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COVID-19, SARS-CoV-2, Molecular Docking, High throughput virtual screening
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Singh S and Florez H. Bioinformatic study to discover natural molecules with activity against COVID-19 [version 1; peer review: 2 approved]. F1000Research 2020, 9:1203 (https://doi.org/10.12688/f1000research.26731.1)
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Research Article
Bioinformatic study to discover natural molecules with activity against COVID-19
[version 1; peer review: 2 approved]
PUBLISHED 06 Oct 2020
OPEN PEER REVIEW
REVIEWER STATUS
This article is included in the Emerging Diseases and Outbreaks gateway.
This article is included in the Coronavirus (COVID-19) collection.
Abstract
Corresponding author:
Hector Florez
Competing interests:
No competing interests were disclosed.
Grant information:
This work was supported by the research group Information Technologies Innovation (ITI) (https://www.itiud.org/), which belongs to the Universidad Distrital Francisco Jose de Caldas located in Bogotá Colombia.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Copyright:
© 2020 Singh S and Florez H.
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
How to cite: Singh S and Florez H. Bioinformatic study to discover natural molecules with activity against COVID-19 [version 1; peer review: 2 approved]. F1000Research 2020, 9:1203 (https://doi.org/10.12688/f1000research.26731.1)
First published: 06 Oct 2020, 9:1203 (https://doi.org/10.12688/f1000research.26731.1)
Latest published: 06 Oct 2020, 9:1203 (https://doi.org/10.12688/f1000research.26731.1)
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Introduction
A novel virus was first found infecting human in China on 30th December, 2019. The causative agent for this disease was found to be severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronavirus disease 2019 (COVID-19) was declared as a pandemic by the World Health Organization on 11th March, 2020. The virus has been responsible for ’locking down’ much of the human race in most countries of the world for several months. The infectivity of this virus is extremely high and the mortality of this disease is around 5–7 percentage of the total infected cases1. However, the infection fatality rate was reported to be 0.5–3.6% and the case fatality rate 2.3–7.2%, as per other reports since it differs from region to region over time2
Coronaviruses consist of an enveloped RNA genome, and these viruses are zoonotic in nature. They cause respiratory, hepatic neurological, and enteric diseases in birds and mammals. As per the recent reports, this disease has claimed 962,634 lives worldwide, infecting 31,175,835 people globally till 22nd September, 20203. SARS-CoV-2 consists of 30 kb nucleotides as its genome4. Two polyproteins essential for replication and transcription, pp1a and pp1ab, are encoded by the SARS CoV-2 replicase gene. The main protease (3C-like protease) or Mpro (a 33.8 kDa protein) is responsible for releasing the functional viral polypeptides from polyproteins after cleaving pp1a and pp1ab4. Therefore, Mpro is identified as an important drug target owing to its functional significance in the life cycle of the virus and also due to the absence of its homologues in the human race4.
A few drugs such as hydroxychloroquine and remdesivir that were found via bioinformatics approach have been reported. Hydroxychloroquine is a drug that is presently used against malaria. But when it was tested on SARS-CoV-2 virus, it showed activity against the virus in vitro. However, it was not found to be effective against the virus in COVID 19 patients during clinical trials5,6. Later, this drug was prohibited from use in certain countries due to it side effects. Remdesivir is an antiviral drug that was found to show activity against the virus and it did inhibit the SARS-CoV-2 virus to certain extent. Remdesivir is presently used against COVID-19 in certain countries7.
In the present work, we carried out high throughput virtual screening (HTVS). It is a rapid way to search for the potential drug against the target. HTVS of Zinc natural database was performed through Python prescription software known as PyRx. This software has in built dock, Vina and Autodock tools along with openbabel. Autodock software utilizes MGLTools that has computer aided drug discovery pipeline. CADD is required for virtual high throughput screening of large databases for probable hits as therapeutic agents. This type of virtual screening helps to dock several ligands on one protein. PyRx is publicly obtainable HTVS software. Analysis and identification of distinct poses visibly and using a scoring algorithm quantitatively forms two basis of docking technique8.
The free binding energy (ΔG) between the protein and the ligands is calculated in docking. The free binding energy, thus calculated, forms the foundations of complex systems of subjects such as molecular biology and biochemistry8.
Methods
Molecule selection
As per our earlier published article8, we found that fexofenadine acetate (Allegra), atovaquone, ethamidindole, baicalin,justicidin D, euphol,curine and glycyrrhetic acid could be used as COVID-19 inhibitory drugs8. In that article, we selected 129 known drugs for drug repurposing, from which we found atovaquone, presently in use for treating malaria and Pneumocystis carinii pneumonia. We had considered another aspect in which we had taken 992 small molecules from Zinc natural database from which fexofenadine acetate (Allegra), an already in use drug for anti-allergic medicine8. Notably, the small molecules from natural database had least binding energy. Therefore, we decided to dock 1100 small molecules (see Extended data9) from the ZINC Natural Products database to identify additional lead molecules against the disease.
Molecular docking
In this study, we utilized a bioinformatic approach that uses predictions for the arrangement of a molecule bounded to another molecule, known as molecular docking10,11. Molecular docking can be performed by two main approaches. The former describes complementation surfaces of the ligand and the protein12. The latter simulates the docking process that calculates the free binding energy ΔG while protein-ligand interaction13–15. We have used PyRx (Python prescription) version 0.8 that performs HTVS. PyRx uses Autodock, ock and Vina as docking tools. Further, MGLTools, providing a computer aided drug discovery or rational drug design pipeline to search large databases to obtain potential leads as therapeutic drugs, is present as inbuilt software in the Autodock tool of PyRx.
The X-ray crystal structure of Mpro (PDB ID: 6M03 (resolution: 2 Å) was considered for our study Å). The database considered for docking was the ZINC Natural Products database. For processing of ligands and macromolecules, the energy of 3D molecules were minimized. The Universal Force Field or UFF was used for the energy minimization of small molecules before docking. Removal of water molecules and insertion of polar hydrogen into the protein crystal structure was carried out in pre-processing of the macromolecule. Later, PDBQT files of ligands and macromolecule were generated using AutoDock version 4.
Molecular docking process
The grid box was as per our previous article, with dimension coordinates of X:29.9242, Y:64.1097, and Z:48.1126 and center coordinates of X:12.2632, Y:12.3998, and Z:5.4737)8. Maximum grid box space was chosen for docking in order to maximize the possibility of finding a good lead molecule owing to the fact that there are still no known molecule to inhibit the virus completely. Therefore, it was thought to be better to maximize the active site space. Docking was performed with the Vina software present in PyRx tool, in which the protein-ligand interaction was studied. Analysis of separate poses were done in the AutoDock and the docked pose with the least binding affinity in kcal/mol was chosen for further analysis.
Conversion of these final docked ligands into PDB format was done using Pymol software version 1.7.4; Discovery studio 4.1 was used to study their interaction. Pymol could also be used for studying the interactions.
In addition, remdesivir, an antiviral agent that was found to act to some extent against this disease, was used as the reference molecule. Hence, it was initially docked with 6M03 in order to know (ΔG) value as the reference range for acquiring the hits.
Results
Docking results
Mpro has six stranded antiparallel beta barrels which provides the substrate binding site between domain I (10–99 residues) and domain II (100–182 residues)16. Dimerization of Mpro is regulated by domain III(198–303 residues) which is a globular cluster of five helices. It is primarily done by forming a salt-bridge interaction between Glu290 of one protomer and Arg4 of the other16. The two molecules domain II of molecule A and the NH2-terminal residues(“N-finger”) of molecule B are arranged perpendicularly to one another to provide a tight dimer for Mpro16. N-finger of each of the two protomers is found to be interacting with Glu166 of the other protomer, therefore, the N-finger is firmly pressed in between domains II and III of the parent monomer and domain II of the other monomer. Dimerization is essential for the catalytic activity of Mpro. Hence, these residues (10 to 99, 100 to 182 and 198 to 303) are essential for increasing the catalytic activity of Mpro16. Docking results for each molecule are given in the Extended data9.
Docking results for top 25 molecules against SARS-CoV-2 Mpro
The free binding energy for our reference molecule, which is remdesivir, was found to be -6.3 kcal/mol and it interacted with Leu272, Leu287, Tyr237, and Asn238 forming six hydrogen bonds with Lys137, Thr199, and Tyr239 residues of the main protease, Mpro (6M03). Figure 1 presents the interaction of remdesivir with Mpro.
Figure 1. Interaction of remdesivir with Mpro.
The best 25 molecules, with binding energies of -8.8 kcal/mol to -8.7 kcal/mol, were shortlisted and are depicted in Table 1.
Table 1. Docking results of top 25 hits against SARS-CoV-2 Mpro.
The results obtained through the molecular docking analysis of some of the presented molecules exhibit different insights that allows asserting whether it is possible to use them for medical treatment of COVID-19.
Ramipril benzyl ester forms two hydrogen bonds with Asp153 as well as Thr304 residues apart from interacting with Asn151, Phe294, Asp295, and Val303 residues of Mpro as presented in Figure 2.
Escholtzine / (S)-stylopine forms two hydrogen bonds with Thr111 and Ile152 apart from interacting with Asp153 residues of Mpro. Its interaction is presented in Figure 3.
3-oxoglycyrrhetinic acid forms two hydrogen bonds with Leu287 as well as Asp289, whereas Glu288, Leu272, and Tyr239 are the other interacting residues of Mpro. Figure 4 presents 3-oxoglycyrrhetinic acid interaction with Mpro.
Lariciresinol forms a hydrogen bond with Arg298 as well as interacts with Phe294, Ile106, and Val104 residues of Mpro. Figure 5 presents the interaction of this molecule with 6M03.
Cochinchinone B forms a hydrogen bond with Arg298 along with interacting with Ile249, Pro252, Leu253, Pro293, Phe294, Val297, and Val303 residues of Mpro. The interaction of this molecule with Mpro is presented in Figure 6
Lactimidomycin interacts with Phe8, Phe305, Val303, Arg298, Phe294, Val297, and Val104. In addition, it forms two hydrogen bonds with Gln110 and Asp295 residues of Mpro as presented in Figure 7.
Cardinalin 4 forms five hydrogen bonds, one with Asp197 and four with Lys5 apart from interacting with other residues of Mpro such as Lys137, Met276, Leu286, Leu287, and Asp289. Its interaction with Mpro is presented in Figure 8
Cinobufagin forms two hydrogen bonds with Thr111 residue and interacts with Val297, Phe294, Arg298, Val303, Phe305, Phe8, and Pro252 residues of Mpro. Figure 9 presents Ccnobufagin interaction with Mpro.
Quillaic acid interacts with Val104, Phe294, Arg298, Val303, and Phe305 residues of 6M03. The quillaic acid interaction with Mpro is presented in Figure 10.
Vitisinol C forms two hydrogen bonds with Asn151 and Arg298 apart from interacting with Gln110, Phe294, Val297, Val303, and Phe8. Figure 11 presents vitisinol C interaction with Mpro.
Citrusinol forms four hydrogen bonds with two residues, Asn151 and Gln110, as well as interacts with Val297, Arg298, and Val303 residues of Mpro. Figure 12 presents its interaction with Mpro.
Segetalin A forms three hydrogen bonds with Gln110, Ser158, and Asp153 and interacting with residues such as Phe294, Arg298, Val303 of Mpro. Figure 13 presents segetalin A interaction with 6M03.
Propafenone dimer interacts with Gln110, Phe294, Arg298, Val303, and Phe305 and forms two hydrogen bonds with Thr111 and Asn151 of Mpro residues as presented in Figure 14
Scopolamine 4-hydroxybenzoate forms hydrogen bonds with Phe294 and Thr304 apart from interacting with Asp295 and Val303 residues of Mpro. Figure 15 presents the scopolamine 4-hydroxybenzoate interaction.
Figure 2. Interaction of ramipril benzyl ester with Mpro.
Figure 3. Interaction of escholtzine / (S)-stylopine with Mpro.
Figure 4. Interaction of 3-oxoglycyrrhetinic acid with Mpro.
Figure 5. Interaction of lariciresinol with Mpro.
Figure 6. Interaction of cochinchinone B with Mpro.
Figure 7. Interaction of lactimidomycin with Mpro.
Figure 8. Interaction of cardinalin 4 with 6M03.
Figure 9. Interaction of cinobufagin with Mpro.
Figure 10. Interaction of quillaic acid with Mpro.
Figure 11. Interaction of vitisinol C with 6M03.
Figure 12. Interaction of citrusinol with 6M03.
Figure 13. Interaction of segetalin A with Mpro.
Figure 14. Interaction of propafenone dimer with Mpro.
Figure 15. Interaction of scopolamine 4-hydroxybenzoate with Mpro.
Discussion
Natural molecules are known derivatives of most of the present drugs in use against many diseases. In this study, we have also found some good leads and most of the 25 selected molecules show medicinal properties. Ramipril benzyl ester (protected form of ramipril), which is an inhibitor of angiotensin-converting enzyme ACE is the host receptor protein where the SARS-CoV-2 spike protein binds. Ramiprilat, which is an active compound of ramipril benzyl ester, is formed on the hydrolysis of the ester group, which later inhibits ACE. This leads to the prevention of the catalytic conversion of angiotensin I to angiotensin II that causes vasodilation17. Stylopine18 and quillaic acid exhibits anti-inflammatory activity19. Cinobufagin, CBF, has antitumor activity as it is known to inhibit the growth of PC3 cells both in vitro and in vivo along with inducing apoptosis of tumor cells as per reports20. It was found that intake of the plant ligands such as lariciresinol reduced endothelial dysfunction and vascular inflammation in post-menopausal women and middle aged/elderly men21. Vitisinol C as per earlier reports was found to exhibit an essential activity against amyloid aggregation, which is present in Vitis vinifera grapevine shoots22.
Segetalins are known to have vasorelaxant activity23. Scopolamine is known as antimuscarinics that blocks the effects of certain natural substance (acetylcholine) on the central nervous system, it is also used to prevent nausea and vomiting caused by motion sickness or medications used during surgery24.
3-oxo-glycyrrhetinic acid has antitumor activity and expectorant (antitussive) properties25. Lactimidomycin has antifungal, antiviral, and anti-cancer properties apart from acting as a direct inhibitor of protein translation inribosomes which is a glutarimide antibiotic derived from the bacterium Streptomyces amphibiosporus26. Cardinalins dinalins 4 exhibit anti-cancer properties. Conchinone B has an antioxidant activity. Rotenone used to treat head lice on humans, scabies, and parasitic mites on chickens, livestock, and pet animals. Propafenone is used for the treatment of irregular heartbeat like atrial fibrillation, paroxysmal supraventricular and tachycardia in order to restore normal heart rhythm for maintaining a regular, steady heartbeat27. These small molecules or potential drugs could be used as therapeutics for COVID-19 disease due to their binding affinity with the viral protein as well as interaction with Mpro. These small molecules were found to interact with the residues involved in enhancing the catalytic activity of Mpro or SARS CoV-2 main protease. Therefore, these could prove to play important role in inhibiting the viral activity. However, there are three small molecules ramipril benzyl ester, propafenone dimer and lariciresinol found from this study that might prove to be useful against this virus especially due to their present medical application, docking score and interaction with Mpro, as illustrated in Figure 16, Figure 17 and Figure 18.
Figure 16. Propafenone dimer interaction image with whole Mpro, main protease(6M03) of SARS-CoV-2.
Figure 17. Lariciresinol interaction image with whole Mpro, main protease(6M03) of SARS-CoV-2.
Figure 18. Ramipril Benzyl Ester interaction image with whole Mpro, main protease(6M03) of SARS-CoV-2.
Conclusions
Ramipril benzyl ester, stylopine, quillaic acid, cinobufagin, vitisinol C, segetalin A, scopolamine, 3-oxo glycyrrhetinic acid, lariciresinol, conchinone B, lactimidomycin, cardinalins 4, and propafenone dimer are 13 molecules that are predicted to form good interaction with Mpro. These molecules showed better binding affinity for Mpro than the reference molecule. Ramipril benzyl ester, apart from binding to the main protease of the virus, was also found to inhibit the virus receptor ACE present in humans. ACE2 is the receptor to which virus spike protein is known to bind to the host. The spike protein and Mpro of the SARS-CoV-2 virus are responsible for the virulence of the virus. Ramipril benzyl ester drug is reported to inhibit ACE and also found to inhibit main protease of SARS-CoV-2 virus from this study. Ramipril benzyl ester is presently used against heart stroke. Ramipril benzyl ester and propafenone are two important small molecules found from our study due to their present medicinal use as drugs for other diseases in human along with lariciresinol due to its medical application as stated in the discussion. As a result, these molecules have medicinal properties which could be used for the treatment of COVID-19 disease after further evaluation. This study presents a significant possibility in finding the cure for the COVID-19 disease from the drugs repurposing point of view. The drug targets mentioned in our study could bring relief to all those who are infected with the disease and these may also act as a prophylactic treatment when tested by the frontline healthcare providers. This study could provide relief to everyone if the drugs mentioned in our study proved to inhibit the virus in COVID19 positive patients since they are known natural derivatives with minimal side effects in individuals. Thus, our study could help in flattening the curve with speedy recovery in COVID -19 positive patients.
Data availability
Source data
The COVID-19 main protease structure was downloaded from the Protein Data Bank, ID 6M03: https://www.rcsb.org/structure/6M03.
Ligands were obtained from the Zinc Natural Products database (http://zinc.docking.org/).
Extended data
Zenodo: Molecular Docking ZINC DB. https://doi.org/10.5281/zenodo.40505769.
This project contains the following extended data:
Docking results for thee 100 best molecules (XLSX).
Interaction_images_viral_protein_molecules (subfolder). (Interaction images of the best 42 molecules.)
Zinc_natural_database_pdbqt_minimized (subfolder). (Molecular docking results for 1100 small molecules from the ZINC Natural Products database.)
Extended data are available under the terms of the Creative Commons Attribution 4.0 International license (CCBY 4.0).
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how to cite this article
Singh S and Florez H. Bioinformatic study to discover natural molecules with activity against COVID-19 [version 1; peer review: 2 approved]. F1000Research 2020, 9:1203 (https://doi.org/10.12688/f1000research.26731.1)
NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.
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PUBLISHED 06 Oct 2020
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How to cite this report:
Pollo-Cattaneo MF. Reviewer Report For: Bioinformatic study to discover natural molecules with activity against COVID-19 [version 1; peer review: 2 approved]. F1000Research 2020, 9:1203 (https://doi.org/10.5256/f1000research.29515.r72544)
The direct URL for this report is:
https://f1000research.com/articles/9-1203/v1#referee-response-72544
https://f1000research.com/articles/9-1203/v1#referee-response-72544
NOTE: it is important to ensure the information in square brackets after the title is included in this citation.
Reviewer Report 23 Oct 2020
Approved
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The article proposes a study for the search of drugs to combat COVID-19. This search will be carried out by virtually screened the Zinc natural database using HTVS tool through molecular docking studies to analyze molecules recommended for the treatment of
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HOW TO CITE THIS REPORT
Pollo-Cattaneo MF. Reviewer Report For: Bioinformatic study to discover natural molecules with activity against COVID-19 [version 1; peer review: 2 approved]. F1000Research 2020, 9:1203 (https://doi.org/10.5256/f1000research.29515.r72544)
The direct URL for this report is:
https://f1000research.com/articles/9-1203/v1#referee-response-72544
https://f1000research.com/articles/9-1203/v1#referee-response-72544
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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How to cite this report:
Ghosh P. Reviewer Report For: Bioinformatic study to discover natural molecules with activity against COVID-19 [version 1; peer review: 2 approved]. F1000Research 2020, 9:1203 (https://doi.org/10.5256/f1000research.29515.r72545)
The direct URL for this report is:
https://f1000research.com/articles/9-1203/v1#referee-response-72545
https://f1000research.com/articles/9-1203/v1#referee-response-72545
NOTE: it is important to ensure the information in square brackets after the title is included in this citation.
Reviewer Report 12 Oct 2020
Approved
VIEWS 19
The manuscript by Singh and Florez presents the discovery of drugs against COVID19 using molecular docking methods.
Major:
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- It might be worthwhile to re-check the binding affinity values for the top
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HOW TO CITE THIS REPORT
Ghosh P. Reviewer Report For: Bioinformatic study to discover natural molecules with activity against COVID-19 [version 1; peer review: 2 approved]. F1000Research 2020, 9:1203 (https://doi.org/10.5256/f1000research.29515.r72545)
The direct URL for this report is:
https://f1000research.com/articles/9-1203/v1#referee-response-72545
https://f1000research.com/articles/9-1203/v1#referee-response-72545
NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.
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