Targeting Natural Plant Metabolites for Hunting SARS-CoV-2 Omicron BA.1 Variant Inhibitors: Extraction, Molecular Docking, Molecular Dynamics, and Physicochemical Properties Study

Round 1

Reviewer 1 Report

This manuscript introduced some potential compounds for SARS-CoV-2 Omicron BA.1 Variant inhibition by using docking and MD. Although some efforts were paid, it is suggested to be rejected in the current form. There are some obvious scientific problems in the simulation calculation of this article, but in view of the workload of the separation and extraction of compounds, it is recommended to submit it to a phytochemical journal.

 

Some major issues:

 

1. Page 2: “Three of them (1,13, and 15) were extracted and annotated from Echium angustifolium aerial parts (Boraginaceae family) via LC-ESI-MS/MS…At this time, Traditional Chinese Medicine (TCM) recommended peach as a contributor in the preven-tion and treatment of COVID-19”

Most of the compounds in this paper were extracted from Prunus persica, but the supporting literature is insufficient. What is the literature basis for the extraction of compounds 1, 13, 15 from E. angustifolium? It is recommended to use "peach" instead of "Prunus persica".

 

2. Some figure and tables in the supporting information are missing numbers

 

3. The structure of compound 15 is not shown.

4. Page 2: delete “because of its high accuracy and capability to create nine various poses of a molecule in-side the protein binding site.”

 

5. Page 6. Fig.1: The corresponding 3D situation should be attached

 

6. Page 7. Fig.2: Why put 50ns, 100ns, 150ns simulations? What is the meaning? Are these simulations independent (0~50ns, 0~100ns, 0~150ns, 0~200ns) or continue with the previous ones (0 ~50~100~150~200ns)?

 

7. Page 8. Fig.3: The RMSD values of several compounds changed drastically. The corresponding time trajectories and representative conformations should be extracted for specific analysis and explanation, and the final stabilized conformation should be given. Otherwise, such results are meaningless. The same is for the analysis of other MD results.

 

8. The RMSD analysis of each trajectory preferably includes protein receptors, ligands, and complexes. The abnormal situation should be further verified or analyzed.

 

9. There are many problems in English expressions.

 

10. Many basic formulas and classical accepted principle explanations occupy most of the space, which is of no practical significance. It is recommended to delete them and replace them by citing references. The article focuses on analyzing the experimental conclusions.

 

11. How is the protein structure used for molecular docking obtained? Please indicate the PDB ID. If there is no PDB ID, please indicate the way to obtain the structure.

 

12. Page 14: 3.6. Molecular Dynamics Simulations: The order of operations should be documented, eg structure preparation should be placed before minimization. How many minimization steps are performed? The sequence and the time of each simulation step prior to the production needs to be clarified. The scientificity of the method is questionable.

 

13. The results of computational experiments have not been fully analyzed, and the experimental data of molecular dynamics are not sufficient to explain the problem. From the existing experimental data, we can only know that these molecules have changed when they interact with proteins. What are the specific changes? Is the change good or bad?

 

Author Response

Manuscript correction requested (cimb-1903255)

Title: Targeting natural plant metabolites for Hunting SARS-CoV-2 Omicron
BA.1 Variant Inhibitors: Extraction, Molecular docking, Molecular dynamics,
and Physicochemical Properties Study

 

We appreciate the editors' and reviewers' effort and thoughtful remarks on our manuscript. We have implemented their comments and suggestions and would like to submit a revised version of the manuscript to the journal for further consideration. Changes in the initial version of the manuscript are marked up using “Track
Changes” function. Below, we also provide a point-by-point response explaining how we have addressed each of the editors' or reviewers' concerns.

 

We hope that the revision is acceptable for publication in your journal.

 

We look forward to the outcome of your evaluation.

Yours sincerely,

 

On behalf of the co-authors

 

Ahmed E. Allam

 

 

Here is a point-by-point response to the reviewers’ comments and concerns.

 
Comments from Reviewer 1

 

This manuscript introduced some potential compounds for SARS-CoV-2 Omicron BA.1 Variant inhibition by using docking and MD. Although some efforts were paid, it is suggested to be rejected in the current form. There are some obvious scientific problems in the simulation calculation of this article, but in view of the workload of the separation and extraction of compounds, it is recommended to submit it to a phytochemical journal.

Some major issues:

1. 1. Page 2: “Three of them (1,13, and 15) were extracted and annotated from Echium angustifolium aerial parts (Boraginaceae family) via LC-ESI-MS/MS…At this time, Traditional Chinese Medicine (TCM) recommended peach as a contributor in the prevention and treatment of COVID-19”

Most of the compounds in this paper were extracted from Prunus persica, but the supporting literature is insufficient. What is the literature basis for the extraction of compounds 1, 13, 15 from E. angustifolium? It is recommended to use "peach" instead of "Prunus persica".

What is the literature basis for the extraction of compounds 1, 13, 15 from E. angustifolium?

Responds;

The pleiotropic activities of phenolic acid derivative compounds especially flavonoids such as their antioxidant and anti-microbial activities, to inhibit key proteins involved in the coronavirus infective cycle, as well as lack of their systemic toxicity, supported the researchers to investigate their potential role against COVID-19 by in silico studies. Consequently, several studies represented the interaction of some flavonoids and phenolic acids with SARS-CoV-2 proteins to combat COVID-19 pandemic [1–7], encouraging us to conduct our research on the phenolic acid derivative compounds of E. angustifolium which were not previously evaluated and peach as well.

1- M. Russo, S. Moccia, C. Spagnuolo, I. Tedesco and G. L. Russo, Roles of flavonoids against coronavirus infection, Chem. Biol. Interact., 2020, 328, 109211, DOI: 10.1016/j.cbi.2020.109211.

2- A. da Silva Antonio, L. S. Wiedemann and V. F. Veiga-Junior, Natural products' role against COVID-19, RSC Advances, 2020, 10(39), 23379–23393, DOI: 10.1039/d0ra03774e.

3- A. G. Omokhua-Uyi and J. Van Staden, Natural product remedies for COVID-19: A focus on safety, S. Afr. J. Bot., 2021, 139, 386–398, DOI: 10.1016/j.sajb.2021.03.012.

4- S. S. El-Hawary, M. A. Rabeh, M. A. Raey, E. M. El-Kadder, M. Sobeh, U. R. Abdelmohsen, A. Albohy, A. M. Andrianov, I. P. Bosko, M. M. Al-Sanea and D. G .El-Kolobby, Metabolomic profiling of three Araucaria species, and their possible potential role against COVID-19, J. Biomol. Struct. Dyn., 2021, 1–3, DOI: 10.1080/07391102.2021.1885494.

5- M. A. A. Ibrahim et al., “Rutin and flavone analogs as prospective SARS-CoV-2 main protease inhibitors: In silico drug discovery study,” J. Mol. Graph. Model., 2021, 105, 107904, DOI: 10.1016/j.jmgm.2021.107904.

6- A. R. Hassan et al., “Chemical constituents from Limonium tubiflorum and their in silico evaluation as potential antiviral agents against SARS-CoV-2,” RSC Advances, 2021, 11(51), 32346–32357, DOI: 10.1039/D1RA05927K.

7- H. A. Hassan et al., “Conducting the RBD of SARS-CoV-2 Omicron Variant with Phytoconstituents from Euphorbia dendroides to Repudiate the Binding of Spike Glycoprotein Using Computational Molecular Search and Simulation Approach,” Molecules, 2022, 27(9), 2929, DOI: 10.3390/molecules27092929.

It is recommended to use "peach" instead of "Prunus persica"

Responds; "peach" is used instead of "Prunus persica" along the manuscript

2. Some figure and tables in the supporting information are missing numbers

Responds;

The missing figures and table numbers were added.

3. 3. The structure of compound 15is not shown.

Responds;

Structure of compound 15 has been already added

4. Page 2: delete “because of its high accuracy and capability to create nine various poses of a molecule in-side the protein binding site.”

Response:

Thank you for bringing this inconsistency to our attention. We have deleted it.

5. Page 6. Fig.1: The corresponding 3D situation should be attached

Response:

Thank you for this comment. We have included it.

6. Page 7. Fig.2: Why put 50ns, 100ns, 150ns simulations? What is the meaning? Are these simulations independent (0~50ns, 0~100ns, 0~150ns, 0~200ns) or continue with the previous ones (0 ~50~100~150~200ns)?

Response:

Thank you for this comment. The main aim of including this figure is to illustrate the fluctuation of binding energy values for the five inspected compounds within the binding cavity of the target protein over different simulation times (50ns, 100ns, 150ns, and 200ns). In some cases, a compound can give, for instance, a high value of binding energy at 50 ns, and then this value drastically decreases at 100ns and further. So, this figure ensures for somewhat the convergence of estimated MM/PBSA binding energies.

 

7. Page 8. Fig.3: The RMSD values of several compounds changed drastically. The corresponding time trajectories and representative conformations should be extracted for specific analysis and explanation, and the final stabilized conformation should be given. Otherwise, such results are meaningless. The same is for the analysis of other MD results.

Response:

Thank you for raising this important point. We agree with this and that's why we have performed a long MD simulation for compounds (200ns) to identify the most stable protein-ligand complex. Furthermore, we have included Fig. 6 to show the protein-ligand structures every 25ns, allowing us to detect the time at which the compound exhibits instability inside the protein binding cavity that causes the RMSD value to change at a defined point. Cmd-3 showed great stability during the whole simulation course (200ns). Cmd-4 from 75ns to the end of the simulation course. Cmd-2 exhibited stability up to 100ns and Cmd-5 up to 125ns. Regarding Cmd-1, we mentioned that it has a negative result with respect to others "In contrast, Cmd-1 showed higher values of RMSD (avg. = 5.53 Å), which reflects the occurrence of some positional or conformational changes".

 

8. The RMSD analysis of each trajectory preferably includes protein receptors, ligands, and complexes. The abnormal situation should be further verified or analyzed.

Response:

Thanks for your valuable comment. We have included Fig. 5 which illustrates the RMSD values of the studied ligand, in addition to the previous Fig. 4 which illustrates the RMSD values for the apo-protein structure and the five complexes.

 

9. There are many problems in English expressions.

Response: Thank you very much for your comment. This manuscript was edited for proper English language, grammar, punctuation, spelling, and overall style by a fluent English speaker. We hope the revised manuscript will meet the requirements of academic publishing in “Current Issues in Molecular Biology”.

 

10. Many basic formulas and classical accepted principle explanations occupy most of the space, which is of no practical significance. It is recommended to delete them and replace them by citing references. The article focuses on analyzing the experimental conclusions.

Response:

Thank you for pointing this out. We have removed the well-known RMSD, RMSF, and Rg equations and provided various references instead.

 

11. How is the protein structure used for molecular docking obtained? Please indicate the PDB ID. If there is no PDB ID, please indicate the way to obtain the structure.

Response:

Thank you for this comment. The ID of the employed protein structure, which was downloaded from the protein data bank (PDB) database, has been mentioned in the methodology section (2.3 protein preparation).

 

12. Page 14: 3.6. Molecular Dynamics Simulations: The order of operations should be documented, eg structure preparation should be placed before minimization. How many minimization steps are performed? The sequence and the time of each simulation step prior to the production needs to be clarified. The scientificity of the method is questionable.

Response:

Thank you for raising these points. We have rearranged the written steps and included more details about the time of minimization and equilibration prior to the final production step.

 

13. The results of computational experiments have not been fully analyzed, and the experimental data of molecular dynamics are not sufficient to explain the problem. From the existing experimental data, we can only know that these molecules have changed when they interact with proteins. What are the specific changes? Is the change good or bad?

Response:

Thank you for mentioning that. MD simulation step is always applied in most research related to the computational investigations of extracted or collected compounds toward the target protein. The value of relative binding energy gives no clear evidence about the behavior of the compound inside the binding cavity of a protein. This is due to the estimation of binding energy value being based on different parameters such as Evdw, Eelec, Egb, Esur, Egas, and Esolv. So, we have included what is called "post-MD analysis" which is used to verify and confirm the potent inhibitors and illustrate their behavior inside the binding site of protein during the simulation. The radius of gyration and root-mean-square deviation can clearly point out the relative stability of Cmd-2, Cmd-3, and Cmd-4 during long MD simulation inside the active site of the protein. In contrast to that, Cmd-1 and, to a lesser extent, Cmd-5.

 

 

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript entitled “Targeting natural plant metabolites for Hunting SARS-CoV-2 Omicron BA.1 Variant Inhibitors: Extraction, Molecular docking, Molecular dynamics, and Physicochemical Properties Study” submitted to the Journal “Current Issues in Molecular Biology” for publication. In the submitted manuscript, the authors isolated various flavonoid compounds from the aerial part of plant E. angustifolium and performed molecular docking and physicochemical studies against SARS-Cov-2 variant. The major issue in the current study is the lack of physical biological evaluation of the compounds extracted from the plant. The studies performed in this manuscript are totally based on computational predictions and physically extracted compounds were not used for their in vitro or in vivo evaluation. Compounds isolated are also very well-known flavonoids and identified previously by other research groups. None of the compounds studied in the current manuscript is found novel. It does not make any sense to perform the extraction and their evaluation just through computational studies. The best way to perform these evaluations is, just download the flavonoid compounds available in the database and perform the molecular binding simulations against SARS-Cov-2 variant. In my opinion, this article needs appropriate modifications and would be well suited in journals based on computational based drug designing. Keeping in mind this major issue, I do not recommend the article to publish in the journal “Current Issues in Molecular Biology”.

 

 

Author Response

Manuscript correction requested (cimb-1903255)

Title: Targeting natural plant metabolites for Hunting SARS-CoV-2 Omicron
BA.1 Variant Inhibitors: Extraction, Molecular docking, Molecular dynamics,
and Physicochemical Properties Study

 

We appreciate the editors' and reviewers' effort and thoughtful remarks on our manuscript. We have implemented their comments and suggestions and would like to submit a revised version of the manuscript to the journal for further consideration. Changes in the initial version of the manuscript are marked up using “Track
Changes” function. Below, we also provide a point-by-point response explaining how we have addressed each of the editors' or reviewers' concerns.

 

We hope that the revision is acceptable for publication in your journal.

 

We look forward to the outcome of your evaluation.

Yours sincerely,

 

On behalf of the co-authors

 

Ahmed E. Allam

 

 

Here is a point-by-point response to the reviewers’ comments and concerns.

 
Comments from Reviewer 2

** “The manuscript entitled “Targeting natural plant metabolites for Hunting SARS-CoV-2 Omicron BA.1 Variant Inhibitors: Extraction, Molecular docking, Molecular dynamics, and Physicochemical Properties Study” submitted to the Journal “Current Issues in Molecular Biology” for publication. In the submitted manuscript, the authors isolated various flavonoid compounds from the aerial part of plant E. angustifolium and performed molecular docking and physicochemical studies against SARS-Cov-2 variant. The major issue in the current study is the lack of physical biological evaluation of the compounds extracted from the plant. The studies performed in this manuscript are totally based on computational predictions and physically extracted compounds were not used for their in vitro or in vivo evaluation. Compounds isolated are also very well-known flavonoids and identified previously by other research groups. None of the compounds studied in the current manuscript is found novel. It does not make any sense to perform the extraction and their evaluation just through computational studies. The best way to perform these evaluations is, just download the flavonoid compounds available in the database and perform the molecular binding simulations against SARS-Cov-2 variant. In my opinion, this article needs appropriate modifications and would be well suited in journals based on computational based drug designing. Keeping in mind this major issue, I do not recommend the article to publish in the journal “Current Issues in Molecular Biology”.”

 

Response:

Thank you very much for comments and suggestions which lead to improvement of our manuscript.

• Regarding the lack of physical biological evaluation of the compounds extracted from the plant; it is known that kind of evaluation (anti-Omicron BA.1 variant) needs advanced tools not easily available in all laboratories. So our plan in this stage was discovering natural phytoconstituents based on computational designing to be further included in in vitro or in vivo evaluation in the near future.
• Regarding the issue '' None of the compounds studied in the current manuscript is found novel ''.

 It is known the isolation from natural products is carried out in a blind manner however, this research contains not only flavonoids but also phenolic acid spermidine derivatives.

 

 

Author Response File: Author Response.docx

Reviewer 3 Report

Using Molecular Docking Simulations, the authors provided new information on chemicals extracted/identified from natural sources as possible therapeutic options to bind CoVID-Omicron Variant.

I would propose to include the following

1. Explain the mechanism of protein of interest (drug target) to be studied and any literature utilized to discover compounds using insilico or other experimental studies.

2. Elucidate potential interactions among compounds and proteins in the absence of the mutations and how chosen ligand's preferred binding site and possible interacting residues (unique and common) participated in stabilizing the complex.

 

Author Response

Manuscript correction requested (cimb-1903255)

Title: Targeting natural plant metabolites for Hunting SARS-CoV-2 Omicron
BA.1 Variant Inhibitors: Extraction, Molecular docking, Molecular dynamics,
and Physicochemical Properties Study

 

We appreciate the editors' and reviewers' effort and thoughtful remarks on our manuscript. We have implemented their comments and suggestions and would like to submit a revised version of the manuscript to the journal for further consideration. Changes in the initial version of the manuscript are marked up using “Track
Changes” function. Below, we also provide a point-by-point response explaining how we have addressed each of the editors' or reviewers' concerns.

 

We hope that the revision is acceptable for publication in your journal.

 

We look forward to the outcome of your evaluation.

Yours sincerely,

 

On behalf of the co-authors

 

Ahmed E. Allam

 

 

Here is a point-by-point response to the reviewers’ comments and concerns.

 
Comments from Reviewer 3

** “Using Molecular Docking Simulations, the authors provided new information on chemicals extracted/identified from natural sources as possible therapeutic options to bind CoVID-Omicron Variant.”

 

Response: Thanks for your appreciation.

 

** “Explain the mechanism of protein of interest (drug target) to be studied and any literature utilized to discover compounds using in silico or other experimental studies.”

 

Response: Thanks for raising these important points. We have addressed all of the raised points in the introduction section and included various recent references.

 

** “Elucidate potential interactions among compounds and proteins in the absence of the mutations and how chosen ligand's preferred binding site and possible interacting residues (unique and common) participated in stabilizing the complex.”

 

Response: Thank you for pointing this out. We agree this significantly strengthens the paper. We have evaluated the potential inhibition of best-reached anti-Omicron drugs against the receptor binding domain (RBD) of the wild-type (WT) SARS-CoV-2 via applying molecular docking calculations. All results are illustrated in Fig. 2 and are well discussed in terms of binding affinity and possible interacting residues. We have also included Table 1, which outlines the mutational sites inside the RBD of Omicron BA.1 and the original residues of the WT SARS-CoV-2.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Most of the issues have been addressed. 

Author Response

Dear reviewer

We appreciate the editors' and reviewers' effort and thoughtful remarks on our manuscript.

Reviewer 2 Report

The authors fail to address the critical issue raised by me in the manuscript. Even, it was not justified by the authors why the isolation of these compounds has been performed. Accordingly, I do not recommend the article to publish in this journal.

Author Response

Dear reviewer

We appreciate the editors' and reviewers' effort and thoughtful remarks on our manuscript.

Natural products are always a very rich source of bioactive compounds, sometimes the isolated compounds are new, others are common and ubiquitous. When we start to isolate the compounds from the mentioned plants, we start it in a blind manner. The resulted compounds are unknown till we do the NMR analyses. Concomitantly we start to discover novel bioactivities for the known compounds. Presence of a pandemic like SARS-COV-2 was the motive beyond our in silico study. Further isolation processes are now already in place to discover other new compounds besides to the enrichment of the previously isolated compounds to complete our study on SARS-COV-2