Aotaphenazine, a rare hydrophenazine, targets topoisomerase II with anticancer efficacy: In silico to in vitro evidence

Ahmed M. Metwaly; Ibrahim H. Eissa; Wael M. Afifi; Eslam B. Elkaeed; Aisha A. Alsfouk; Ibrahim M. Ibrahim; Mohamed S. Abdelfattah

doi:10.1371/journal.pone.0338135

Peer Review History

Original SubmissionJuly 21, 2025
27 Oct 2025 Decision Letter - Abdullahi Ibrahim Uba, Editor Dear Dr. Metwaly, Based on the reviewers’ comments, the manuscript requires minor revision before it can be considered for publication. The experimental section should be expanded with sufficient methodological details and appropriate citations. The figure and table legends need to be more detailed and self-explanatory, and proper citations for the chemical characterization of Aotaphenazine (¹H NMR, ¹³C NMR, and MS data) should be provided. Figures 2 and 7 should be improved—specifically by including both 2D and 3D representations for DOXO and Aotaphenazine in complex with DNA-Topo II, and by clarifying images C1–C3. The cell cycle and apoptosis assay section should be revised to include control cell data for comparison with the Aotaphenazine-treated MDA-MB-231 cells. Finally, the conclusion section should be concise and clearly summarize the in vitro findings. plosone@plos.org . 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Yes ******** 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 Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: Dear Author, I have gone through the manuscript, it is fine in majority of aspects. There are few aspects that needs attention like: 1. The experimental section needs to be more elaborate with proper references. 2. In the references, the authors may provide full details of the authors instead of mentioning et al. 3. Under conclusions the details of in vitro studies should be clearly discussed and it should be brief. 4. The legends to the figures and tables should be more elaborate and self explanatory. 5. Under cell cycle and apoptosis assay, only Aotaphenazine treated MDA-MB-231 cells are shown. Why the control cell data is not shown in the figure. Reviewer #2: Reviewer Comments Give the citation reference for the characterization of Aotaphenazine’s chemical structure, like 1HNMR/13CNMR & MS analysis. Fig. 2: Include the two-dimensional pictures for each reference DOXO and Aotaphenazine using the DNA-Topo II complex, supported with each 3D graphic to better explain the many types of amino acid interactions. Fig. 7: Images C1, C2, and C3 require further clarification. ****** what does this mean? ). 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Revision 1
5 Nov 2025 Author Response Dear editors, Firstly, the authors would like to sincerely thank the Editor and Reviewers for their valuable time, constructive feedback, and insightful comments. Their suggestions have greatly contributed to improving the clarity, rigor, and overall quality of our manuscript. We have carefully revised the paper in accordance with all recommendations as detailed below: Reviewer #1: Dear Author, I have gone through the manuscript, it is fine in majority of aspects. There are few aspects that needs attention like: 1. The experimental section needs to be more elaborate with proper references. Response: We thank the reviewer for this valuable comment. The Experimental Section has been expanded with detailed methodological descriptions and appropriate literature references. 2. In the references, the authors may provide full details of the authors instead of mentioning et al. Response: We thank the reviewer for this helpful suggestion. The reference list has been revised to include the full author names for all citations, replacing et al. where applicable, in accordance with the journal’s referencing guidelines 3. Under conclusions the details of in vitro studies should be clearly discussed and it should be brief. Response: We appreciate the reviewer’s helpful comment. The conclusion has been revised to briefly summarize the in vitro findings, including Topo II inhibition, cytotoxicity in cancer and normal cells, and flow cytometry results, providing a clearer and more focused summary. 4. The legends to the figures and tables should be more elaborate and self explanatory. Response: We sincerely thank the reviewer for this valuable suggestion. All figure and table legends have been thoroughly revised to include detailed experimental descriptions, key parameters, and concise interpretations 5. Under cell cycle and apoptosis assay, only Aotaphenazine treated MDA-MB-231 cells are shown. Why the control cell data is not shown in the figure. Response: We thank the reviewer for this important observation. In the revised version, the control (untreated) MDA-MB-231 cell data have now been included alongside the aotaphenazine-treated samples in both the cell cycle and apoptosis figures. This addition allows for a clear visual comparison between treated and control groups. Reviewer #2: Reviewer Comments Give the citation reference for the characterization of Aotaphenazine’s chemical structure, like 1HNMR/13CNMR & MS analysis. Response: We thank the reviewer for this valuable suggestion. The reference detailing the full spectroscopic characterization of aotaphenazine (¹H NMR, ¹³C NMR, and MS data) has now been cited in the revised manuscript as Reference [38], which reports its isolation and structural elucidation by Prof. Masami Ishibashi and co-workers. Notably, one of the co-authors of that study, Dr. Mohamed S. Abdelfattah, who was the first author of the isolation paper, is also a co-author of the present manuscript. Fig. 2: Include the two-dimensional pictures for each reference DOXO and Aotaphenazine using the DNA-Topo II complex, supported with each 3D graphic to better explain the many types of amino acid interactions. Response: We thank the reviewer for this excellent suggestion. The revised Figure 2 now includes both the (2D) and (3D) interaction diagrams for doxorubicin (DOXO) and aotaphenazine bound to the DNA–Topo II complex. The updated panels clearly illustrate hydrogen bonding, π–π stacking, and hydrophobic interactions with key amino acid residues and nucleobases, providing a more comprehensive visualization of the binding modes. Fig. 7: Images C1, C2, and C3 require further clarification. Response: We thank the reviewer for this helpful observation. The legend and figure description for Figure 7 have been revised to provide clearer explanations of images C1, C2, and C3 Attachments Attachment Submitted filename: Response.docx https://doi.org/10.1371/journal.pone.0338135.r002
19 Nov 2025 Decision Letter - Abdullahi Ibrahim Uba, Editor Aotaphenazine, a Rare Hydrophenazine, Targets Topoisomerase II with Anticancer Efficacy: In Silico to In Vitro Evidence PONE-D-25-39652R1 Dear Dr. Metwaly, 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. For questions related to billing, please contact billing support . 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, Abdullahi Ibrahim Uba Academic Editor PLOS ONE Reviewers' comments: Reviewer's Responses to Questions Comments to the Author Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions??> Reviewer #1: Yes Reviewer #2: Yes ****** 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 Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes Reviewer #2: Yes ****** Reviewer #1: Dear Author, I have gone through the manuscript. Now after incoroprating the modifications suggested by the reivewer's, the manuscript looks much better. It may be accepted for publication. Reviewer #2: (No Response) ******** what does this mean? https://doi.org/10.1371/journal.pone.0338135.r003
Formally Accepted
Acceptance Letter - Abdullahi Ibrahim Uba, Editor PONE-D-25-39652R1 PLOS ONE Dear Dr. Metwaly, 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 You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days 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. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. 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 Dr. Abdullahi Ibrahim Uba Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0338135.r004

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