Elucidating the inhibitory mechanism of Zika virus NS2B-NS3 protease with dipeptide inhibitors: Insights from molecular docking and molecular dynamics simulations

Shahid Ullah; Farhan Ullah; Wajeeha Rahman; Anees Ullah; Sultan Haider; Cao Yueguang

doi:10.1371/journal.pone.0307902

Peer Review History

Original SubmissionMarch 13, 2024
3 Apr 2024 Decision Letter - Ahmed A. Al-Karmalawy, Editor PONE-D-24-08994Elucidating the Inhibitory Mechanism of Zika Virus NS2B-NS3 Protease with Tripeptide Inhibitors: Insights from Molecular Docking and Molecular Dynamics Simulations.PLOS ONE Dear Dr. Ullah, 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. Please submit your revised manuscript by May 18 2024 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. 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). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Please note that PLOS ONE has specific guidelines on code sharing for submissions in which author-generated code underpins the findings in the manuscript. In these cases, all author-generated code must be made available without restrictions upon publication of the work. Please review our guidelines at https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-code and ensure that your code is shared in a way that follows best practice and facilitates reproducibility and reuse." 3. Please provide a complete Data Availability Statement in the submission form, ensuring you include all necessary access information or a reason for why you are unable to make your data freely accessible. If your research concerns only data provided within your submission, please write "All data are in the manuscript and/or supporting information files" as your Data Availability Statement. 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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A ****** 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: No Reviewer #2: Yes ****** 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: Authors of the presented manuscript evaluated the anti-ZIKV potentiality of singular tripeptide small molecule through its binding affinity towards viral protease with sole computational approach. The manuscript can be considered relevant in its field. However, several comments and suggestions are addressed as follows: 1. Authors investigated singular molecule against singular ZIKV biotarget. This can be considered a too consisted investigation to identify potential hits/leads for anti-ZIKV therapeutics. Investigating a series of compound would be more beneficial for developing comparative structure-activity relationship that would guide further optimization and development. 2. Adopting a positive control is advised within the computational study. This would benchmark the furnished predicted molecular aspects of the investigated molecule. Testing against positive control chosen as market or reference anti-ZIKV protease agent is advised to be done. 3. Within the molecular docking section, brief introduction about the targets’ topology and catalytic domain/binding site should be presented prior the results presentation. Cartoon and/or surface representations for the pocket and the key structural and functional features of the protein should be highlighted. This would allow the reader to grasp the differential docking findings across the investigated compounds. 4. Authors should provide compound-residue interactions in terms of the hydrogen bonding and hydrophobic contacts. However, hydrogen binding should be presented within hydrogen bond distances as well as bond angles since hydrogen bond depend on both. Authors should mention the Hydrogen bond angles as well as their distances, since the strength of hydrogen bonding is based on both parameters in a way to ensure the adequacy of optimum hydrogen bonding. 5. Authors are advised to provide the molecular dynamics free binding energies using MM-GBSA of MM-PBSA calculation for the computational simulations. These calculations would highlight the nature of the binding interactions in terms of predominant binding energy terms (i.e. ΔGelectrostatic and ΔGvan der Waal) as well as quantify the predicted polar solvation penalties against the compound binding. 6. Authors should provide snapshots for the simulated ligand-target complex across specific time intervals (e.g. 0ns, 100ns, and 200ns) to track the conformational and orientation changes for the simulated ligand and vicinal residues across the MD simulation runs. 7. Based on the study results, what are the take-away messages. Authors are advised to highlight the suggested structural modifications that would improve the compounds’ biological activities based on the in silico findings. These insights would be beneficial for guiding further lead optimization and development. 8. Finally, concerning the conclusion, authors are advised to elaborate more on the future of this work? Will you broaden the scope to other targets and/or microorganism? What are the study limitations and what approaches could be conducted to further address them? Reviewer #2: COMMENTS TO AUTHOR: The authors of the provided article on Understanding How Tripeptide Inhibitors Block the Zika Virus NS2B-NS3 Protease: Findings from Molecular Docking and Molecular Dynamics Simulation rephrase. This manuscript holds significance and adds value to the field, with some recommendations for prior publication. Few suggestions and comments are presented: 1. Ensure the formatting of the text in the manuscript is correct. Check space everywhere, Ex family [1, 2]. Space should be there. 2. The author utilized Schrödinger Release for ligand preparation and PyRx/Autodock Vina for molecular docking. What was the rationale for employing different software tools for these tasks…? 3. In figure 2 author should provide clear figure/ structure. 4. In silico word should be italic font. Check everywhere in the manuscript. 5. The authors should to include both the angles and distances of hydrogen bonds, as the effectiveness of hydrogen bonding relies on both factors, ensuring the adequacy of optimal hydrogen bonding. 6. The authors should provide additional details regarding the utilization of tripeptide inhibitors in this study, explaining the rationale behind their choice, in a single paragraph. ****** 6. 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: Yes Reviewer #2: No ******** [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.] 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0307902.r001
Revision 1
14 Jun 2024 Author Response Thank you and the reviewers for your time and valuable comments. Indeed, we have taken more time to carefully address and implement all comments, significantly improving our article. All changes have been highlighted for your review. 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: Authors of the presented manuscript evaluated the anti-ZIKV potentiality of singular tripeptide small molecule through its binding affinity towards viral protease with sole computational approach. The manuscript can be considered relevant in its field. However, several comments and suggestions are addressed as follows: 1. Authors investigated singular molecule against singular ZIKV biotarget. This can be considered a too consisted investigation to identify potential hits/leads for anti-ZIKV therapeutics. Investigating a series of compound would be more beneficial for developing comparative structure-activity relationship that would guide further optimization and development. Answer: Thank you for this direction. To address the mentioned concern, we have expanded our investigation significantly. In addition to the originally evaluated tripeptide small molecule, we included a dipeptide as a control to provide a comparative basis. Furthermore, we performed docking studies on a library of 200 compounds. From these, we selected the top 10 compounds based on their binding affinities and other relevant computational parameters. Changes Made: • Expanded Compound Library: We expanded our computational screening to include 200 compounds, providing a broader scope for potential anti-ZIKV activity. • Control Compound: A dipeptide was included as a control to enhance the comparative analysis and provide insights into the structure-activity relationship (SAR). • Top 10 Compounds Selection: We identified the top 10 compounds with the highest binding affinities and favorable interactions with the ZIKV protease. Detailed analyses of these compounds are included in the revised manuscript. • These changes have significantly strengthened our study, allowing for a more comprehensive evaluation of potential anti-ZIKV therapeutics and providing a robust framework for future optimization and development. We hope that these revisions meet your expectations and we are grateful for your valuable feedback which has improved the quality and relevance of our manuscript. 2. Adopting a positive control is advised within the computational study. This would benchmark the furnished predicted molecular aspects of the investigated molecule. Testing against positive control chosen as market or reference anti-ZIKV protease agent is advised to be done. Answer: We have expanded our investigation by including a broader range of compounds and a positive control. The specific changes we made regarding this specific control are: • Control Compound: We included a dipeptide as a control to enhance the comparative analysis and gain better insights into the structure-activity relationship (SAR). • Positive Control: We incorporated a known anti-ZIKV protease inhibitor as a positive control. This established benchmark allows us to compare the predicted molecular aspects of our investigated molecules against a reference compound, providing a more robust validation of our computational findings. 3. Within the molecular docking section, brief introduction about the targets’ topology and catalytic domain/binding site should be presented prior the results presentation. Cartoon and/or surface representations for the pocket and the key structural and functional features of the protein should be highlighted. This would allow the reader to grasp the differential docking findings across the investigated compounds. Answer: By repeating experiment and properly understanding the detailed topology and the specific features of the binding sites in these proteins, we better interpreted the differential docking results observed across the investigated compounds. The illustration has been designed as per instructed by author (cartoon) and surface representations provide a visual understanding of the pockets, showing how compounds interacted with these sites based on their size, shape, and chemical properties. 4. Authors should provide compound-residue interactions in terms of the hydrogen bonding and hydrophobic contacts. However, hydrogen binding should be presented within hydrogen bond distances as well as bond angles since hydrogen bond depend on both. Authors should mention the Hydrogen bond angles as well as their distances, since the strength of hydrogen bonding is based on both parameters in a way to ensure the adequacy of optimum hydrogen bonding. Answer: thanks for this comments. We have provided all the above mention requirements. Which are clearly illustration in the figures. 5. Authors are advised to provide the molecular dynamics free binding energies using MM-GBSA of MM-PBSA calculation for the computational simulations. These calculations would highlight the nature of the binding interactions in terms of predominant binding energy terms (i.e. ΔGelectrostatic and ΔGvan der Waal) as well as quantify the predicted polar solvation penalties against the compound binding. Answer: Answer: The required calculations of free binding energies using MM-GBSA of MM-PBSA are presented in table 2. 6. Authors should provide snapshots for the simulated ligand-target complex across specific time intervals (e.g. 0ns, 100ns, and 200ns) to track the conformational and orientation changes for the simulated ligand and vicinal residues across the MD simulation runs. Answer: Thank you very much for this comments, kindly see the bellow snapshots of the need time intervals. 7. Based on the study results, what are the take-away messages? Authors are advised to highlight the suggested structural modifications that would improve the compounds’ biological activities based on the in silico findings. These insights would be beneficial for guiding further lead optimization and development. Answer: The key take-away messages from this study are Identification of a Promising Therapeutic Target, Potential Candidate Ligand, Confirmation of Binding Affinity and Structural Modifications for Enhanced Activity. By highlighting the suggested structural modifications based on in silico findings, we have provided valuable insights for guiding future lead optimization efforts, ultimately aiming to develop more potent and selective compounds for combating ZIKV infection. 8. Finally, concerning the conclusion, authors are advised to elaborate more on the future of this work? Will you broaden the scope to other targets and/or microorganism? What are the study limitations and what approaches could be conducted to further address them? Answer: Future prospective have been discussed according to author’s requirement. Reviewer #2: COMMENTS TO AUTHOR: The authors of the provided article on Understanding How Tripeptide Inhibitors Block the Zika Virus NS2B-NS3 Protease: Findings from Molecular Docking and Molecular Dynamics Simulation rephrase. This manuscript holds significance and adds value to the field, with some recommendations for prior publication. Few suggestions and comments are presented: 1. Ensure the formatting of the text in the manuscript is correct. Check space everywhere, Ex family [1, 2]. Space should be there. Answer: Thank you very much for deep insight and recommendation of the article for publication. We have properly reviewed and corrections are made accordingly. 2. The author utilized Schrödinger Release for ligand preparation and PyRx/Autodock Vina for molecular docking. What was the rationale for employing different software tools for these tasks…? Answer: Considering the above point only pyrx/ autodock vina was used for molecular docking. 3. In figure 2 author should provide clear figure/ structure. Answer: Properly reviewed and corrections are made accordingly. 4. In silico word should be italic font. Check everywhere in the manuscript. Answer: Properly reviewed and corrections are made accordingly. 5. The authors should to include both the angles and distances of hydrogen bonds, as the effectiveness of hydrogen bonding relies on both factors, ensuring the adequacy of optimal hydrogen bonding. Answer: The above mention requirements are clearly illustration in all figures. 6. The authors should provide additional details regarding the utilization of tripeptide inhibitors in this study, explaining the rationale behind their choice, in a single paragraph. Answer: The inhibitor used in the article is dipeptide and it significances are highlighted in the manuscript as per mentioned accordingly. Attachments Attachment Submitted filename: Revised_Comments.docx https://doi.org/10.1371/journal.pone.0307902.r002
24 Jun 2024 Decision Letter - Ahmed A. Al-Karmalawy, Editor PONE-D-24-08994R1Elucidating the Inhibitory Mechanism of Zika Virus NS2B-NS3 Protease with Dipeptide Inhibitors: Insights from Molecular Docking and Molecular Dynamics Simulations.PLOS ONE Dear Dr. Ullah, 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. ============================== Please submit your revised manuscript by Aug 08 2024 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. 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). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Ahmed A. Al-Karmalawy, PhD Academic Editor PLOS ONE Journal Requirements: 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. [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: (No Response) 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 #1: (No Response) Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) 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: (No Response) 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: (No Response) 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 authors responded to almost all comments and suggestions. Still proper annotation for the ligand-target interaction is required. The authors annotated for the hydrogen distances only without annotating for the hydrogen bond angles. Hydrogen binding should be presented as both distances and bond angles since hydrogen bond depend on both as to ensure the adequacy of optimum hydrogen bonding Reviewer #2: The author responds to all comments given by reviewer now the article is appropriate for publication ****** 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: Yes Reviewer #2: No ******** [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.] 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0307902.r003
Revision 2
9 Jul 2024 Author Response Reviewer #1: The authors responded to almost all comments and suggestions. Still proper annotation for the ligand-target interaction is required. The authors annotated for the hydrogen distances only without annotating for the hydrogen bond angles. Hydrogen binding should be presented as both distances and bond angles since hydrogen bond depend on both as to ensure the adequacy of optimum hydrogen bonding Answer: We value your feedback and recommendations about the ligand-target interaction annotation, especially about the need to include hydrogen bond angles. We agree that angle and distance criteria do define hydrogen bonds; these two factors together govern the specificity and strength of the interactions. Our investigation originally concentrated on hydrogen bond distances to give a clear picture of the interactions. However, a more complete and accurate representation of the binding interactions will be provided by including hydrogen bond angles. The angle between the donor atom, the hydrogen atom, and the acceptor atom is the standard way to measure the hydrogen bond angle (D-H...A). This angle usually falls between 150° and 180° for the best hydrogen bonding. The angle criteria filter the hydrogen bonds mentioned in the manuscript. For the sake of visualization, we mentioned the angle criteria in the figure legend and in the manuscript. 2nd Reviewer Thanks you very much for taking your time and accepting our manuscript. Attachments Attachment Submitted filename: Response to the reviewer.docx https://doi.org/10.1371/journal.pone.0307902.r004
15 Jul 2024 Decision Letter - Ahmed A. Al-Karmalawy, Editor Elucidating the Inhibitory Mechanism of Zika Virus NS2B-NS3 Protease with Dipeptide Inhibitors: Insights from Molecular Docking and Molecular Dynamics Simulations. PONE-D-24-08994R2 Dear Dr. Ullah, 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. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Ahmed A. Al-Karmalawy, PhD Academic Editor PLOS ONE 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: (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: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) ****** 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: (No Response) ****** 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: (No Response) ****** 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: (No Response) ****** 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: Yes ******** https://doi.org/10.1371/journal.pone.0307902.r005
Formally Accepted
30 Jul 2024 Acceptance Letter - Ahmed A. Al-Karmalawy, Editor PONE-D-24-08994R2 PLOS ONE Dear Dr. Ullah, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Associate Professor Ahmed A. Al-Karmalawy Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0307902.r006

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