PONE-D-19-16606

In silico Identification of Angiotensin Converting Enzyme inhibitory Peptides from MRJP1

PLOS ONE

Dear Dr Sehgal,

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Kind regards,

Ghulam Md Ashraf, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. 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 manuscript submitted by Rana Tahir et al depicted "In silico Identification of Angiotensin Converting Enzyme inhibitory Peptides from MRJP1", established a systematic computational workflow to screen ACE inhibitor by Homology modeling, Molecular dynamic and docking, and predicted peptide “EALPHVPIFDR” has higher anti-hypertensive activity, and we looking forward to some deeper and new-finding based on that. This work is innovative and interesting. And the methods are also standard. In conclusion, this paper can be accepted, but there are still some places need to be revised:

1:Is the time of 30ns too short for protein structure stabilization and optimization using molecular dynamics?

2. It is suggested to incorporate the flow chart of the applied methodology for better understanding.

3: Authors stated that "The values of RMSD and RMSF denoted that the MRJP1 has rare loop structures at corresponding residues", What is the basis of this argument? References need to be updated.

4. The label of Figure 5 is difficult to see particularly yellow color. Change it to visible labeling and yellow color to any other one

5. Authors stated that "Mainly, PubChem, CDK-fingerprint, XLogP, electrotopological state atom type and auto-correlation descriptors were implemented..." and "while tri- peptide QSAR model was developed primarily by KlekotaRoth fingerprint count, PubChem fingerprint, CDK graph only fingerprint and extended fingerprint descriptors....". What is the basis of selecting these different descriptors for di-peptide or tri-peptide? References need to be updated. It is recommended to add a detailed description for modelling process of SVM, including feature engineeing (how to select or exclude features) and relevant diagrams.

6. Auther stated that "The peptide “EALPHVPIFDR” showed strong binding affinity and higher anti-hypertensive activity along with the global energy of -58.29 and docking score of 9590". The authors are off to a good start, however, this study requires additional experiments, such as implementing MD on complex structure from docking and then computing

binding free energy by MMPBSA. It would be better if auther supplement the simple biological test in vitro.

Reviewer #2: The authors present a purely computational study to identify Novel Angiotensin‐Converting Enzyme Inhibitory Peptides Derived from honey protein MRJP1. They tried to make a reliable structural model of MRJP1. Later, the top ranked derived peptides was docked against MRJP1 to analyse the molecular interactions. The fact that several modeling services have been employed, as well as their combination with MD refinement denote considerable care. However, the underlying problem remains the lack of independent confirmation of the docking poses, which is possibly the weakest link.

Major issues:

1) My main problem with this manuscript is that I do not understand who might take advantage of the reported findings: i) Biochemists/Molecular biologists might be tempted to validate In vitro the top ranked peptides if they have the system in place already, and peptides need to be synthesized and to make it proteolytic resistant which is the major challenge in peptide designing. ii) Computational fellows might want the presented pipeline to be validated before applying the method to other systems and iii) Chemists as well will not start any optimization before an experimental evidence.

2) The other major concern relates with their homology modeling approach. When they had an x-ray resolved crystal structure of Major Royal Jelly Protein 1 Oligomer readily available (PDB ID: 5YYL against their mentioned uniprot sequence ID: O18330) then there was absolutely no need to perform homology modeling, MD refinement and later model validation analysis. They should revise the study by taking into account the crystal structure and perform further analysis. Later, they should perform MD simulation of MRJP1 with/without bound peptide to examine the considerable influence of bound peptide. They should also perform the actual MM-GBSA calculations to better explore the binding free energy calculations in the presence of explicit solvent. The only docking conformations are not enough to estimate the most plausible interactions.

3) Another comment on their work relates with a suggestion if one considers a homology model. On what basis a 30ns timescale was selected? A careful reflection on RMSD revealed a gradual expansion even after 30 ns. In general, especially to estimate the protein’s stability, a simulation must be long enough to converge the dynamics of interest and exhibit equilibrium sampling. Everything depends on what you're trying to study, and depends again on the size of the system. For example, two small proteins with only one domain could perhaps be studied in a matter of tens or hundreds of nanoseconds. Complexes of multi-domain proteins may require microseconds or milliseconds depending on the time scale of domain motion. If there was a need of homology modeling (provided the template with >70% identity), they should have examined a backbone stability unless RMSD show convergence up to 10 ns at least. Later, they should perform clustering analysis and take the most representative conformation (with RMSD < 1Å) from the largest cluster to declare it a reasonable model. To better understand the timescale, author should consider this article:

Zwier, M.C. and Chong, L.T., 2010. Reaching biological timescales with all-atom molecular dynamics simulations. Current opinion in pharmacology, 10(6), pp.745-752.

4) I really enjoyed reading their discussion. After considering all above points in their revised study, the authors should compare their findings with the previously reported ACE inhibitory peptides. Quite a few articles are already published which have the same methodology as authors presented in their study. For example:

Yu, Z., Fan, Y., Zhao, W., Ding, L., Li, J. and Liu, J., 2018. Novel Angiotensin‐Converting Enzyme Inhibitory Peptides Derived from Oncorhynchus mykiss Nebulin: Virtual Screening and In Silico Molecular Docking Study. Journal of food science, 83(9), pp.2375-2383.

Yu, Z., Chen, Y., Zhao, W., Li, J., Liu, J. and Chen, F., 2018. Identification and molecular docking study of novel angiotensin‐converting enzyme inhibitory peptides from Salmo salar using in silico methods. Journal of the science of food and agriculture, 98(10), pp.3907-3914.

Vukic, V.R., Vukic, D.V., Milanovic, S.D., Ilicic, M.D., Kanuric, K.G. and Johnson, M.S., 2017. In silico identification of milk antihypertensive di-and tripeptides involved in angiotensin I–converting enzyme inhibitory activity. Nutrition research, 46, pp.22-30.

Others include:

Yu, Z., Wu, S., Zhao, W., Ding, L., Shiuan, D., Chen, F., Li, J. and Liu, J., 2018. Identification and the molecular mechanism of a novel myosin-derived ACE inhibitory peptide. Food & function, 9(1), pp.364-370.

Wang, C., Tu, M., Wu, D., Chen, H., Chen, C., Wang, Z. and Jiang, L., 2018. Identification of an ACE-Inhibitory Peptide from Walnut Protein and Its Evaluation of the Inhibitory Mechanism. International journal of molecular sciences, 19(4), p.1156.

Tu, M., Wang, C., Chen, C., Zhang, R., Liu, H., Lu, W., Jiang, L. and Du, M., 2018. Identification of a novel ACE-inhibitory peptide from casein and evaluation of the inhibitory mechanisms. Food chemistry, 256, pp.98-104.

Yu, Z., Fan, Y., Zhao, W., Ding, L., Li, J. and Liu, J., 2018. Novel Angiotensin‐Converting Enzyme Inhibitory Peptides Derived from Oncorhynchus mykiss Nebulin: Virtual Screening and In Silico Molecular Docking Study. Journal of food science, 83(9), pp.2375-2383.

By providing a comparison with the previously reported ACE inhibitory peptides, a fruitful discussion can be presented.

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Reviewer #1: No

Reviewer #2: Yes: Muhammad Usman Mirza

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

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PONE-D-19-16606R1

In silico Identification of Angiotensin Converting Enzyme inhibitory Peptides from MRJP1

PLOS ONE

Dear Dr. Sehgal,

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.

We would appreciate receiving your revised manuscript by 25th November 2019. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Ghulam Md Ashraf, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

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, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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 manuscript submitted by Rana Tahir et al depicted "In silico Identification of Angiotensin Converting Enzyme inhibitory Peptides from MRJP1", established a systematic computational workflow to screen ACE inhibitor by Homology modeling, Molecular dynamic and docking, and predicted peptide “EALPHVPIFDR” has higher anti-hypertensive activity, and we looking forward to some deeper and new-finding based on that. This work is innovative and interesting. And the methods are also standard. The response is basically reasonable. In conclusion, this paper can be accepted.

Reviewer #2: The authors improved the manuscript but I think authors simply overlooked one major concern as follows (2nd comment in my first revision):

1. "2). The other major concern relates with their homology modeling approach. When they had an x-ray resolved crystal structure of Major Royal Jelly Protein 1 Oligomer readily available (PDB ID: 5YYL against their mentioned uniprot sequence ID: O18330) then there was absolutely no need to perform homology modeling, MD refinement and later model validation analysis. They should revise the study by taking into account the crystal structure and perform further analysis. Later, they should perform MD simulation of MRJP1 with/without bound peptide to examine the considerable influence of bound peptide. They should also perform the actual MM-GBSA calculations to better explore the binding free energy calculations in the presence of explicit solvent. The only docking conformations are not enough to estimate the most plausible interactions"

Once MD simulations has performed then its easy to calculate the MM-GBSA/MM-PBSA. Authors can refer a tutorial from the link below as they used AMBER 14 simulation package:

https://ambermd.org/tutorials/advanced/tutorial3/py_script/section2.htm

2. The advantage of having a crystal structure in this study (as it is readily available) will significantly improved MD simulation analysis in terms of overall stability with bound peptide and binding free energy calculations.

The manuscript should publish after the incorporation of above mentioned suggestions/comments.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Muhammad Usman Mirza

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

In silico Identification of Angiotensin Converting Enzyme inhibitory Peptides from MRJP1

PONE-D-19-16606R2

Dear Dr. Sehgal,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Ghulam Md Ashraf, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

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, so please note any specific errors here.

Reviewer #2: Yes

**********

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 #2: Although the manuscript lack the important binding free energy calculations after MD simulations but the presented data is still publishable. In future studies, the authors should include MM-GBSA/PBSA calculations as an integral part after simple more unpredictable docking studies.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No