key: cord-305511-gdpxvqkk
authors: Dave, Gaurav S.; Rakholiya, Kalpna D.; Kaneria, Mital J.; Galvadiya, Bhemji P.; Vyas, Sudhanshu R.; Kanbi, Vaktabhai H.; Patel, Manubhai P.
title: High affinity interaction of Solanum tuberosum and Brassica juncea residue smoke water compounds with proteins involved in coronavirus infection
date: 2020-08-11
journal: Phytother Res
DOI: 10.1002/ptr.6796
sha: 
doc_id: 305511
cord_uid: gdpxvqkk

The world is in an immediate need of treatment for coronavirus disease (COVID‐19). Chronic exposure of hydroxychloroquine in the treatment of COVID‐19 may have multiple adverse effects on human physiology, such as cardiac arrhythmias. Natural compounds need to be evaluated as treatment and preventive agents in coronavirus infection. A total of 30 compounds of Solanum tuberosum and Brassica juncea residue smoke water were selected for the virtual screening against SARS‐CoV‐1, SARS‐CoV‐2 and cellular proteins involved in the mechanism of infection. Docking analysis identified lead molecules with favorable binding energy, number of poses and hydrogen bond interactions, which indicates the effective modulation of ACE2 and TMPRSS2 receptors. Results indicated (a) curcumenol, (b) N‐desmethylselegiline, (c) phentermine and (d) sphingolipid derivatives as a selective and potent candidates in comparison to hydroxychloroquine for COVID‐19 treatment. Our in silico findings, therefore, warrant further in vitro validations of the selected compounds for the discovery of novel preventive and therapeutic drug against SARS‐CoV‐2 infection.

the rate of transmission, and post-infection remedies are to be developed to prevent mortality. The treatment given to infected patients based on patient's symptoms and broad-spectrum antiviral drugs is the only option till new vaccine or therapeutic agents would be available throughout the world.

A specific set of proteins from human and SARS-CoV are involved in the infection mechanism. Human transmembrane serine proteases type II (TMPRSS2) and angiotensin-converting enzyme 2 (ACE2) play a main role in SARS-CoV-S protein binding in infection and entry into the human host cell (Jia et al., 2009; Shulla et al., 2011) . Similarly, RNA-dependent RNA polymerase (RdRp) presents a promising target due to its essential role in RNA synthesis and replication of RNA viruses (Shannon et al., 2020) . Virus encoded proteases are actively involved in SARS-CoV entry in host cells, and papain-like protease (PLpro) is responsible in virus replication for the cleavages of replicase to release Nsp1, Nsp2 and Nsp3 (Harcourt et al., 2004) . 3C-like main protease (3CLpro) directly mediates the maturation of Nsps, which is essential in the life cycle of the virus . This can be the founding target to investigate the structural and catalytic mechanisms of SARS-CoV-2 3CLpro for drug development. Earlier, small peptide and molecules have been investigated as an inhibitor of SARS-CoV and SARS-CoV-2 3CLpro . Two major proteins of COVID-19, that is, RNA dependent RNA polymerase (RdRp) (Elfiky, 2020) and SARS-CoV-2 Spike protein (SARS-CoV-2-S) (Bhattacharya et al., 2020) are the main proteins involved in attachment and infection cycle in human cell host. Whereas ACE2 and TMPRSS2 are involved in the mechanism of entry of COVID-19 in host cell (Hoffmann et al., 2020) .

Molecular modulation by lead molecules can be utilized to target COVID-19 through different proteins of virus and human to avert the infectivity of the virus. Through the tools of Bioinformatics and in silico methodologies, drug designing is accelerated to vanish the SARS-CoV-2 infection.

Currently, repurposing of hydroxychloroquine (HCQ) as a treatment of SARS-CoV-2 is in practice in India (COVID-19, 2020) and in the rest of the world. Repurposing potential of HCQ against SARS-CoV-2 needs to be evaluated through in vivo and in vitro experiments thoroughly. In search of a superior drug (highly specific and minimum side effects) than HCQ, exploration of additional resources have to be performed.

Plants are a remarkable source of bioactive compounds, which require the in silico and wet lab experiments for its exploitation as a treatment for SARS-CoV-2. Smoke water of plant material is responsible for various physiological and biochemical activities in plant (Salomon et al., 2017) . Solanum tuberosum and Brassica juncea are regular food crops all over the world and possess remarkable pharmacological potential (Bontempo et al., 2015; Lee et al., 2014; Rakholiya et al., 2011) . In view of the above, the present research evaluates the in silico interaction study of reported molecules from Solanum tuberosum and Brassica juncea residue smoke water (Dave et al., 2018) docked with TMPRSS2, PLpro, SARS-CoV-2 3CLpro, SARS-CoVRdRp, SARS-CoV-2-S and ACE2 to prevent/cure COVID-19.

Phytoconstituents that possess remarkable pharmacological activity were selected as reported from Solanum tuberosum and Brassica juncea residue smoke water (Dave et al., 2018) and Ligand library was prepared of potential molecules to be used for the docking study with human, SARS-CoV and SARS-CoV-2 proteins as given in Table 1 (Sakai et al., 2014) , SARS-CoV-S in complex with ACE2 (PDB: 2AJF) (Li et al., 2005; Micholas & Jeremy, 2020) , PLpro (PDB: 3E9S) , SARS-CoV-2 3CLpro (PDB: 6LU7) , SARS-CoVRdRp (PDB: 6NUR) (Elfiky, 2020) , SARS-CoV-2 spike glycoprotein structure (SARS-CoV-2-S) (PDB: 6VSB) (Grifoni et al., 2020) , binding complex of human ACE2 and RBD (PDB: 6VW1) (Qiu et al., 2020) , SARS-CoV-2-S (closed) (PDB: 6VXX) (Walls et al., 2020) , SARS-CoV-2-S with one S B (open) (PDB: 6VYB) (Walls et al., 2020) and Human angiotensin-converting enzyme 2 (ACE2) (PDB: 1R42) (Jia et al., 2009) are selected for the present study as target proteins ( Figure 1 ). All water molecules were removed, and in the final stage, hydrogen atoms were added to the receptor molecule. For the ligand preparations, SMILES was obtained from PubChem and was further translated to mol file using "Open babel" Translator A Molecular Mechanics (MM) method UFF was used for refining initial geometries, using the "Clean Geometry" option in the ArgusLab Software. The active site was defined from the coordinates of the ligand in the original PDB files Protein for the receptor protein. Residues that lie within 5 Å unit area of ligand that interact with it through their side chain were identified and were considered as Active site residues. The docking between receptor and ligand was performed using the "Dock a ligand" command. A spacing of 0.4 Å between the grid points was used. Binding site box size was set to (15 × 15 × 15 Å) so as to encompass the entire active site. Molecular docking was implemented on 1Z8G, 2AJF, 3E9S, 6LU7, 6NUR, 6VSB, 6VW1, 6VXX, 6VYB and 1R42 receptor against ligands using Argus Lab 4.0.1 (Mark A. Thompson, Planaria Software LLC, Seattle, WA, http://www.arguslab.com) to find the reasonable binding geometries and to explore the protein-ligand interactions.

Docking of the protein-ligand complex was mainly targeted only on the predicted active site. Docking simulations were performed by selecting "Argus Dock" as the docking engine and their relative stabilities were evaluated using molecular dynamics, and their binding affinities, using free energy simulation, pose and time. Singletrajectory method was used for the binding energy calculation. The selected residues of the receptor were defined to be a part of the binding site and ligand-protein interaction visualized by Biovia Discovery studio.

T A B L E 1 Molecular structure and reported pharmacological functions or properties of selected compounds for virtual screening Sr. no. Name Structure Application/property 1 a Curcumenol Anti-inflammatory, Anti-tumor and hepatoprotective (Lo et al., 2015) 2 a N-Desmethylselegiline Treatment of Parkinson's disease (Mizuta et al., 2000) 3 b Phentermine Appetite control (Baumann et al., 2000) 4 a Monodesmethylpheniramine Antihistamines are used in treatment of allergies (Kelmenson et al., 2013) (Cui et al., 2018) 6 b (5alpha,8beta,9beta)-5,9-Epoxy-3,6-megastigmadien-8-ol

Constituent of Passifloraedulis (passion fruit) (Yannai, 2003) Sr. no. Name Structure Application/property 15 a Î ± -9(10)-EpODE Plant and algae fatty acid derivative (Richardson et al., 2017) 16 a Methylthiobenzoic acid (4-Methylthiobenzoic Acid) Anti nephrotoxicity, antioxidant (Husain et al., 1996) 17 a Polidocanol Sclerosing agent (Goldman & Guex, 2017) 18 b 3-Methylbutyraldehyde oxime Plant derivative (Sørensen et al., 2018) 19 b 6-Hydroxy pseudo oxynicotine Nicotine metabolite (Ganas et al., 2008) 20 b 8-Isoquinoline methanamine Isoquinoline derivative (You et al., 2019) 21 b Desethyletomidate Etomidate derivative and 11β-Hydroxylase inhibitor (Pejo et al., 2016) 22 b DMPO Spin trapping reagent for free radical detection (Kalyanaraman et al., 1994) 23 b Gallic acid Natural phenolic compound (Kahkeshani et al., 2019) 24 b N-(2-Methylpropyl) acetamide Acetamide derivative found alcoholic beverages (Yannai, 2003) 

Sphingolipid derivative for anti-inflammatory action (Vasconcelos et al., 2017) 26 b N-methyl hexanamide Hexamide derivative (Sha et al., 1995) 27 b Phytosphingosine Antimicrobial and anti-inflammatory natural compound (Pavicic et al., 2007) 28 b Pinidine Piperidine alkaloid (Virjamo et al., 2010) 29 b Sphinganine Sphingolipid derivative (Christie & Han, 2012) 

The field of molecular docking has emerged during the last three decades and now is becoming an integral aspect in drug discovery and development area. Molecular docking is utilized for the prediction of protein-ligand complexes, which is composed of two components: a search algorithm, an algorithm that creates possible protein-ligand complex geometries, and thus performs the process of "pose generation," and a scoring function that predicts the binding affinity of the ligand to the protein based on the complex geometry. Binding energies are the most widely used mode of measuring binding affinity of a ligand. In the present study, binding energy of the best pose was recorded as docking results of the selected compounds, carried out by Argus lab in Table 2 .

From the virtual screening, with the proteins of human and SARScoronavirus, involved in the mechanism of infection, three most potent compounds were found, namely (a) curcumenol, (b) N-desmethylselegiline and (c) Phentermine exhibited promising activity based on the docking T A B L E 2 Docking score against selected receptors using ArgusLab of screened compounds score (Table 2) . Moreover, Monodesmethylpheniramine and N-(3Ehexadecenoyl)-deoxysphing-4-enine-1-sulfonate also possess remarkable activity with high specificity and selectivity towards the selective proteins ( Walls and Asn61 with covalent bond (Figure 2c ).

In addition, other compounds brought promising results of binding activity against the studied proteins, such as (5alpha,8beta, 9beta)-5,9-Epoxy-3,6-megastigmadien-8-ol, (R)-2,3-Dihydroxypropane- (Li et al., 2003) . Blocking the interaction between host ACE2 and SARS-CoV-S can be utilized as a strategy to prevent the COVID-19 infection in host cell. Human ACE2 recognition as a receptor by SARS-CoV-2-S is due to its structural similarity and sequence conservation with SARS-CoV-S glycoproteins (Walls et al., 2020) . Moreover, structural studies of ACE2 in complex with SARS-CoV-2-S, with human and other species may contribute to understanding interspecies transmission and receptor usage of COVID-19 (Qiu et al., 2020) . In SARS-CoV infection, ACE2 is released from human airway epithelial cells, an important initial site of infection, where it retains terminal carboxypeptidase activity as well as its ability to bind the virus, in addition, ACE2 must be cell attached to function as a SARS-CoV receptor (Jia et al., 2009) . Previously, it was demonstrated that TMPRSS2 mediated ACE2 cleavage increases SARS-CoV-S-mediated entry in host cell (Heurich et al., 2014; Shulla et al., 2011) . Therefore, targeting the TMPRSS2 is also important as a preventive strategy against COVID-19 infection.

Curcumenol was studied earlier for its interaction with human serum albumin through the docking study , We have found N-desmethylselegiline is a metabolite of selegiline (Monoamine oxidase-B inhibitor) used for the treatment of Parkinson's disease (Heinonen et al., 1994) and protects neurons through stimulating expression of the neurotrophic factors (Mizuta et al., 2000) . N-Desmethylselegiline interacted with all the studied proteins with remarkable docking score, mainly TMPRSS2, SARS-CoV-2-S, PLpro, RdRp and ACE2 are involved in the mechanism of infection of SARS-CoV-2 in human (Hoffmann et al., 2020) . Spike protein cleaved into S1 and S2 by the host cell, TMPRSS2, and other proteases, and S1 binds with host cell surface receptors, whereas S2 mediates virus-cell and cell-cell membrane fusion .

Spike structural integrity and cleavage activation play a key role in virus invasion and virulence . TMPRSS2 activates SARS-CoV infection through spike protein of virus in host cell, so the inhibition of TMPRSS2 can prevent the coronaviruses entry into host cells (Glowacka et al., 2011) . Recently, to target SARS-CoV-2, many

anti-viral and anti-bacterial drugs are virtually screened to inhibit TMPRSS2 . Present results complement the protease specific action of the drug, binding to PLpro showed the preventive effect/inhibitory effect to virus infection, encouraged the use as an ACE2 inhibitor as reflected from the docking score.

Phentermine has been approved by the Food and Drug Administration (FDA) as appetite suppressants for the treatment of obesity (Connolly et al., 1997) . Phentermine effects involve dopamine neurons, which increase extracellular dopamine and involve in appetite control (Baumann et al., 2000) . Phentermine showed interaction with minimum energy level with TMPRSS2, PLpro. RdRp and ACE2. Phentermine was found to bind with all the studied protein structures of human and SARS-CoV, as well as its complexes, compliant with its potency and selectivity to block the coronavirus infection in human. in vitro, as well as in controlled or uncontrolled clinical studies (Gautret et al., 2020; Liu et al., 2020; Yao et al., 2020) . Moreover, HCQ has been recommended in India to treat suspected and confirmed cases of COVID-19 (COVID-19, 2020) . In addition, HCQ use has to be balanced against the risk of cardiac arrhythmia (Chen et al., 2006) , whereas curcumenol and N-desmethylselegiline have not been reported of side effects on heart function. In contrast, Phentermine is associated with valvular heart disease-upon chronic exposure (Connolly et al., 1997) . Apart from the best three molecules found in the present study, other molecules from natural sources like (5alpha,8beta,9beta)-5,9-Epoxy-3,6-megastigmadien-8-ol, 

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The authors declare no conflicts of interest.

https://orcid.org/0000-0001-5317-9217Mital J. Kaneria https://orcid.org/0000-0003-4498-6003