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v1

Pharmacoinformatics and molecular dynamic simulation studies reveal

potential inhibitors of SARS-CoV-2 main protease 3CLpro

Mubarak A. Alamria*, Muhammad Tahir ul Qamarb, Safar M. Alqahtania

a
Department of Pharmaceutical Chemistry, College of Pharmacy, Prince Sattam Bin Abdulaziz

University, P.O. Box 11323, Alkarj, Saudi Arabia.

b
College of Life Science and Technology, Guangxi University, Nanning 530004, P. R.

China

*Corresponding author: [email protected]

© 2020 by the author(s). Distributed under a Creative Commons CC BY license.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 February 2020 doi:10.20944/preprints202002.0308.v1

Abstract

The SARS-CoV-2 was confirmed to cause the regional outbreak of coronavirus disease 2019

(COVID-19) in Wuhan, China. The 3C-like protease (3CLpro), an essential enzyme for viral

replication, is a valid target to compacts SARS-CoV and MERS-CoV. In this research, an

integrated library consisting of 1000 compounds from Asinex Focused Covalent (AFCL)

library and 16 FDA-approved protease inhibitors were screened against SARS-CoV-2

3CLpro. Top compounds with significant docking scores and making stable interactions with

catalytic dyad residues were obtained. The screening results in identification of compound

621 from AFCL library as well as Paritaprevir and Simeprevir from FDA-approved protease

inhibitors as potential inhibitors of SARS-CoV-2 3CLpro. The mechanism and dynamic

stability of binding between the identified compounds and SARS-CoV-2 3CLpro were

characterized using 50 nanoseconds (ns) molecular dynamic (MD) simulation approach. The

identified compounds are potential inhibitors worthy of further development as SARS-CoV-

2 3CLpro inhibitors/drugs. Importantly, the identified FDA-approved therapeutics could be

ready for clinical trials to treat infected patients and help to curb the COVID-19.

Keywords: SARS-CoV-2; COVID-19; 3CL protease; Molecular docking; Molecular

dynamics and simulations

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Introduction

In last 2 decades, several pathogens spilled over and causes outbreaks. Among them, emergence

and reemergence of coronavirus related epidemics widely spread fatal respiratory illness 1.

Coronaviruses are enveloped RNA viruses that are distributed broadly among humans, other

mammals, and birds and that cause respiratory, enteric, hepatic, and neurologic diseases 2. Six

coronavirus species are known to cause human disease. Four viruses 229E, OC43, NL63, and

HKU1 are prevalent and typically cause common cold symptoms in immunocompetent

individuals. The two other strains severe acute respiratory syndrome coronavirus (SARS-CoV)

and Middle East respiratory syndrome coronavirus (MERS-CoV) are zoonotic in origin and have
3, 4
been linked to sometimes fatal illness . The SARS-CoV emerged from Guangdong, China in

2003 and affected over 8000 individuals with 774 associated deaths. The MERS-CoV emerged

from Saudi Arabia was first reported in 2012 with global mortality rate of 35% (WHO:

https://www.who.int/).

On December 12, 2019, Wuhan Municipal Health Commission (WMHC), China reported

27 cases of viral pneumonia with 7 of them being critically ill. All of them had history of exposure

linked to the Huanan Seafood Wholesale Market where also sold poultry, bats, and snakes 5. The

viral pneumonia outbreak was not caused by SARS-CoV, MERS-CoV, influenza virus, or

adenovirus as determined by laboratory tests 6. A new strain of coronavirus come to limelight 7, 8.

On December 30th, The World Health Organization (WHO) temporarily named the viral

pneumonia causing pathogen 2019 novel coronavirus (2019-nCoV). On January 30th, 2020, the

WHO announced a Public Health Emergency of International Concern (PHEIC) for the 2019-

nCoV outbreak. On the Feb 12th, 2020, the WHO permanently named the 2019-nCoV pathogen

SARS-CoV-2 and the causing disease into coronavirus disease 2019 (COVID-2019). By February

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19th, 2020, the death toll reached 2009, with 74,284 laboratory-confirmed cases and 5248

suspected cases. The SARS-CoV-2 also widely spread to over 20 different countries.

Recent studies showed that the SARS-CoV-2 belongs to the beta-corona-virus family and

it is closely related to SARS-CoV coronavirus 9. Similar to other beta-corona-virsues, SARS-CoV-

2 produces an 800-kDa polypeptide upon transcription of its genome 5. This polypeptide is

proteolytically cleaved to generate various proteins 5, 9. The proteolytic processing is mediated by

papain-like protease (PLpro) and 3-chymotrypsin- like protease (3CLpro). 3CLpro cleaves the

polyprotein at 11 distinct sites to generate many of the non-structural proteins which are important

in viral replication. Thus, this protease plays a critical role in replication of virus 10, 11. Structure-

based activity studies and various high-throughput studies have identified distinct inhibitors of

SARS-CoV and MERS-CoV 3CLpro. Thus, it is essential to identify novel inhibitors of SARS-

CoV-2 3CLpro.

Traditional methods for identification of inhibitors are expensive and time consuming.

Therefore, the use of in silico techniques for identification of inhibitors has gained importance in

recent years 12, 13. The available small molecule database could be utilised for either ligand- based
14
or structure based molecular modelling and effective identification of inhibitors . Moreover,

3CLpro is highly conserved across coronaviruses, therefore, there is a potential target for
14
identification of compounds that could have broad spectrum anti-viral activity . In this

contribution, a combined virtual screening approaches, molecular docking and molecular dynamic

simulation were utilized to explore potential inhibitors of SARS-CoV-2 3CLpro enzyme as anti-

SARS-CoV-2 drugs.

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Materials and Methods

Sequence and Structural Alignment Analysis

A multiple sequence and structure alignment analysis was carried out to find out the

evolutionary conserved functional residues among SARS-CoV-2, SARS-CoV and MERS-CoV

which could be further targeted as probable targets for the discovery of drug hits. Sequence and

3D structures of SARS-CoV-2 (PDB ID: 6LU7), SRAS-CoV (PDB ID: 2A5I) and MERS-CoV

(PDB ID: 5WKK) 3CLpro were retrieved from protein data bank (PDB). The 3CLpro sequences

were aligned using Mega v6.0 15. To ensure broad spectrum relevance of these protein targets,

conserved functional residues recognition within active pockets were analyzed through structural

alignment as well. Structural alignment/superposition analysis was done using PyMOL tool 16.

Chemical libraries preparation

Two chemical libraries were obtained; the commercially available Asinex Focused Covalent

(AFCL) library, which consist of 1000 molecules, was retrieved from (http://www.asinex.com/)

and FDA-approved protease inhibitors including 16 anti-HIV and anti-Hepatitis C antiviral agents

were downloaded individually from Pubchem (https://pubchem.ncbi.nlm.nih.gov/) in SDF format.

Discovery studio visualizer 17 was used to combined both libraries in one SDF file.

Structure-based virtual screening

The chemical libraries were screened against the 3CLpro active site within SARS-CoV-2 structure

(PDB ID: 6LU7) using Autodock vina in PyRx program 18. The chemical compounds were initially
19
imported into OpenBable tools in PyRx for energy minimization . The latter, was used also to

convert the compounds’ SDF files into PDBQT files. The grid box which represent the docking

search area, was set to cover the active site of 3CLpro. Compounds were ranked based on their

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docking scores in Kcal/mole. Autdock tools 1.5.6 program 20 was used to convert the PyRx output

files into PDB files. The molecular interactions and binding modes of top poses were determined

using Discovery studio Visualizer 17 and Pymol programs 16.

Molecular docking

The docking was performed for candidate compounds against the SARS-CoV-2 3CLpro structure

using Autodock vina 1.1.2 program 21. Discovery studio Visualizer was used initially to prepare

the protein PDB file. Autodock tool 1.5.6 was used to add the polar hydrogens to the protein

structure and to convert the PDB files into PDBQT. The same program was used also to obtain the

three-dimensional grid box for docking simulation in which the box with size of 24x22x26 was

centered using the following dimension; -1.549 x 2.454 x 7.117 to cover the active site along with

the essential residues within the binding pocket. Discovery studio Visualizer and Pymol 1.3.

programs were used for data analysis.

The predicted inhibitory constant (pKi) was calculated using the following equation 22-24 :

pKi = 10 [Binding energy score  1.366]

Molecular dynamic (MD) simulation

The structure of SARS-CoV-2 3CLpro and candidate molecules were prepared for MD simulation

using Chimera 1.14 25. The MD simulation of 3CLpro-inhibitor complexes were carried out at 50
26
ns using Gromacs 2018.1 package using the OPLS-AA/L force field. The parameters of

candidate inhibitors were generated by Swissparam online server (http://www.swissparam.ch/) 27.

The simulation started by solvating the 3CLpro-inhibitor complexes in triclinic box using TIP3P

water model. The counter ions were added to the neutralized the system. Periodic boundary

conditions were used. The system was energy minimized using a steepest decent algorithm with a

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maximum step size of 0.01nm and tolerance of 1000kJ/mol/nm. The system was then equilibrated

using NVT and NPT ensemble for 100 ps. Finally, 50 ns production MD was performed for the

system. The trajectories were set to be generated every 2 fs and save every 2ps. The 3CLpro-

inhibitor complexes’ results were analyzed for root mean square deviation (RMSD), root mean

square fluctuations (RMSF), radius of gyration (Rg) and bond potential energy (BPE).

Results and discussion

Analysis of 3CLpro for conserveness among coronaviruses

The sequence alignment showed that the 3CLpro enzyme of new SARS-CoV-2 is 96.08% and

51.82% identical to SRAS (PDB: 2A5I) and MERS CoV (PDB: 5WKK) respectively. The

sequence alignment also revealed that catalytic dyad residues His41, Cys145 of 3CLpro are

conserved among SARS-CoV-2, SRAS-nCoV and MERS-CoV. Furthermore, the structural

alignment/superposition of all 3 coronaviruses 3CLpro revealed conserved catalytic dyad residues

His41, Cys145, inlaid at exactly same position in the binding pocket with an average RMSD nearly

0.12 Å as shown in Figure 1. Sequence and structural alignment clearly demonstrated that

conserved functional residues within the active pockets of 3CLpro among SARS-CoV-2, SRAS-

nCoV and MERS-CoV.

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Figure 1: (A) Ribbon representation shown for the overlaying of SARS-CoV-2 3CLpro (red) (PDB
ID: 6LU7), SARS-CoV 3CLpro (yellow) bound to an azapeptide covalent inhibitor (green) (PDB:
2A5I) and MERS-CoV 3CLpro (blue) bound to GC813 (pink). (B) Ribbon representation shown
the catalytic dyad His41, Cys145 within SARS-CoV-2 3CLpro and SARS-CoV 3CLpro in yellow
and red sticks, respectively. Catalytic dyad His42, Cys148 within MERS-CoV 3CLpro shown as
blue sticks.

Structure-based virtual screening

The generated structure was used to perform the structure-based virtual screening against an

integrated library of 1016 compounds including 1000 covalent protease inhibitors and 16 FDA-

approved protease inhibitors. The latter approach was applied to make use of both de novo drug

design as well as the drug repurposing strategies. The screening of both databases reveals three

potential inhibitors of SARS-CoV-2 3CLpro, namely, 621, Paritaprevir and Simeprevir with high

binding energy scores of -13.3, -8.8 and -8.78 (Table 1). The predicted inhibitory constants (pKi)

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based on the autodock vina docking scores showed that compound 621, Paritaprevir and

Simeprevir could inhibit the SARS-CoV-2 3CLpro with pKi values of 0.00013, 0.36 and 0.37 µM.

Interestingly, Paritaprevir and Simeprevir are acylsulfonamide FDA-approved drugs that act as

anti-hepatitis C by targeting the NS3/4A protease 28. Up to our knowledge, there were no clinical

data available regarding using of these two compounds to treat SARS-CoV-2, MERS-CoV or

SARA-CoV.

Table 1: Chemical structures, binding energy scores, predicted inhibitory constant (pKi) and
molecular interactions of identified candidate compounds.

Name Chemical Structure Binding pKi H-bond Hydrophobic

Energy (µM)
Score
(Kcal/mole)
621 -13.3 0.00013 Gly143 and His41
Cys145

Paritaprevir -8.8 0.36 Gly143 and Thr45,

Cys145 Met165 and
Pro168

Simeprevir -8.78 0.37 Gly143 and Pro168

Gln0189

Molecular interaction and binding mode

In order to understand the mechanism of interaction of these compounds with SARS-CoV-2

3CLpro, an unbiased docking of these compounds into the active site of SARS-CoV-2 3CLpro

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enzyme was performed. Interestingly, the three compounds adapt the same binding mode within

the binding pocket between catalytic dyad His41, Cys145, Figure 2. Furthermore, the distance

between the reactive group within each compound was 4.1, 4.1 and 4.9Å for, compound 621,

Paritaprevir and Simeprevir, respectively. While compound 621 and Paritaprevir were found to

form hydrogen bond with Gly143 and Cys145, Simeprevir was found to form hydrogen bonds

with Gly143 and Gln 189.

Figure 2: Binding mode and molecular interaction of identified candidate compounds with SARS-
CoV-2 3CL protease (PDB ID: 6LU7). (A), (B) and (C) showed the binding mode of compound
621, Paritaprevir and Simeprevir, respectively within the active site of SARS-CoV-2 3CL protease.
The catalytic dyad His41, Cys145 within the active site is shown in orange sticks. (D), (E) and (F)
showed molecular interaction of compound 621, Paritaprevir and Simeprevir, respectively with
SARS-CoV-2 3CL protease.

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Molecular dynamic simulation

To confirm the docking results and get more insight into the stability of ligand-protein complex,

MD simulations were carried out for each SARS-CoV-2 3CLpro-inhibitor complex in the solvated

states at 50 ns. The results of MD simulations have been examined on the basis of root mean square

deviation (RMSD), root mean square fluctuation (RMSF) and radius of gyration values as a

function of time.

Root mean square deviation (RMSD)

The RMSD measures the direct changes in the protein from the initial coordinates. The RMSD

values of the protein backbone in complex with the three potential inhibitors were computed with

respect to the initial structure as a frame reference (0 to 50 ns). The RMSD values steadily

increased from 0 to 5 ns, and reached equilibration after that throughout the simulation period,

especially for Paritaprevir and Simeprevir. The RMSD value for Compound 621 showed

oscillations between 29 to 32 ns indicting that the compound was adapting another conformation

within the binding pocket (Figure 3). The average RMSD values for the last 40 minutes for 621,

Paritaprevir and Simeprevir were 0.32 ±0.03, 0.30±0.02 and 0.35 ±0.04 Å, respectively.

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Figure 3: Plot of root mean square deviation (RMSD) of C–Cα–N backbone vs. simulation time
for solvated SARS-CoV-2 3CL protease in complex with the three candidate compounds during
50 ns molecular dynamics simulations.

Root mean square fluctuation (RMSF)

To explore the local protein flexibility, the time average of RMSF values of the 306 amino acids

of SARS-CoV-2 3CLpro protein in presence of the three inhibitors over simulation period were

calculated. The RMSF values for the three complexes suggested that the catalytic dyad residues

(His41 and Cys148) showed less fluctuation in all complexes (Figure 4). The average RMSF

values were 0.11± 0.04, 0.12±0.012 and 0.15 ±0.07 Å for compound 621, Paritaprevir and

Simeprevir, respectively.

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Figure 4: The root mean square fluctuation (RMSF) values of SARS-CoV-2 3CL protease in
complex with the three candidate compounds were plotted against residue numbers.

Radius of gyration (Rg)

The radius of gyration (Rg) of the protein is associated with its size and compactness. The Rg

values of three complexes were found to be 2.16 nm at the initial state. The Rg values of the

complex of protein with Paritaprevir and Simeprevir were stabilized after initial increase at 5 ns

supporting that the systems have reached equilibrium state. In the other hand, the Rg value for

compound 621 was decreased from 10 ns to 40 ns and then it slightly increases up to 50 ns. The

latter, indicates that the binding of 621 to the protein stabilized it secondary structure (Figure 5).

The MD simulation results confirmed the stability of all identified compounds at the active site of

SARS-CoV-2 3CLpro.

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Figure 5: Plot of radius of gyration (Rg) during 50ns MD simulation of SARS-CoV-2 3CL
protease in complex with the three candidate compounds.

Conclusion

In the current study, pharmacoinformatics and molecular dynamic approaches were unitized to

identified inhibitors of SARS-CoV-2 3CLpro as treatments for the new outbreak coronavirus

disease 2019. Three compounds, compound 621, Paritaprevir and Simeprevir, were identified as

potential inhibitors of SARS-CoV-2 3CLpro enzyme with predicted inhibitory constant in low

micromolar concentration range. The binding affinity, mechanism and stability of binding of these

compounds to SARS-CoV-2 3CLpro were confirmed by molecular docking and molecular dynamic

simulation. Compound 621 could be used as a seed for de novo drug design of potential inhibitors

to target the 3CLpro enzyme of SARS-CoV-2 as well as MERS-CoV and SARS-CoV. The clinical

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agents, Paritaprevir and Simeprevir may also play a role in expediting the drug discovery process

and be tested in clinical trials as a treatment for coronavirus disease 2019.

Acknowledgment

Mubarak A. Alamri and Safar M. Alqahtani would like to thank Prince Sattam Bin Abdulaziz

University for providing the necessary tools to conduct this research.

Conflict of interest

Authors have no conflict of interest to declare.

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