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PONE-D-23-17723Evaluation of Anticancer Potential of Tetracene-5,12-Dione (A01) and Pyrimidine-2,4-Dione (A02) via Caspase 3 and Lactate Dehydrogenase cytotoxicity investigationsPLOS ONE

Dear Dr. Ejaz,

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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: Yes

Reviewer #2: Partly

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #3: No

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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

Reviewer #3: Yes

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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: I think authors should add the images of the in vitro and the other activities and the graphical abstract for the same. I recommend to add the more references in the manuscript. overall it was good manuscript.

Reviewer #2: The current study has aimed to use pyrimidine and pyrazole-based compounds against Caspase-3 , NF-Kappa-b , and p53 proteins using breast cancer and cervical cancer cell line experiments combined with molecular docking and dynamic simulation analysis. Two compounds were assessed in this study, compound A01, and A02 and they both sound to have good cytotoxic potential. However, there are some serious corrections demanded in the following manuscript:

1. The abstract dose not differentiate clearly between the results of other groups and the results of the current studies.

2. lines :53-55, the sentence in line 53 can have confusing interpretations.” the dysfunctional operation of deoxyribonucleic acid is primarily to blame for its development (DNA)” . It is not ideal to say that dysfunction of DNA is the primarily reason for development of cancer, but differential expression level of certain genes in cancer cells as a result of certain dysfunctional and altered cellular pathways, proteins and transcription factors, have been associated with the progression, differentiation and development of different kinds of cancer. Please provide a related reference for line 53-55.

3. Lines 63-64: “ Caspases 3, p53, and NF-kappa-B are potential therapeutic

targets for malignancy “. The authors should specify that these proteins have been suggested for what kind of cancer? Reference 8 is talking about bladder cancer; can the findings of a single study be implied on all types of cancers in general? Please provide more references that have reported the importance of these proteins in other types of cancer as well.

4. Lines 67-68: the authors have explained about the role of the three targets specified in lines 63-64 in cancer, while each protein has a specific role in apoptotic pathway, cell cycle, DNA repair and response to inflammation, and finally in lines 67-68 it is written that they are possible targets for design of anti-cancer agents. There is two problems with these sentences: first, if the current study aims to target DNA repair pathway, talking about the benefits of other pathways such as inflammation is not beneficial, the authors should try to design inhibitors against a specific set of proteins that are known to function in a certain pathway that is associated with progression of certain types of cancers. Second, the authors should try to define which pathway and target is suitable for design of anti-cancer agents in what type of cancer. Targeting a general pathway might not lead to optimum results in all types of cancer. Therefore, it is important to focus on a specific type or types of cancers and specific pathways for design of new anticancer agents.

5. Lines 86-89, based on reference 19, the three suggested targets could have potential to be investigated for further development of new anticancer inhibitors in cervical cancer and breast cancer. Therefore, in lines 87-89 , it should be specified what type of cancer could be potentially treated if the Caspase3, p53, and NF-B proteins get targeted for inhibition. Based on the cell lines selected for this study, it is suggested to add references that have analyzed the function and role of these proteins in breast cancer and cervical cancer.

6. It is not clear in the introduction either the authors are aiming to target PARP protein or Caspase3, p53, and NF-B factors. References 20 and 23 are claiming that pyrimidine and pyrazole-based compounds have potential to inhibit the EGFR structure. Therefore, the authors should clarify based on references if the pyrimidine and pyrazole-based compounds have the potential to disturb the function of these specified proteins if any previous study is available about it.

7. In method section part 3., the authors should try not to use first-person pronouns “we” frequently.

8. Lines 336-338, the docking results of compounds A01 and A02 with Caspase-3, NF-Kappa-B and p53 does not sound to be significant, docking scores in ranges of -5 kcal/mol to -8 kcal/mol are pretty insignificant and not enough to make a strong conclusion over the binding affinity and potential of these compounds with following proteins and can just be used to make a prediction about their possible binding modes with the structures of specified proteins.

9. Line 442-443, its not clear that the “significant binding affinity” of compound A01 with NF-Kappa-B and A02 with p53 is based on previous docking results or molecular dynamic simulation.

Also, it is better to say that compound A02 was shown to have a relative binding affinity with p53 protein. The sentence “compound A02 (A02) was discovered to significantly inhibit P53” is a bit more exaggeration compared to what the docking results demonstrated.

10. Is figure 14 showing the fluctuations of NF-Kappa-B protein in complex mode with compound A01?

11. Figure 15 should clarify the type of protein used for MD analysis.

12. Why did the authors not perform MD simulation on Caspase-3 with compounds A01 and A02?

13. As the docking results were not significant, the MD simulation is expected to be performed for both compound A01, A02 with all three respective proteins (Caspase-3, NF-KB, p53). There was not such significant gap in the docking results of the following compounds with the selected proteins. Therefore, it not convincing enough to claim compound A01 had higher affinity with NF-KB and A02 with p53 protein and so the authors decided to perform MD simulation only on them.

Reviewer #3: Authors of the manuscript entitled “Evaluation of Anticancer Potential of Tetracene-5,12-Dione (A01) and Pyrimidine-2,4Dione (A02) via Caspase 3 and Lactate Dehydrogenase cytotoxicity investigations” introduced both in vitro and in silico investigation regarding the anti-cancer activity of two compounds against different cancerous biotargets. Despite promising findings, the manuscript requires further improvements within several aspects as being highlighted within the provided suggestions and comments:

1. Authors focused on two compounds for their whole study without providing proper introduction (even no actual figure of both compounds except 3D representation within the docking studies). Moreover, the rational for adopting these two particular compounds for investigation is not clear. why among other reported compounds the authors chose these two particular compounds. a better explanation should be highlighted.

2. Within the in vitro cytotoxicity activity assay, the rational for adopting these three cancerous cell line should be highlighted. Whether the authors used these cells for extensive target expression or based on reported citation, either of which should be stated.

3. In line 204, authors should be consistent regarding the annotation of the compounds' cytotoxic activity, either use IC50 or GI50.

4. In sections 3.1.2 and 3.1.3, Levels of statistical significance should be presented within the context and at Figures 2 and 3. Similar to figure 3, the LDH analysis for the control samples should also be represented in Figure 2.

5. IC50 graphs for the investigated compounds should be presented within the supplementary data.

6. Regarding the DFT analysis, authors should provide potential energy surface (PES) scan to allocate the most energetically stabilized geometries. this can be achieved by varying a dihedral angle rotation from 0° to 360° in 10° stepwise rotation while allowing the rest of the molecule to undergo free iterations.

7. The PDB file of both NF-Kappa B and TP53 are without co-crystallized bound ligand. Authors should provide a rational for adopting a particular binding site where molecular modelling simulation were performed.

8. Superimposed binding modes of co-crystallized ligand and their respective redocked poses should be presented at least within the supplementary data.

9. Within the docking results section, author should provide brief description regarding the topology and protein ternary structure of the adopted biotargets prior introducing the docking results. Providing valuable information regarding binding site surface and reported key binding/catalytic residues would help readers to track docking findings.

10. Authors provided comparative data regarding ligand binding modes through both highlighted polar hydrogen bonds 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, since the strength of hydrogen bonding is based on both parameters in a way to ensure the adequacy of optimum hydrogen bonding. Additionally, the authors should mention the distances for the Hydrophobic interactions.

11. In addition to protein’s RMSD, the authors should provide RMSD trajectories for the sole simulated ligands furnishing information regarding ligand-pocket accommodation (ligand was maintained within the pocket or not) as well as relevant ligand's conformational changes as time evolves across the simulation time. Moreover, the sole ligand RMSDs would confirm protein convergence and system stability at the end of the simulation runs in case of ligands' RMSD never exceeds 2-fold the RMSDs of their bounded protein.

12. Overlay of the initial and final frames would provide insights regarding the orientation/conformation changes for both the protein and bounded ligands as well as the conserved and reformed ligand-amino acid bindings and close-range contacts.

13. Notably, Figure 15 showed the apo RMSDs being steadier and with minimal fluctuations as compared to those of the liganded/holo TP53 protein. The latter would question the stability of A02 at the target binding site. Analysis of the above described sole ligand RMSDs as well as overlaid trajectories would speculate such assumptions.

14. Through the RMSF analysis, authors should illustrate trajectories for apo protein as well. This approach would better highlight the impact of compound’s binding on target through pinpointing flexible and immobile patterns for the protein ternary structures and amino acids in reference to the unliganded form. Difference RMSF (ΔRMSF = RMSFApo-Holo) could also be adopted (please refer to doi: 10.1016/j.bmcl.2019.02.031 and doi: 10.1016/j.bmcl.2019.02.031).

15. Findings from the MM-GBSA free binding energy calculations as well as the constituting energy terms (VDW, electrostatic, solvation, SASA) should be represented in Figure or Table for better tracking of binding affinity results.

16. At least in silico ADME_TOX analysis for investigated compounds should be provided through exploring the drug-likeness profile and several pharmacokinetic parameters of these investigated compounds. Findings would be valuable for guiding future drug optimization & development strategies

17. Authors should elaborate more on the discussion section through presenting comparative findings from reported literature studies that investigated other close related compounds against target proteins.

18. Finally, within the discussion sections, authors should highlight the takeaway messages that would be adopted in future lead optimization and development base on the docking and molecular dynamics studies. Prospective/recommended structure modifications to improve the compound’s binding and interactions, as well as pharmacokinetics should be provided within the discussion and conclusion sections.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Evaluation of Anticancer Potential of Tetracene-5,12-Dione (A01) and Pyrimidine-2,4-Dione (A02) via Caspase 3 and Lactate Dehydrogenase cytotoxicity investigations

PONE-D-23-17723R1

Dear Dr. Ejaz,

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,

Ahmed A. Al-Karmalawy, Ph.D.

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 #2: All comments have been addressed

Reviewer #3: (No Response)

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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: Yes

Reviewer #3: Yes

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

Reviewer #2: Yes

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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

Reviewer #3: Yes

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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: All comments and concerns mentioned for the authors have been addressed accordingly very well as suggested.

Reviewer #3: Authors adequately provided responses and committed manuscript modifications as per recommendations and suggestions.

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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

Reviewer #3: Yes

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