Journal of Biomolecular Structure and Dynamics

ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20

Computational studies of drug repurposing and synergism of lopinavir, oseltamivir and
ritonavir binding with SARS-CoV-2 Protease against COVID-19

Nisha Muralidharan, R. Sakthivel, D. Velmurugan & M. Michael Gromiha

To cite this article: Nisha Muralidharan, R. Sakthivel, D. Velmurugan & M. Michael Gromiha (2020): Computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with SARS-CoV-2 Protease against COVID-19, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2020.1752802
To link to this article: https://doi.org/10.1080/07391102.2020.1752802
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Running title: Synergism of lopinavir, oseltamivir and ritonavir binding

Computational studies of drug repurposing and synergism of lopinavir, oseltamivir and ritonavir binding with SARS-CoV-2 Protease against COVID-19
Nisha Muralidharan1, R. Sakthivel1, D. Velmurugan2 and M. Michael Gromiha1,*

1Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India

2School of Bioengineering, Department of Biotechnology, SRM University, Chennai, India

*To whom correspondence should be addressed Tel: +914422574138 Fax: +914422574102 E-mail:
[email protected]

Abstract
A novel coronavirus (SARS-CoV-2) has caused a major outbreak in humans all over the world, and it is the latest pandemic in the series of other infectious diseases. The concept of drug repurposing has been used successfully for many years for known diseases. Considering the emergency and urgency, drug repurposing concept is being explored for coronavirus disease (COVID-19) as well. Recently, the combination of three known drugs, lopinavir, oseltamivir and ritonavir has been proposed to control the virulence to a great extent in COVID-19 affected patients within 48 hours.
Hence, we tried to understand the effect of synergism of these drugs against the SARS-CoV-2 protease using sequential docking studies. As a result, combination of three drugs showed a better binding energy than that of individual drugs. Further, the complex was subjected to molecular dynamics simulations to get insights into the stability of the complex, considering the simultaneous interactions between three drugs and the protein. The protein complexed with three drugs remained stable during the simulations. Hence, these drugs can be explored further for drug repurposing against the successful inhibition of COVID-19.

Keywords: SARS-CoV-2; COVID-19; drug repurposing; molecular docking; molecular dynamics simulations; protease

1. Introduction

A novel coronavirus, formally named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused coronavirus disease 2019 (COVID-19) worldwide, and it is the latest pandemic in the series of other infectious diseases including avian flu, Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). According to the World Health Organization, SARS-CoV-2 has caused an initial outbreak in Wuhan, China in the end of December 2019 and affected 82,000 people approximately and caused deaths of around 3,000 people till date in that country alone. Rapidly, it has become a global pandemic and spread to other countries such as Republic of Korea, Thailand, Iran, Italy, United States of America, India, etc., affecting more than 800,000 people with around 40,000 deaths worldwide (World Health Organization data as reported by national authorities by 10 AM CET 01 April 2020. https://www.who.int/docs/default- source/coronaviruse/situation-reports/20200401-sitrep-72-covid-19.pdf?sfvrsn=3dd8971b_2).
Hence, it is the utmost public health emergency at present.

The concept of drug repurposing is widely used nowadays to identify potential drugs for various diseases. It has gained an enormous attention for the ability to reuse already available drugs for various diseases than they have been originally developed for specific diseases. Many drugs have multiple protein targets and many diseases share overlapping molecular pathways (Hodos et al., 2016). In such cases, reusing drugs for more than one purpose and finding their new uses can significantly reduce the cost, time and risks of the drug development process using fast-growing computational approaches (Xue et al., 2018). This concept of drug repurposing has been used successfully for many years for known diseases. For example, favipiravir, an approved influenza virus drug, and sofosbuvir, a hepatitis C virus drug have a strong repurposing potential against Ebola and Zika viruses (Mercorelli, Palù, & Loregian, 2018). Different drugs such as nelfinavir, lopinavir, oseltamivir, atazanavir and ritonavir have been used to cure MERS and SARS (Dobson et al., 2015; Lv, Chu, & Wang, 2015). Similarly, several drug repurposing options are being considered and are under investigation to control the COVID-19 as there is an urgent requirement for a strong drug or combination of drugs to combat the disease (Shanmugam et al., 2020). Recently, a combination of three drugs, lopinavir, oseltamivir and ritonavir has been proposed to control the virulence to a great extent in the COVID-19 affected patients within 48 hours (The Scientist dated February 3, 2020. https://www.the-scientist.com/news-opinion/flu-and-anti-hiv-drugs-show-efficacy-against- coronavirus-67052.). Lopinavir and ritonavir have been originally developed for HIV, while oseltamivir has been developed for influenza virus.

CoVs encode proteases such as papain-like protease (PLpro) and main protease (Mpro) (Thiel et al., 2003), which are involved in the proteolytic processing of the polyproteins into individual non- structural proteins (nsps) to control viral gene expression and replication (Xue et al., 2008). Though both proteases involve in the proteolytic processing, nsps generated by the main protease play a major role in the viral replication (Zhao, Weber, & Yang, 2013). Mpro is a homodimer with three structural domains, domain I, II and III (Anand et al., 2002). Mpro is highly conserved among Coronaviridae members such as SARS-CoV and MERS-CoV, exhibiting 40% – 44% of sequence homology (Yang et al., 2003). The substrate-binding site is located in the Cys-His catalytic dyad located in a cleft between domain I and II (Shi, Wei, & Song, 2004). Chen et al., (2006) reported that dimerization of the two protomer is important; however, only one protomer is active at a time.

Hence, Mpro has emerged as the most potential antiviral target because of its main role in self maturation and subsequent maturation of polyproteins.

In this study, we tried to understand the mechanism of the proposed drugs for COVID-19. Generally, the combination of drugs is more effective in combating certain viruses like HIV and coronavirus than individual ones (Pirrone et al., 2011). Hence, we explored the effect of synergism of the drugs, lopinavir, oseltamivir and ritonavir against the SARS-CoV-2 protease. We performed molecular docking and molecular dynamics (MD) simulations to understand the interaction between the ligand and protein, considering the simultaneous interactions between three drugs and the protein.

2. Materials and Methods

2.1 Protein and ligand structure preparation

The structure of SARS-CoV-2 protease (6LU7.pdb) available in Protein Data Bank (http://www.rcsb.org/) (Burley et al., 2019), was used as a receptor. The SMILES of the three drugs lopinavir, oseltamivir and ritonavir were obtained from DrugBank (Wishart et al., 2018) and their accession numbers are DB01601, DB00198, and DB00503 respectively. These SMILES were converted to PDB format with 3D coordinates using OpenBabel (O’Boyle et al., 2011), an open source chemical toolbox for the interconversion of chemical structures. The structures of the drugs lopinavir, oseltamivir and ritonavir are shown in Figure 1.

2.2 Molecular docking

Docking between the protein and ligand was performed using AutoDock 4.2 (Morris et al., 2009). The protein was processed by removing the unrelated complex molecule, removing water, adding hydrogens and Kollman charges. The Gasteiger charges are assigned and rotatable bonds are detected for the ligand. The prepared protein and ligand structures were saved in PDBQT file format. Initially, since the binding sites were unknown, we performed induced fit docking for all three drugs against the protein individually. For that, the Autogrid was set to the entire molecule with the default grid spacing of 0.375Å and by adjusting the grid size in x,y,z directions. After analysing the binding regions of three drugs, the simultaneous effect of these three drugs together with the protein was performed through sequential docking. For sequential docking, the Autogrid size was set to particular binding regions of each drug with the default grid spacing. Lamarckian Genetic Algorithm (GA 4.2) was used for the docking, which gives the top 10 estimated free energy of binding score for each trial. The protein–ligand interactions were analysed and rendering was done through Discovery Studio (Accelrys, 2018). The docked structures were used as models for the MD simulations.

2.3 Molecular dynamics simulations

Two MD simulations were carried out for this study – one with only free protein and another one is a complex of the protein with three drugs. Antechamber and parmchk modules of Amber16 (Case et al., 2016) were used for generating preparatory files. MD simulations of 100 ns were performed for the complex with three ligands and the free protein using FF14SB and Generalised Amber Force Field (GAFF) in Amber16. The complex and the free protein were protonated and counter ions (Na+) were added appropriately to make the total charge zero. The molecules were solvated using TIP3PBOX water model with the edge of the box at least 10 Å away from solute molecules. Throughout the simulation, each complex system is maintained at the temperature of 300 K with constant pressure. Energy minimization was done for 50,000 steps. The MD simulation was carried out through the Particle Mesh Ewald Molecular Dynamics (PMEMD) of Amber16. The trajectories were collected for every nano second to get insights into the interactions at the atomistic level. Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) were calculated by the CPPTRAJ (Roe & Cheatham, 2013) module of Amber tool. The plots were made using XMGRACE software package (Turner, 2005).

3. Results and discussion

3.1 Individual and sequential docking of three drugs against SARS-CoV-2 protease

Molecular docking of the three drugs, lopinavir, oseltamivir and ritonavir, with the SARS- CoV-2 protease were performed and individually, these drugs showed a binding energy of -4.1 kcal/mol, -4.65 kcal/mol, -5.11 kcal/mol, respectively. The docked ligand molecules with the protease are shown in Figure 2. The hydrogen bonding and hydrophobic interactions between SARS-CoV-2 protease and the three considered drugs obtained with individual docking are presented in Table 1A and B. Li et al., (2020) reported a similar trend of the presence of three binding pockets in SARS CoV protease, which confirms our studies.

However, through sequential docking, binding of three drugs simultaneously with the protein showed a significant improvement in the binding energy to -8.32 kcal/mol. This shows that the combination of drugs is more effective than considering each drug separately. The result of the sequential docking is shown in Figure 3. Table 1A and B includes the hydrogen bonding and hydrophobic interactions between SARS-CoV-2 protease and the considered drugs obtained with sequential docking. We observed that hydrophobic interactions are dominant in SARS-CoV-2 protease – lopinavir complex whereas both hydrogen bonding and hydrophobic interactions are important in other two complexes (SARS-CoV-2 protease with oseltamivir and ritonavir). Further, several hydrogen bonding and hydrophobic interactions are lost during sequential docking compared with individual docking, which reveals that the interaction energy is not additive. Note that NZ ofLys100 forms a salt bridge with O4 of oseltamivir and a cation-π interaction is formed between ND1 of His41 and ring 3 in ritonavir in both individual and sequential docking. The root mean square deviation of all heavy atoms in the ligands, lopinavir, oseltamivir, and ritonavir between individual and sequential docking are 0.0Å, 0.91Å and 1.54Å, respectively.

3.2 Root-mean square deviation (RMSD)

To examine the change in the protein dynamics and the conformational stability of the protein-ligand complex, the free protein and the protein complexed with three drugs were subjected to 100 ns MD simulations. CPPTRAJ module of Amber was employed to measure the RMSD and RMSF. RMSD was calculated at an interval of 1ns for the free protein and the complex. The variation of RMSD of the free protein predominates in the range from 1.2 to 3Å. However, for the complex, it ranges from 1 to 2.1Å, which was lower compared to the free protein. Lower RMSD value of the complex indicates its stability with three drugs and provided a suitable basis for our study. Figure 4 shows the RMSD value of Cα atoms of the protein at various nanoseconds.

In order to find out the stability of the three ligands, RMSD graph was plotted for three ligands individually (Figure 5). Lopinavir showed deviations during the initial simulations till about 20 ns, but over the time, it was stable around 2Å. Oseltamivir was stable throughout the simulations at 1Å. For ritonavir, there were deviations till 65 ns from 1 to 2.5Å, and after that, it maintained stable at 3Å. RMSD graph of all three ligands showed stability during the simulations.

3.3 Root-mean square fluctuation (RMSF)

RMSF was measured with respect to Cα atom of each amino acid residues and the plot of RMSF was used to depict the fluctuations at the residue level. RMSF plot in Figure 6 exhibited a similar trend of residue fluctuation profile for both free protein and the complex with an average RMSF of 1.5Å. This trend in the RMSF plot for the complex indicates that binding of three drugs to the protein was stable and had no major effect on the flexibility of the protein throughout the simulations.

Conclusions

Our analysis through computational methods revealed that the binding energy of the combination of drugs against the protein is stronger than that of each drug docked against the protein individually. RMSD of the protein-ligand complex has maintained the stability at around 2Å and RMSD of three drugs complexed with the protein are in the favorable range of within 3Å and has remained stable during the simulations. The backbone atoms of the complex and free protein show similar RMSF, indicating the stability of the three drugs binding to the protein. Hence, the ABT-538 combination of lopinavir, oseltamivir and ritonavir are highly effective against SARS-CoV-2 protease, and these drugs can be explored further for drug repurposing against the successful inhibition of COVID-19.

Acknowledgements

We thank the Department of Biotechnology and Indian Institute of Technology Madras for computational facilities. We wish to acknowledge Dr. C. Ramakrishnan, Dr. S. Anusuya, S. Akila Parvathy Dharshini and Dr. A. Mary Thangakani for fruitful discussions. The work is partially supported by the Department of Biotechnology, Government of India to MMG.

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