binding.

Nelfinavir was non-mutagenic, whereas all bioactive lignans showed mutagenic activity. Niranthin, diphyllin and phillygenin showed a medium hERG inhibition risk, whereas bicyclol showed the lowest risk (Table 2).

The binding energies obtained from docking 6LU7 with the nelfinavir, niranthin, diphyllin, phillygenin, and bicyclol were -8.3, -6.1, -8.1,-6.6, and -6.5 kcal/mol, respectively. Diphyllin showed the highest negative binding energy, whereas niranthin showed the lowest (Table 3).

The results of docking analysis (Table 4 and Figure 1) showed that the nelfinavir interacts with protein 6LU7 via two hydrogen bonds. The amino acid residues of the 6LU7 that interact with nelfinavir are GLN A: 110. Diphyllin scored the highest number of hydrogen bond interactions with 6LU7 via four bonds, and amino acid residues of the protein involved in the interaction are HIS A: 41, CYS A: 145, HIS A: 163, and GLU A: 166. Niranthin scored the lowest hydrogen bond interaction with protein by one hydrogen bond via HIS A: 163 amino acid. Phillygenin forms two hydrogen bonds via amino acids GLY A: 143 and GLU A: 166, whereas bicyclol forms three hydrogen bonds via HIS A: 41, CYS A: 145, and GLU A:166.

Conclusions

The results of the present study conclude that niranthin, diphyllin, phillygenin and bicyclol have an affinity towards the molecular target of COVID-19. Nelfinavir may represent a potential treatment option, and niranthin, diphyllin, phillygenin and bicyclol may be recommended as most potential inhibitors of COVID-19 Mpro. 

Conflict of Interest

Nil

Acknowledgements

The Authors are thankful to Dr. Pramod Gadad, Principal of KLE College of Pharmacy, Nipani, and Dr. Sunil S. Jalalpure, Principal, KLE College of Pharmacy, Belagavi, for providing support, motivation, and guidance to complete the present study.