Novel Coronavirus (SARS-CoV-2) Main Protease: Molecular docking of Puerarin as a Potential inhibitor

SARS-CoV-2 a single stranded RNA virus which triggered the global pandemic Coronavirus Disease-2019 (COVID-2019). It has infected about 2,844,712 patients and brought forth mortality rate to about 201,315 among 216 countries as cited by WHO. Drugs including Chloroquine and Hydroxychloroquine derivatives are being administered in most urgent cases; although, with probable side effects to people with metabolic disorders. Thus, unavailability of authorized drugs and treatment for this pandemic demands the research world to discover natural compounds with potency to cure it. This paper assesses the iso�avonoid puerarin from Pueraria lobata as a possible inhibitor of the main protease of SARS-COV-2 (Mpro) via in silico approach, for example molecular docking, Lipinski’s rule of �ve and toxicity prediction (ADME). Puerarin revealed high binding a�nity with the target site of SARS-CoV-2 main protease. This compound slightly meets the criteria of Lipinski’s rule and does not possess properties that could cause adverse effects in humans thus, making puerarin a potential drug candidate to investigate for its usage against COVID-19.

From late December 2019, the symptoms such as high fever, cough, tiredness, phlegm production, di culty in breathing started to increase as cluster of pneumonia were noticed and recognized as betacoronavirus in Wuhan, Hubei Province, China (Wang et al., 2020).The beta-coronavirus was primarily and o cially termed as 2019-novel coronavirus (2019-nCoV) and coronavirus 2019  respectively by WHO while the International Committee of Coronavirus Study Group (CSG) suggested the usage of the term SARS-CoV-2 (Guo et al., 2020).Through the sequential analysis study of the virus, bat was thought as the biological host of the virus.This virus is transmitted between humans by binding to angiotensin converting enzyme-2 receptor (Zhou et al., 2020).
Various kinds of medications have been suggested which are largely antiviral drugs and are currently being used clinically (Gautret et al., 2020;Huang et al., 2020).In the meantime, chloroquine phosphate and hydroxychloroquine sulphate are being administered for the urgent treatment of COVID-19 by the recommendations gotten from in vitro and some clinical studies data although the Food and Drug Administration (FDA) speci ed that these drugs are not yet accepted due to some recent experiments revealing that hydroxychloroquine can result in deleterious and reasonable austere effects in individuals who are already being treated for metabolic disorders.Furthermore, treatment of hydroxychloroquine was perceived to impede in ammatory cytokines (Guastelgname and Vallone et al., 2020).
Due to the side effects hydroxychloroquine have on the viral proteins associated in the life cycle of the virus, a new effective drug candidate with less side effects needs to be established.In silico studies of chemically synthetic drugs for example Darunavir have been reported (Khan et al., 2020).Also, several treatment target sites for handling COVID-19 have been established, SARS-CoV-2 main protease was preferred as a result of the potential in handling CoV-2 patients and stopping the multiplying process of the virus (Jin et al., 2020).
Phytomolecules are rapidly produced, cheap and are right candidate for compositions of drugs of interest.This model of repositioning phytocompounds in drug discovery will accelerate the process.Thus, the exploration for effective natural compounds with promising ability to inhibit the main protease.(Khaerunnisa et al., 2020).Puereria lobata also known as Kudza is a fast developing perennial vine arising from China.The plant is eatable with health sustaining bene ts of Kudzu arising from its owers and roots, which comprise numerous phytomolecules such as iso avones and saponins (Li et al. 2010).Puereria lobata was reported to have anti-viral property against HIV-1 replication by inhibiting the entry of HIV-1 into target cells (Mediouni et al. 2018).The bioactive compound in Puereria lobata is puerarin.Puerarin possesses many pharmacological activities, comprising anti-diabetic, anti-cancer, antiin ammatory, and anti-viral activities (Wu et al., 2013;Gan and Yin, 2015;Zhu et al., 2015;Ullah et al., 2018).Puerarin has been reported to have antiviral properties against Porcine epidemic diarrhea virus.As a result of these interests, this work engages the molecular docking analysis of puerarin against SARS-CoV-2 main protease.

Molecular Interaction of Puerarin with SARS-CoV-2 main protease
Target preparation and Docking SARS-CoV-2 main protease 3D structure was downloaded from Protein Data Bank (PDB with ID number 6W63).To monitor the interaction modes of puerarin with SARS-CoV-2 main protease, the Maestro Molecular Modeling platform (version 11.5) by Schrödinger, LLC was used.The Protein Preparation Wizard module was used to prepare the structure.The Glide receptor grids constructed based on the cocrystalized ligand-binding sites in the Glide application (Glide, version) of Maestro.The center of each grid was arranged at the centroid of the crystalized ligand-binding site which is set with inner (acceptable space for the ligand center) and outer (search space surrounded all the ligand atoms) box sizes of 10 and 20 Å, respectively by virtual screening work ow (Bagherzadeh et al., 2020).The extra precision (XP) Work ow in Maestro was used to dock puerarin with SARS-CoV-2 main protease.During the molecular docking full exibility of the protein and puerarin were considered.The maestro software was used to determine protein-ligand interaction.
ADMET predictions ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) analyses establishes the pharmacokinetics of a drug molecule.Swissadme (http://www.swissadme.ch) and admetSAR (predated.bmdrc.kr)servers were used in this study to predict and describe signi cant druglikeness such as mutagenicity, toxicological dosage level and pharmacologically relevant properties of the compounds (Elekofehinti et al., 2018).

Results
The 3D structure of the puerarin and lopinavir was modeled and used as a target for docking simulation against SARS-CoV-2 (Figure 1).Puerarin and lopinavir structures (Figure 2) were downloaded from pubmedchem and prepared for the docking (3D) using Maestro Molecular Modeling platform.
The molecular docking scores and binding a nity of puerarin with -OH, puerarin with -O and lopinavir (standard protease inhibitor) with SARS-CoV-2 protease produced negative values for free energy -8.070 and -4.658 Kcal/mol as well as lopinavir with -8.081 Kcal/mol in the grid box, indicating high interaction with the binding pocket (Table 1).
Puerarin with -OH revealed good docking score and higher binding interaction found rooted into the binding cavity of SARS-CoV-2 displaying all the major interaction (Figure 3) compared with lopinavir a protease inhibitor (Figure 5).Also, lopinavir showed a good docking pose with SARS-CoV-2 than puerarin with -O which exhibited the lowest binding energy (Figure 4). 2 shows that puerarin slightly meet all the requirements of the rule of ve.

Data presented in Table
Puerarin has H-bond donor value of 6.Data as predicted using admetSAR server revealed the prospective ADME pro les of the puerarin.ADMET properties, as derived from admetSAR server, reveal that puerarin had no signi cant ADME properties that could cause antagonistic effects in humans (Table 3).It was predicted to have high human intestinal absorption (HIA), not blood brain barrier permeant, inhibits CYP2C19, CYP2C9 and CYP3A4 cytochromes, and plasma protein binding value was found to be 54.383253.Skin and MDCK (Madin Darby canine kidney) permeability values was -4.6011 and 3.99199 respectively.The result in this study (AMES Test) shows puerarin is not a potential mutagen and non-carcinogen.

Discussion
With the aim of combating the high death rate caused by SARS-CoV-2 virus, numerous compounds are already in trial to give antidote to this terrible disease plague.Numerous scholars have gone on several studies that can be employed to derive various therapeutic substitute in the cure of SARS-CoV-2 virus (Bagherzadeh et al., 2020).Furthermore, hydroxychloroquine is being given to emergency cases, though, it was reported to elevate the hydrogen ion concentration of protease and documented as a possible SARS-CoV-2 inhibitors (Bagherzadeh et al., 2020;Wang et al., 2020).Although, the use of hydroxychloroquine is not without severe side effects but effective, there is need to provide alternative measures with little or no side effects.Hence, the use of natural plant products such as puerarin are safe, cheap, readily available with no known side effects.
Puerarin, a potent bioactive compounds extracted from Pueraria lobata was used in this study due to its overwhelming properties.This compound was analyzed using molecular simulation tools against SARS-CoV-2 main protease virus showing a high binding a nity (-8.070Kcal/mol) with the active site residues when compared to lopinavir, a protease inhibitor with binding energy value of -8.081 Kcal/mol.Although, some bioactive compounds reported to inhibit SARS-CoV-2 protease was not capable to prove their binding a nity when likened to puerarin.Examples of such compounds include quercetin -8.58 Kcal/mol, demethoxycurcumin -8.17 Kcal/mol and kaempferol -9.41 Kcal/mol, respectively (Khaerunnisa et al., 2020).Thus indicating that puerarin has a better inhibitory property against SARS-CoV-2 protease.
Lipinski's rule of ve is a technique used to assess drug-likeness of a chemical compound to ascertain whether a chemical compound has certain biological activities or pharmacological properties that would make it a possible effective oral drug in humans (Lipinski, 2004).It also aids in illustrating high possibility of failure or success due to drug-likeness of molecules ful lling the underlying rules such as; molecular mass ≤ 500 Dalton, high lipophilicity, which is represented as LogP, should be less than 5, hydrogen bond donors not greater than 5, hydrogen bond acceptor not more than 10 and molar refractivity should be between 40-130 (Lipinski, 2004;Oyinloye et al., 2019).Puerarin revealed exceptional druggability properties which is con rm as a result of its molar refractivity which indicates the its ability to pass through speci c biomembranes amid weak or strong interactions.
Furthermore, the lipophilicity of puerarin reveals its characteristics for oral absorption.Though, puerarin slightly obey the Lipinski rule of ve, it can act as a drug candidate which is laudable of testing in biological systems.
The ADME data, predicted that puerarin had no signi cant ADME properties that can lead to serious side effects in humans.It is also a non-inhibitor of p-gp inhibition indicating the ability to liberate phosphate group from adenosine triphosphate and concurrent bonding of adenosine diphosphate to glycoprotein.The capability of puerarin to in ltrate blood brain barrier is of remarkable bene t, as it suggests a therapeutic effect against glucose -neurotoxicity.The prediction of human absorption via Caco-2 based penetration assay is routinely performed during drug development.However, puerarin could satisfy as a good drug as it has easy passage through the blood brain barrier, human intestinal absorption and Caco-2 penetration.Puerarin is also not mutagen neither carcinogenic as indicated in the data present.
The safety and toxicity of medicinal plants remains a giant concern to health practitioners particularly modulation of cytochrome P450 enzyme family (CYP) and P-glycoprotein (P-gp) due to the therapeutic consequence they have, as CYPs are the most recognized enzymes involved in biotransformation of drug.(Elekofehinti et al., 2018).The predicted inhibition of CYP2C9, CYP2C19, and CYP3A4 by puerarin, hence proposes its signi cant role in drug metabolism.Puerarin does not halt the metabolism of numerous bene cial drugs through the inhibition of CYP2C19, oxidation of steroids, fatty acids and biotransformation of drugs in addition to the synthesis and degradation of hormones via the inhibition of CYP3A4.Taken together, puerarin has good binding a nity with SARS-CoV-2 slightly meeting the Lipinski's rule of ve and has good ADME toxicity pro le.The ADMET prediction revealed puerarin as a safe compound which can be given as a drug.Although, the properties are considerable in silico data, additional ndings from in vitro, clinical and preclinical investigations addressing SARS-CoV-2 ought to be deliberated on.

Declarations Con ict of Interest
There is no con ict of interest expressed by the authors.

Figures
Figure 1 The Figure 2

.
Zhou P, Yang X, Wang X, Hu B, Zhang L, Zhang W, Si H, Zhu Y, Li B, et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature, 2020; 579(7798), 270-273 Zhu C, Wu Y, Jiang Z, Zheng C, Wang L, Yang X, et al.Dietary soy iso avone attenuated growth performance and intestinal barrier functions in weaned piglets challenged with lipopolysaccharide.Int

Table 2 :
evaluation of the oral drug-likeness puerarin using Lipinski's rule of five filters HBD; Hydrogen bond donor; HBA: Hydrogen bond acceptor