Eucalyptol (1,8 Cineole) from Eucalyptus As COVID-19 Mpro Inhibitor

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 31 March 2020 doi:10.20944/preprints202003.0455.v1

Eucalyptol (1,8 cineole) from eucalyptus as COVID-19 Mpro inhibitor. However, essential oil a potential inhibitor of further research is necessary to investigate COVID 19 corona virus infection by their potential medicinal use.

Molecular docking studies

Arun Dev Sharma* and Inderjeet Kaur Keywords: COVID-19, Essential oil, Eucalyptol, Molecular docking PG dept of Biotechnology, Lyallpur Khalsa College Jalandhar

*Corresponding author, e mail: [email protected] Graphical abstract

Abstract

Background: COVID-19, a member of corona virus family is spreading its tentacles across the world due to lack of drugs at present. Associated with its infection are cough, fever and respiratory problems causes more than 15% mortality worldwide. It is caused by a positive, single stranded RNA virus from the enveloped coronaviruse family. However, the main viral proteinase (Mpro/3CLpro) has recently been regarded as a suitable target for drug design against SARS infection due to its vital role in polyproteins processing necessary for coronavirus reproduction. Objectives: The present in silico study was designed to evaluate the effect of Eucalyptol (1,8 cineole), a essential oil component from eucalyptus oil, on Mpro by docking study. Methods: In the present study, molecular docking studies were conducted by using 1- click dock and swiss dock tools. Protein interaction mode was calculated by Protein Interactions Calculator. Results: The calculated parameters such as RMSD, binding energy, and binding site similarity indicated effective binding of eucalyptol to COVID-19 proteinase. Active site prediction further validated the role of active site residues in ligand binding. PIC results indicated that, Mpro/eucalyptol complexes forms hydrophobic interactions, hydrogen bond interactions and strong ionic interactions. Conclusions: Therefore, eucalyptol may represent potential treatment potential to act

Introduction represents a potential target for the

COVID-19 is easily transmissible and it has inhibition of CoV replication [Lu, 2020]. It already been spread worldwide. Symptoms was observed that genome of CoV encodes are flu-like and can include fever, muscle two proteins ppla and pplb which are and body aches, coughing, and sore throat. involved in spike, membrane, envelop, Symptoms may appear 5-6 days after nucleoprotein, replicase, and polymerase infection. As of March 20h, 2020, over activity of viruses. This function is 243,000 cases of COVID-19 have been performed by main protease (Mpro/3CLpro) confirmed worldwide, over 10,000 of which (Liu and Wang, 2020). The Mpro has 3 have resulted in death. At present, no structural domains; domain I (residues 8 - specific therapies for COVID-19 are 101) and domain II (residues 102 - 184) both available and research regarding the have beta barrel motifs representing treatment of COVID-19 are infancy. chymotrypsin catalytic domain and domain However, the measures that have been III (residues 185 - 200) with a helical implemented remain limited to preventive structure participates in dimerization of and supportive therapies, designed to protein and active enzyme production. prevent further complications and organ Given its vital role in polyprotein processing damage [Morales et al., 2020]. Some and virus maturation, Mpro is considered to preliminary studies have investigated be a suitable target for viral inhibitor potential combinations that include anti development as an approach toward SARS.

malarial drug chlorouinone, and anti-HIV Environmental factors can greatly influence vaccines can be used to treat COVID-19 the secretion of secondary metabolites from infections. A separate investigation aromatic plants. Therefore, great attention performed by Lu (2020) indicated that has been paid to the secondary metabolites among 4 tested drugs (nelfinavir, secreted by plants that may be developed as pitavastatin, perampanel, and praziquantel), medicines [Xang et al., 2018]. Among nelfinavir was identified as the best potential several compounds, essential oils, from inhibitor against COVID-19. medicinal plants, have been reported to have

The main protease (Mpro)/chymotrypsin- antiviral bioactivities [Zakaryan et al., like protease (3CLpro) from COVID-19, 2017]. Essential oils are highly volatile substances in a mixture of terpenes,

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 31 March 2020 doi:10.20944/preprints202003.0455.v1

oxygenated derivatives and other aromatic Lipinski’s rule of five, which proposes that compounds. Eucalyptol (1,8 cineole) is the molecules with poor permeation and oral principal component found in eucalyptus oil absorption have molecular weights > 500, C from all eucalyptus plants (Goodger et al., logP > 5, more than 5 hydrogen-bond 2016). Eucalyptol is a natural organic donors, and more than 10 acceptor groups compound that is a colorless liquid. It is a Adherence with Lipinski’s rule of five as cyclic ether and a monoterpenoid. In the calculated using SWISSADME prediction present study, we investigated eucalyptol as (http://www.swissadme.ch/). potential inhibitor candidates for COVID-19 Binding Mode of Docked Complexes Mpro. The findings of the present study will provide other researchers with opportunities The docked complex structure output format to identify the right drug to combat COVID- was submitted into the Protein Interactions 19. Calculator (PIC) webserver (http://pic.mbu.iisc .ernet.in/) in order to Material and methods map the interaction of the resulting docked Proteins/Macromolecules complex [27]. The parameters such as number of hydrogen bonds, number of COVID-19 3CLpro/Mpro structures were hydrophobic residues, and number of obtained from PDB (https://www.rcsb.org/). aromatic and ionic interactions were The native ligand for 3clpro/Mpro structures considered in interpreting the strength of the was Eucalyptol (1,8 Cineole). interaction Ligand and Drug Scan Determination of Active Sites The 3-dimensional (3D) structure of The amino acids in the active site of a eucalyptol (CID 278) was obtained from protein were determined using the PubChem Computed Atlas for Surface Topography of (https://pubchem.ncbi.nlm.nih.gov/). Proteins (CASTp) PubChem is a chemical substance and (http://sts.bioe.uic.edu/castp/index.html?201 biological activities repository consisting of l). three databases, including substance, compound, and bioassay databases. Drug- Molecular Docking like properties were calculated using

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 31 March 2020 doi:10.20944/preprints202003.0455.v1

The docking study of the compound over 19 provides a nice path to identify potential COVID-19 Mpro was studied using 1-click drug candidates to prevent infection. As docking (https://mcule.com/apps/1-click- cited by Liu and Wang (2020), proteases docking/) and swiss doc represent key targets for the inhibition virus (http://www.swissdock.ch/ ) softwares. replication, and the protein sequences of the SARS-CoV Mpro and the 2019-nCoV Mpro Result and Discussion are 96% identical, hence host proteases can Computational and structural biology be used as potential therapeutic targets. We methods have accelerated the discovery of followed the structural biology aspects novel drugs used to treat viral diseases. which focus on the availability and retrieval Corona viruses (CoVs) are a group of of a main protease (Mpro) or 3C-like viruses that infect animals and humans. CoV protease (3CLpro) receptor structure from infections affect animals in various ways PDB database. The ligand (Eucalyptol) was like: respiratory, fever, cold, digestive, and docked to main protease (Mpro) or 3C-like liver systems of humans and animals. It is protease (3CLpro) using 1-click and swiss the main protease (Mpro) found in the CoV dock softwares. Table 1 shows the structure associated with the severe acute respiratory of ligand and amino acids found in the syndrome (SARS), which can be accessed in active site pockets of Mpro. PDB and was suggested to be a potential Previously several ligands and drug drug target for 2019-nCov [Lu, 2020). In candidate compounds have been selected, as many viruses, proteases play essential roles per criteria of Lipinski’s rule of five. So the in viral replication; therefore, proteases are ligands that did not incur more than 2 often used as protein targets during the violations of Lipinski’s rule could be used in development of antiviral therapeutics. In molecular docking experiments with the CoV, the Mpro protein is involved in virus target protein. The drug scanning results proteolytic maturation and has been (Table 1) show that eucalyptol, ligand used examined as a potential target protein by in this study, was accepted by Lipinski’s inhibiting the cleavage of the viral rule of five. 1-click docking tool that was polyprotein to prevent the spread of used to find out interaction of inhibitor i.e infection. The invention of the eucalyptol (1,8 cineole) with Mpro/3CLpro Mpro/3CLpro protease structure in COVID- protein revealed 4 different poses based on

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 31 March 2020 doi:10.20944/preprints202003.0455.v1

and phenolics from essential oils, have been reported to show antiviral bioactivities [Im et al., 2015; Zakarayan et al., 2017]. We investigated that eucalyptol (1,8 cineole) as potential inhibitor of the COVID-19 Mpro. Hydroxy groups (-OH), ketone groups (=O) and ether groups (-O-) in eucalyptol compounds are predicted to play roles amino acid residue interactions at the active site of COVID-19 Mpro.

the dock score and the pose with highest negative values indicated maximum binding affinity (Table 3). The results of Swiss Dock are shown in Table 3. The results of Swiss Dock showed full fitness and Gibbs free energy predicts docking interactions. The eucalyptol showed full fitness of-2291.07 kcal/mol and estimated ΔG of-6.04 kcal/mol within active site amino acids PHE138 and HIS61 of Mpro/3CLpro proteins of COVID- 19 (Fig. 1). Located at the interface between

domains I and II, the 2 conserved residues His61 and PHE138 form the catalytic dyad RMSD data indicated good affinity of ligand of Mpro. CAST-P server also revealed the with protein structure with local and global presence of PHE138 as active site residue of RMSD values 5.09 (Fig. 2). Once we cavity 3 in Mpro protein (Table 1). Several observed that the eucalyptol could compounds, such as flavonoids, terpenoids potentially bind to Mpro/3CLpro protein, the

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hydrophobic interactions with ALA7,PRO52,TRP207,LEU29,TRY126, PRO184; hydrogen bond interactions with M4,V18,L30,D10,T16; and ionic

next step was to know the binding mode. In interactions with particular, we have used Protein Interactions LYS3,ASP34,ARG38,HIS163 as shown in Calculator (PIC) to recognize the Table 2. These residues may be considered interactions within the bound complexes. In as key or critical and may play a major role structural bioinformatics, predicting protein- in the protein protein-interaction and might protein interactions which stabilize the inhibit the formation of the beta-barrel motif tertiary and quaternary structures is an of Mpro/3CLpro. Further studies may help important task. For the top best four to understand the role of these residues in Mpro/eucalyptol complexes with the best drug binding mechanism. Structural cluster size were subjected to PIC server and flexibility is one of the important physical the binding mode (interactions) of each properties that affect protein conformation peptide are given in Table 4. PIC identified and function. Whereas high increase in interactions such as hydrophobic residues kinetics energy and protein flexibility can interactions, ionic interactions, hydrogen disrupt non covalent interactions as in bonds, aromatic-aromatic interactions and thermal denaturation; a sharp decrease in aromatic–sulphur interactions within the flexibility can also cause protein peptide-protein complexes. According to the denaturation as seen in cold denaturation. PIC server results as shown in Table 4, Therefore, proteins need an essential amount Mpro/eucalyptol complexes forms of flexibility to carry out their native

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 31 March 2020 doi:10.20944/preprints202003.0455.v1

function at physiological conditions. In this Acknowledgment

context, an inhibitor by binding to a protein ADS want to thank management for this can alter its flexibility and decrease its support. enzymatic activity. Finally, lack of wet-lab Conflict of interest validation is a drawback in our research and we expect computational biology analysis Authors declares no conflict of interest and its integration with wet-lab data can be Compliance with Ethical Standards productive in the determination of potential The authors declare that they have no anti-Mpro/3CLpro components. conflict of interest. This article does not contain any studies involving animals or Conclusion human participants performed by any of the authors Due to non approved drugs at present Currently, COVID-19 has emerged in the Author contributions

human population, in China, and is a ADS: designed the study and prepared potential threat to global health, worldwide. manuscript Currently, the main target for COVID-19 IJK: designed the study and prepared treatment primarily act on the main protease manuscript (Mpro). The aim of this study was to References examine Eucayptol (1,8 cineole) from Rodríguez-Morales P., Alfonso J., eucalyptus essential oil that may be used to MacGregor K., Kanagarajah S., Dipti P., inhibit the COVID-19 infection pathway. Schlagenhauf. 2020. Going global – Travel Eucalyptol has high binding affinity and and the 2019 novel coronavirus. Travel lowest binding energies. Therefore, we Med. Infect. Dis., 33, doi: suggested that Eucalytol may represent https://doi.org/10.1016/j.tmaid.2020.101578. potential treatment options, and found in medicinal plants that may act as potential Lu H., 2020. Drug treatment options for the inhibitors of COVID-19 Mpro. However, 2019-new coronavirus (2019-nCoV). further studies should be conducted for the Biosci. Trends, 2020, validation of these compounds using in vitro doi:10.5582/bst.01020. and in vivo models to pave a way for these Liu X., Wang X.J., 2020. Potential inhibitors compounds in drug discovery. against 2019-nCoV coronavirus M protease

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 31 March 2020 doi:10.20944/preprints202003.0455.v1

from clinically 13 of 14 approved medicine. J. Genet. Genomics, , doi: 10.1016/j.jgg.2020.02.001. Yang L., Wen K.S., Ruan X., Zhao Y.X., Wei F., Wang Q., 2018. Response of plant secondary metabolites to environmental factors. Molecules, 23, 1–26, 2018, doi: 10.3390/molecules23040762. Zakaryan H., Arabyan E., Oo A., Zandi K., 2017. Flavonoids: promising natural compounds against viral infections. Arch. Virol., 162, 2539–2551, , doi: 10.1007/s00705-017-3417-y. Goodger J.Q.D., Senaratne S.L., Nicolle D., Woodrow I.E., 2016. Correction: Foliar Essential Oil Glands of Eucalyptus Subgenus Eucalyptus (Myrtac eae) Are a Rich Source of Flavonoids and Related Non-Volatile Constituents. PLOS ONE 11(5): e0155568. https://doi.org/10.1371/journal.po ne.0155568

Im K., Kim J., Min H., 2015. Ginseng, the natural effectual antiviral: Protective effects of Koran Red Ginseng against viral infection. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/P MC5052424/