key: cord-0714508-6wy1lwgr
authors: Baildya, Nabajyoti; Ghosh, Narendra Nath; Chattopadhyay, Asoke P.
title: Inhibitory activity of hydroxychloroquine on COVID-19 main protease: An insight from MD-simulation studies
date: 2020-06-02
journal: J Mol Struct
DOI: 10.1016/j.molstruc.2020.128595
sha: 39e6363dbef3d5479a4d4c94c2dc088975a5d3de
doc_id: 714508
cord_uid: 6wy1lwgr

The present work is an investigation to test hydroxychloroquine as an inhibitor for the COVID-19 main protease. Molecular docking studies revealed a high docking score and interaction energies and decent level of docking within the cavity in protease moiety. Molecular dynamics simulations also lead to the evaluation of conformational energies, average H-bonding distance, RMSD plots etc. Large RMSD fluctuations for the first 2 ns seem to provide the conformational and rotational changes associated with the drug molecule when it comes into the vicinity on the protease matrix. Snapshots of structural changes with respect to time vividly indicates that drug molecule has a profound impact on the binding sites as well as overall geometry of the protease moiety. On the whole, hydroxyxhloroquine confers good inhibitory response to COVID-19 main protease. We hope the present study should help workers in the field to develop potential vaccines and therapeutics against the novel coronavirus.

From the last two decades infectious virus diseases like Lassa, Ebola, Zika have created a great burden to human, social and economical health. Along with that, coronaviruses like the severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV) and highly pathogenic influenza show their rampage. But somehow mankind recovered from those attacks by use of proper medicines. From December 2019, a deadly virus viz. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) created an uproar worldwide known as COVID-19. Observing the devastating impact of COVID-19 and its huge mortality rate, the World Health Organization (WHO) has declared this outbreak as a pandemic on 12th March, 2020 [1] . Till 12th March, 2020, globally 125048 people were infected and 4613 people were dead [1] .

The first coronavirus B814 was found in 1965 in human embryonic tracheal organ cultures [2] . These viruses can cause disease in many animal species like rats, mice, dogs, cats, chickens etc [3] . Severe acute respiratory syndrome (SARS) originated from animal corona viruses was emerged in 2002-2003 from southern China, with outbreaks in 29 countries in North and South America, Europe and Asia [3] [4] [5] [6] . Coronaviruses have positive sense singlestandard RNA which are enveloped within fatty lipid molecules. These are mainly categorised into four groups viz. α-COV, β-COV, γ-COV and δ-COV, while a clear idea about SARS-COV-2 remains unknown.

The spiky ball type SARS-COV-2 first attacks human protein within the surface cell and then increases its activity. In recent days, from December 2019, emergency situation arising from novel coronavirus (SARS-COV-2) outbreaks reportedly started from Wuhan, China and spread throughout the world rapidly [7] [8] . Its outbreak has created a pandemic and challenges the scientific community to design and discover the ultimate treatment strategy against this deadly virus (SARS-CoV-2). Till date, there is no antiviral therapy or vaccine available in the market which can efficiently combat the infection caused by this virus. SARS-CoV-2 uses the viral surface spike glycoprotein to bind to the host cell. After infecting the host cell with its RNA, it forces the host cell to bend and leave free copies of the virus created within the cell (a process known as "budding"). So the virus specific molecular interaction with the host cell represents a promising therapeutic target for identifying SARS-CoV-2 antiviral drugs. With the available experimental techniques, such selection is a difficult task as they offer lowresolution data to study the necessary interactions. In this regard potential in silico methods can be applied to select promising drug molecules with fewer trials and errors effectively reducing time and cost of researchers. The purpose of this work is to investigate the potential subunits of vaccines against the novel Coronavirus. Using detailed 3D-structures of relevant biomolecules and drug compounds, available bioinformatic techniques such as molecular docking may be applied to quickly identify promising and potential drug candidates. This can be followed with atomistic molecular dynamics or similar simulation methods in order to fully unravel underlying interactions responsible for drug-receptor binding and resulting structural modifications, leading to change in activity of the biological system. During clinical trials, some known drugs showed potent effect on this disease by curing a few COVID-19 patients [11] . But the actual mechanism or mode of action of those drugs are still unknown. Azithromycine, which can be used for the treatment of Zika virus [9, 10] , was applied to prevent COVID-19 along with hydroxychloroquine [11] . Chloroquine, an antimalarial drug, is known to possess antiviral activity [12] . This was also applied to COVID-19 patients [13, 14] . Chloroquine phosphate has shown positive activity on COVID-19 [15] .

Though both chloroquine and hydroxychloroquine have some toxic effects, and their use in high doses can create major risks to the patients [16] , under the present situation there is not enough information to find out other potent drugs with immediate effect. (EMC) (PubChem SID: 46507606) have been taken from PUBCHEM. The structure of the protease was cleaned by removing all water molecules and by removing hetero groups using UCSF Chimera (ver 1.12) [17] and then the docking simulations were done by selecting best binding sites using AutoDock Vina package [18] . Auto Dock Tools was used to construct the initial structures to run the docking simulations.

The molecular dynamics (MD) simulation studies were carried out on the minimum energy configurations obtained from the docking studies using GROMACS (Version 2018.5) [19] with the CHARMM36-mar2019 force-field [20] using TIP3P model [21] . The ligand (HCQ) topology and parameters were generated using CHARMM General Force Field server. A cubical box was generated where the protease-HCQ complex was at least 1 nm from the edges of the box to maintain at least 2 nm distance between two successive images of the complex using periodic boundary conditions. Four Na + ions were added to maintain charge neutrality of the system. First, energy minimization was carried out until the maximum force becomes less than 10 kJmol -1 nm -1 . The steepest descent algorithm was used followed by conjugate gradient protocol. Then the system was equilibrated for 100 ps using isochoricisothermal (NVT) equilibration at 300K. The time step was taken 2 fs. This was followed by equilibration at isothermal-isobaric or NPT ensemble at 300 K for 100 ps. Modified

Berendsen thermostat was used for the NPT ensemble. Here also the time step was kept 2 fs.

For both NVT and NPT equilibration, electrostatic and van der Waals interaction cut off were kept at 1.0 nm. Long range interactions were calculated using smooth particle mesh Ewald (PME) method [22] . The equilibrated ensembles were finally subjected to MD simulation for 10 ns, with electrostatic and van der Waals cut off as before. PME method was used to calculate long range electrostatic interactions. A modified Berendsen thermostat and a Parinello-Rahman barostat were used with reference temperature and pressure at 300K and 1 bar respectively. Snapshots of the trajectory were saved every 1 ns for each case.

For protease-HCQ systems, structural trajectories were calculated using trjconv tool. The trjconv tool of GROMACS was used to recentre the protein and other molecules within the cubical box. van der Waals interaction, electrostatic energy, interaction energy values etc were calculated using gmx energy tool of GROMACS. RMSD plots were done using xmgrace plotting tool.

Required PDB file for the crystal structure of COVID-19 main protease (PDB ID: 6LU7) was downloaded from protein data bank and the drugs for screening was obtained from PubChem.

The drugs which are used for screening of molecular docking are either anti-malerial or anti HIV agents. CQ, HCQ, LV and EMC were used for screening and docking was made with protease of COVID-19. The docking power of drugs on the co-crystal of COVID-19 was evaluated by measuring docking score. We find a docking score of -6.3 for hydroxychloroquine (HCQ) which is quite high compared to the other tested drugs. The docking score of other drugs are shown in Table 1 . Table 2 represents the conformational energy and total energy of the systems along with the number of H-bonds and average distance of H-bonds. The value of conformational energy is more in case of composite system which insert more flexibility within the molecule. Again total energy for the protease is more than the composite system after MD simulations, which suggests that the drug makes the system less stable. This is also corroborated by the RMSD plot in Fig Fig. 5f shows the net deviation in the structure of the protease from t = 0 ns to t = 10 ns of the protease backbone. After the first 3 nanoseconds, RMSD plot shows a huge deviation which is reflected in Fig.   5b . Possibly, drug enters into closer proximity of the protease moiety in this period. After 7 ns the RMSD fluctuation appears to stabilize, indicating equilibration of the geometry.

The present work involves study of interaction between hydroxychloroquine, a known antimalarial and a putative anti-SARS-COV-2 drug, and the protease of the latter. Molecular docking study reveals that HCQ docked at the binding site due to high electrostatic interactions, H-bonds etc. MD simulation study shows there is a robust change in the main protease of SARS-COV-2. RMSD data shows that there is a remarkable structural change in the protease moiety at 2 ns, around the time the drug begins closer interaction with the protease. The drug-protease system attains equilibrium at around 7 ns. The RMSD plot also shows that the main protease backbone gets destabilized following interaction with the drug molecule. It is interesting that the number of H-bonds between the drug and protease increases as drug-protease complex forms, while the average H-bonding distance shows the opposite trend. The present molecular docking and MD simulation studies suggest that the drug hydroxychloroquine may have considerable effect on structure protease of SARS-COV-2, which may lead to significant inhibitory effect on the latter. 

Research and Development on Therapeutic Agents and Vaccines for COVID-19 and Related Human Coronavirus Diseases

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History and Recent Advances in Coronavirus Discovery

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A novel coronavirus associated with severe acute respiratory syndrome

Coronavirus as a possible cause of severe acute respiratory syndrome

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Zika virus cell tropism in the developing human brain and inhibition by azithromycin

Azithromycin Inhibits the Replication of Zika Virus

Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial

Design and Synthesis of Hydroxyferroquine Derivatives with Antimalarial and Antiviral Activities

Chloroquine and hydroxychloroquine as available weapons to fight COVID-19

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Breakthrough: Chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated pneumonia in clinical studies

Recommendations on screening for chloroquine and hydroxychloroquine retinopathy

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GROMACS: A message-passing parallel molecular dynamics implementation

CHARMM36 united atom chain model for lipids and surfactants

CHARMM TIP3P water model suppresses peptide folding by solvating the unfolded state

Optimization of parameters for molecular dynamics simulation using smooth particle-mesh Ewald in GROMACS 4.5

The authors wish to acknowledge infrastructural support received from the Dept. of Chemistry, University of Kalyani, and from the Dept. of Chemistry, University of Gour Banga, Malda.This research did not receive any special grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data is available on request to the corresponding author

• Molecular Docking studies reveals that hydroxychloroquine (HCQ) possesses a high docking score and interaction energy compared to other small drug molecule with the COVID-19 main protease.• Atomistic Molecular Dynamics simulation indicates considerable interaction between HCQ and COVID-19 protease • RMSD plot suggests complex formation between HCQ and COVID-19 protease after 2 ns • Equilibration of binding occurs between HCQ and COVID-19 protease between 7 to 10 ns • Large structural change occurs in the COVID-19 protease following binding with HCQ between 2 to 7 ns, which may be responsible for change in activity of the former after binding with HCQ

☐√The authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: