PONE-D-21-32120Reconstruction of the unbinding pathways of noncovalent SARS-COV and SARS-COV-2 3CLpro inhibitors using Unbiased Molecular DynamicsPLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

5. Review Comments to the Author

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Reviewer #1: In the article entitled “Reconstruction of the unbinding pathways of noncovalent SARS-COV and SARSCOV-2 3CLpro inhibitors using Unbiased Molecular Dynamics” the authors performed an in silico analysis of the unbinding process for two SARS-CoV and two SARS-CoV-2 3CLpro inhibitors. While the idea of the research seems good and very useful and the research seems to be performed correctly, the discussion is lacking; there are several issues that require attention and need to be corrected and more thoroughly explained before the article is accepted:

1. Capitalization of abbreviations “SARS-CoV” and “SARS-CoV-2” needs to be corrected in the title, as well as in the text. Additionally, capitalization of other words, such as “viruses” in the abstract needs to be corrected.

2. Also, there seems to be an issue with font type and size which is inconsistent.

3. Throughout the manuscript, English should be corrected e.g. the sentence (lines 35-36): “Coronaviruses (CoVs), which have the largest genome among RNA viruses, are enveloped by single-stranded RNA viruses.” does not make any sense.

4. The sentence (lines 71-72): “On the other hand, investigation of the unbinding pathways of particular inhibitors, protein-ligand key interactions, and ligand's effectiveness or weakness can be obtained.” needs to be rephrased, it does not make a lot of sense.

5. The sentence (line 73): “Ultimately, the obtained results will be presented as a fully atomistic scenario [17].” needs to be rephrased (and font changed).

6. The sentence (lines 86-90): “Since, half-maximal inhibitory concentration (IC50) of SARS-COV 3CLpro inhibitors are measured experimentally before, and the 3CLpro of SARS-COV and SARS-CoV-2 are approximately the same, our research team for participating in the global solidarity trial decided to examine some noncovalent SARS-CoV and SARS-CoV-2 protease inhibitors' unbinding pathways by the SuMD, Comparatively.” is very confusing and needs to be rephrased. Also, what does “approximately the same” even mean? Additional clarification is needed and does this influence the binding site?

7. There is no discussion and comparison of the obtained results with results of other papers, have other researchers performed similar studies, are the conclusions the same, is there any difference in results or a new insight?

8. What are the differences between the SARS-CoV and SARS-CoV-2 3CLpro binding sites? How does this reflect on binding of the studied inhibitors and their selectivity? If there are not any differences, it should also be said.

9. In figures, such as Figure 3 l, m and n, should the word “state (S)” be “bound state”?

10. Figures in general are of low quality, I don’t know if this is a consequence of converting to the PDF format or not.

11. Can the authors explain the “free energy” term in e.g. Figure 2 m, n, o, is it the ∆GBIND or something else?

12. Can the authors also explain how come in some cases (e.g. Figure 3.c) the total interaction energy goes as low as -350 kJ/mol, what does this interaction energy represent?

13. In line 255, what does the expression “the almost deep part” mean?

14. In the Conclusion, English is especially poor: the sentence (lines 336-338): “All fragments of inhibitors did not interact seriously at specific residues in the binding pocket, so this factor was sufficient to weaken critical interactions, resulting in rapid unbinding.” is very confusing and has no meaning.

15. The same goes for the sentence after that (lines 338-341): “These almost free fragments could compete with other fragments for interactions with essential residues. Even if they did not enter into competitive interactions, they could change inhibitors' position and brought the inhibitor closer to the exit path by their rotations.” which needs to be rephrased.

16. The sentence (lines 353-355): “In conclusion, the next series of noncovalent inhibitors should be designed so that they can occupy all S regions of the binding pocket to make maximum noncovalent interactions.” Does not say anything new, this is common knowledge – the inhibitors should fill out the binding site as much as possible. Are there any concrete suggestions the authors have for development of future 3CLpro inhibitors, which interactions are good and should stay, which interactions are bad and should be corrected etc.?

Reviewer #2: The article "Reconstruction of the unbinding pathways of noncovalent SARS-COV and SARS-COV-2 3CLpro inhibitors using Unbiased Molecular Dynamics" is an interesting paper in which pure classical Molecular Dynamics Simulations have been employed to investigate the unbinding process pathways of two noncovalent inhibitors of SARS-COV and SARS-COV-2. Authors have used the PDB IDs: 3V3M, 4MDS, 6W63 and 5RF7 and they have explored the inhibitors comparing the protein-inhibitor interaction by means of unbiased molecular dynamics (UMD) simulations. Authors have found that when the inhibitor occupies all space of the binding pocket, water molecules cannot penetrate under the ligand, and important inhibitor-protein interactions do not become water-mediated. In other words, the inhibitor must occupy all S region to develop a proper binding, otherwise, water molecules can occupy this space and important interactions can get lost.

Comments:

1) The methodology of this work is correct, the analysis is well supported, and the results are partially interesting.

2) The SuMD MD simulation is interesting to unbind the inhibitor from the active site because the center of masses distance is increased in each window. Perhaps, for pure classical Molecular Dynamic Simulations, just 500 ps could be not enough to equilibrate the system. Have the authors tried to increase the simulation time by window to avoid possible equilibration problems?

3) The structure of the paper and the results are very clear. There are four systems: two inhibitors and two proteins (3CL of SAR-CoV and SARS-CoV-2). For each system, three replicas unbinding the inhibitor (from 25 to 65 ns). For the results, same figures and same analysis to compare the results properly. My major criticism about this paper is that the main goals from the results: on one hand, when the inhibitor is unbinding, water molecules are coming in the active site, so, when the inhibitor occupy the S region, the binding should be better because less water molecules are affecting the inhibitor-enzyme interaction, so the main idea is that “water molecules played a functional role in all unbinding mechanisms by interfering and breaking important protein-inhibitor interactions”, which is something very well known in from many other works On the other hand, the inhibitor interacts such as independent fragments with different parts of the proteins. I think these findings are not very novel, however, some results are very interesting such as Figures 2, 4 and 6, parts m, n and o. These maps should be explained by the authors more carefully.

4) The bibliography is insufficient. There are many QM/MM Molecular Dynamic simulations and classical simulations of many inhibitors of the 3CL enzyme of SAR-CoV and SARS-CoV-2 which must be included by the authors.

5) As minor points:

1) The structure of the inhibitors should be provided in the introduction section. In addition, the structure of the active site of the complex protein-inhibitor highlighting the critical distances should be showed as well.

2) Page 9, line 178 “j and k” looks like a typo mistake

3) Footnote of Figure 3. Cut-offs are 0.35 and 0.5 angstroms? I think there is a mistake is those data.

Reviewer #3: The manuscript ID PONE-D-21-32120 entitled “Reconstruction of the unbinding pathways of noncovalent SARS-COV and SARS-COV-2 3CLpro inhibitors using Unbiased Molecular Dynamics” is quite a good study. The main protease (3CLpro) is one of the essential components of the SARS-COVs viral life cycle, which makes it an interesting target for overpowering Viruses. Although many covalent and noncovalent inhibitors have been designed to inhibit this molecular target, none have gained FDA approval as a drug. Because of the high rate of COVID-19 pandemic development, in addition to laboratory research, we require in silico methods to accelerate rational drug design. The unbinding pathways of two SARS-COV and SARS-COV-2 noncovalent inhibitors with the PDB IDs: 3V3M, 4MDS, 6W63, 5RF7 were explored from a comparative perspective using the unbiased molecular dynamics (UMD) simulations. We uncovered common weak points for selected inhibitors that could not have significant interactions with a binding pocket at specific residues by all their fragments. So water molecules entered the free binding S regions and weakened protein-inhibitor key interactions gradually. Finally, the authors believed that these results will help to design new potent inhibitors against SARS-CoV-2.

I appreciate the authors for their great effort of enclosing all the supplementary materials. The videos are really nice.

However, the following queries should be addressed before submitting a revision

1) In the abstract, the author provided more general info instead of the results of the study. In addition, the conclusion and methods are not appropriate---make it clear

2) In the introduction section, the author might start with SARS first, then SARS-2 to follow the uniformity of the title of the study.

3) Page 8 – lines 46-49: “The domains I and II have 46 antiparallel β barrel structures responsible for the catalytic reaction, while domain III has α-helices (Fig 1a, b). Since the dimeric form of Mpro is catalytically active, intermolecular interactions between the helical domains play an essential role in activating the enzyme [8, 9]. ------ It is not clear. What is the role of domain III?

4) What is unbiased molecular dynamics (UMD) and its advantages? Brief up 1 or 2 lines.

5) This line seems to be meaningless – “MD simulation has been a standard method of predicting hidden information in many science fields since the 1950s”----what is meant by hidden info by MD simulations?

6) What is the correlation of the above lines and “in judging candidate drugs, the bioavailability, selectivity, metabolic properties, and binding affinity of the designed inhibitor to its target protein are all important? [16].”

7) It is not an appropriate word “ligand's effectiveness or weakness can be obtained”. It may be ligands interacting efficiently with the target

8) These lines are a bit confusing or meaningless “Since, half-maximal inhibitory concentration (IC50) of SARS-COV 3CLpro inhibitors are measured experimentally before, and the 3CLpro of SARS-COV and SARS CoV-2 are approximately the same, our research team for participating in the global solidarity trial decided to examine some noncovalent SARS-CoV and SARS-CoV-2 protease inhibitors' unbinding pathways by the SuMD, Comparatively.” From MD authors defining IC50 value?

9) This is unclear: we performed 12 simulations, three replicas for each complex, by the SuMD method with some modifications---what are 12 simulations? Whether author’s starts from 1ps to 500ps or 1ps to 1500 ps defined as replicas? Because the following line authors mentioned, “------to extend the next 500 ps simulation”.

10) Reference missing for UCSF Chimera

11) Author may improve the discussion because in the results and discussion section authors described only results.

12) How do the authors create a free energy plot? Which package?

13) In all the figures, authors may reduce the thickness of the lines to get better visualization---figures are not in good resolution

14) Conclusion should be improved.

15) The language of the manuscript should meet the journal’s adequate standard

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PONE-D-21-32120R1Reconstruction of the unbinding pathways of noncovalent SARS-CoV and SARS-CoV-2 3CLpro inhibitors using Unbiased Molecular DynamicsPLOS ONE

Dear Dr. Aryapour,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR: You may want to upload a letter of editing from a professional agency or native-English speaker in addition to the reviewer's comments because reviewers have expressed a concern about the language part.

==============================

Please submit your revised manuscript by Feb 04 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Kind regards,

Chandrabose Selvaraj, Ph.D.

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have significantly improved the manuscript and added further discussion of their results. There are still significant language issues that need to be corrected throughout the manuscript (with e.g. lines 39-40 „Coronaviruses (CoVs), an RNA virus with the largest genome, are enveloped by a single-stranded RNA virus.“ – coronaviruses are enveloped RNA viruses, they are not enveloped BY a single stranded RNA virus; and lines 99-101: “So our research team, for participating in the solidarity clinical trial for COVID-19 treatments, decided to examine some noncovalent SARS-CoV and SARS-CoV-2 protease inhibitors' unbinding pathways by the SuMD, Comparatively.“ which is still grammatically very incorrect). Additionally, my comment about the lack of discussion and comparison with results from other researchers is still standing. The authors have improved their discussion, but they have obtained a lot of new information which could have been more thoroughly discussed and compared, it would further increase the significance and value of their work. Nonetheless, even in the present state, I believe that this research should be accepted for publishing.

Since the PLOS ONE’s states “PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision.”, I must ask the authors to find someone sufficiently proficient in English to proofread the article and correct all grammatical errors.

Reviewer #2: 1) Answer #5-1:

All fragments of each inhibitor are existence in the figures of the result section. If we change the location of these pictures, the readership of this article may get board to switch to the introduction section and again back to the result section. The active site domain of the protein is explained in Figure1, and a binding pocket of the complex protein-inhibitor is picturizing separately in Figures 2, 4, 6, and 8.

Comment#5-1:

I still think that a scheme of the inhibitors in the introduction section would increase the clarity of the study.

2) Answer #3:

For the "On the other hand, the inhibitor interacts such as independent fragments with different parts of the proteins. I think these findings are not very novel" we explained in the answer of Comment #16 of reviewe1. Also, we added a more detailed explanation of the Free energy landscape plots in the method section.

From the free energy landscapes, would be possible to predict a free energy of binding value of each system?

3) The initial Molecular Dynamic Simulations should be longer to obtain an equilibrated configuration of the proposed models. Before SuMDs, the system should be very well equilibrated. You can see many studies about 3CL enzyme in which classical MD of microsecond timescales is performed before to study binding or reaction processes. The initial protocol of this work does not allow, perhaps, to explore important configurations of the enzyme that should be considered in the study. Authors should explore this way carefully for future studies.

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Reconstruction of the unbinding pathways of noncovalent SARS-CoV and SARS-CoV-2 3CLpro inhibitors using Unbiased Molecular Dynamics

PONE-D-21-32120R2

Dear Dr. Aryapour,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Chandrabose Selvaraj, Ph.D.

Academic Editor

PLOS ONE

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Reviewer #1: All comments have been addressed

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Reviewer #1: Yes