Development of Acridone Derivatives: Targeting c-MYC Transcription in Triple-Negative Breast Cancer with Inhibitory Potential

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

First of all, I would like to thank you for inviting me to review the manuscript entitled: 'Development and antioxidant activity of acridone  derivatives: Targeting c-MYC transcription in triple negative breast cancer with inhibitory potential’. The aim of the study was to employs fragment-based growth techniques to design and synthesize novel acridone derivatives, thereby enhancing the structural diversity of molecules acting as G4 stabilizers. The general conclusions demonstrate that  acridone derivatives could stabilize c-MYC G4 structure. Among which N8 shows promising effects and deserves further investigation. Thus I recommend publication after some major issues have been addressed:

Major:
1.  I believed that the introduction is too long. Part 1.1. is good, however 1.2.-1.4 should be shortened. Some of the information should moved to discussion.
2. The quality of Figure 3 should be improved.
3. Also, in discussion section please add paragraph related to clinical and practical aspects of the study. How we can applicate your results into practice?, why your work is valuable in the field?
4. Please, provide limitations and strengths of the study, especially in context of study design. I suggest that this information be included in the last paragraph of the discussion as a continuation of this part.

Minor points:
1.  Minor modification of the grammar and punctuation is required.


General: interesting, well-conducted work.

Comments on the Quality of English Language

Minor modification of the grammar and punctuation is required.

Author Response

Dear Reviewer,

I would like to express my sincere gratitude for taking the time to review our manuscript. Your valuable insights and constructive feedback are highly appreciated.

We sincerely appreciate your recognition of our work, and your professional insights have presented valuable challenges to enhance the quality of our research. Under your guidance, we are diligently addressing both major and minor issues you highlighted to ensure that our paper achieves the highest standards in both structure and language. Below is our point-by-point response:

Major:

Question1.  I believed that the introduction is too long. Part 1.1. is good, however 1.2.-1.4 should be shortened. Some of the information should moved to discussion.

Answer1: Thank you for your suggestions. We have simplified the content of sections 1.2 -1.4, moved some relevant content to the discussion section.

Question2. The quality of Figure 3 should be improved.

Answer 2: Thank you for your advice. We have maximized the resolution of Figure 3 to enhance the quality as much as possible.

Question 3. Also, in discussion section please add paragraph related to clinical and practical aspects of the study. How we can applicate your results into practice?, why your work is valuable in the field?

Answer 3: Thank you for your suggestions. We have incorporated relevant content into the penultimate paragraph of the discussion section, focusing on the relevance of the study results in clinical and practical applications. We discuss how the findings of this study can be applied in real-world scenarios, emphasizing the practical value of the research and its significance in the field of TNBC treatment. We outline the potential applications of our work in actual clinical settings and explain the reasons behind the value of this study in the respective domain.

Question 4. Please, provide limitations and strengths of the study, especially in context of study design. I suggest that this information be included in the last paragraph of the discussion as a continuation of this part.

Answer 4: Thank you very much for your review and suggestions. We have incorporated discussions on the limitations and strengths of the study, particularly focusing on the aspects of the research design, in the last paragraph of the discussion. Additionally, we have provided insights into future research plans.  This section aims to provide readers with a more comprehensive understanding while highlighting the credibility and limitations of our research.

Minor points:

Question 1.  Minor modification of the grammar and punctuation is required.

Answer 1: Thank you very much for your guidance. We have enlisted a native English speaker to review our manuscript. They have identified and corrected some grammar and punctuation errors, as well as improved the expression of certain content. We believe these revisions will further enhance the quality of the manuscript.

Once again, thank you for your valuable time and professional guidance.

Sincerely,

Fanhao Meng

Reviewer 2 Report

Comments and Suggestions for Authors

In the current manuscript, Liang et al. aimed to discover novel small compounds that potentially inhibit c-MYC transcript via stabilization of G-quadruplex (G4) structure, resulting in anti-tumor effects for breast cancer cells. Given previous reports and their virtual screening showing acridone could work as a ligand by docking into the G4 structure of c-MYC, they designed and synthesized 29 acridone derivatives, investigating their therapeutical effects using human breast cancer cell line, MDA-MB-231 cells. The molecular characterizations of screened compounds were further investigated by QSAR analysis and molecular docking. The methods, outcomes, and corresponding discussion of computer-aided drug designing and screening are well written in the manuscript. The primary weakness of the current study is that it is not clear whether inhibition of c-MYC underly examined anti-tumor effects (i.e., ROS production and cytotoxic effects) in MDA-MB-231 cells treated with studied compounds. In addition, the listed items need to be addressed.

 

Major point:

Given the results of molecular docking-based selectivity assessment (Figure 4), the authors mention the possibility that L20, N1, N3, N6, and N7 may be targeting alternative pathways beyond MYC, inhibiting tumor cell proliferation. Indeed, it seems that G4 structure targeting compounds could show anti-tumor effects via multi-targeting properties due to a wide range of genes harboring G4 structure. However, since the authors aim to investigate DNA-G4 structure-mediated cMYC inhibitors in the current study, it is important to assess the direct effects of cMYC suppression in the cells treated with synthesized acridone derivatives. The authors need to examine gene and protein levels of cMYC expression in the cells treated by studied compounds and compare them to an established G4-interactive compound targeting the c-MYC (i.e. CX-3543). Further, cMYC plays its oncogenic function through the induction of its target genes. Thus, analyzing the expression of cMYC-target genes in the in vitro screening setting could be an alternative approach to clarify targeting specificity and its associated biological function.

 

Minor points:

-      Please describe what kind of vehicle was used for studied compounds, when they were administrated to the cultured cells.

-      It is highly recommended to include cMYC in the heatmap shown in Figure 4 as a positive control of selectivity assessment of DNA-G4 structure. In addition, please add a simple explanation for the values of Flow Z sore in the figure legend.

-      Table 1 - Please explain what the numbers indicated next to EC50 and IC50 represent.

-      The two-tailed Student’s t-test is not accurate for the data sets shown in Figure 3. ANOVA followed by Dunnett's test should be the better statistical approach.

 

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Dear Reviewer,

We sincerely appreciate your thoughtful and constructive feedback on our manuscript, titled "Discovery of Acridone Derivatives as Potential c-MYC Inhibitors in Breast Cancer Cells through G-Quadruplex Stabilization." We are grateful for the time and effort you dedicated to reviewing our work. Below is our detailed response to your comments:

Major point:

Given the results of molecular docking-based selectivity assessment (Figure 4), the authors mention the possibility that L20, N1, N3, N6, and N7 may be targeting alternative pathways beyond MYC, inhibiting tumor cell proliferation. Indeed, it seems that G4 structure targeting compounds could show anti-tumor effects via multi-targeting properties due to a wide range of genes harboring G4 structure. However, since the authors aim to investigate DNA-G4 structure-mediated cMYC inhibitors in the current study, it is important to assess the direct effects of cMYC suppression in the cells treated with synthesized acridone derivatives. The authors need to examine gene and protein levels of cMYC expression in the cells treated by studied compounds and compare them to an established G4-interactive compound targeting the c-MYC (i.e. CX-3543). Further, cMYC plays its oncogenic function through the induction of its target genes. Thus, analyzing the expression of cMYC-target genes in the in vitro screening setting could be an alternative approach to clarify targeting specificity and its associated biological function.

Answer：

Minor points:

Question1：Please describe what kind of vehicle was used for studied compounds, when they were administrated to the cultured cells.

Answer1：Thank you for your suggestion. The compounds in our study were initially dissolved in PBS to 10 m, and then further diluted to the respective concentrations with complete culture medium when added to the cultured cells. We have also added this detail to the "Methods" section of the manuscript.

Question2：It is highly recommended to include cMYC in the heatmap shown in Figure 4 as a positive control of selectivity assessment of DNA-G4 structure. In addition, please add a simple explanation for the values of Flow Z sore in the figure legend.

Answer2：Thank you for your suggestion. In fact, the heat map in Figure 4 already contains information about c-MYC as a positive control. The reason is that we used the result of selectivity score instead of docking result to draw the heat map, and the calculation formula for selectivity is as follows:

Selective score(i)=10^(DockingScore|i-cMYC|)

In the formula, i is the target with G4 structure other than c-MYC, and this formula is based on the calculation method of docking scoring of MOE software. We have added this part to the experimental method, as section 2.9. Compare to the docking scoring, the selective score can more intuitively display the selectivity of small molecules towards different targets in the heat map results. The docking scores of all compounds towards the DNA-G4 target have been added to the supplementary file as Table S3, which is the raw data for calculating the selectivity score.

In addition, the explanation for Z-score has been added to the figure legend.（page13, line 405）

Question3: Table 1 - Please explain what the numbers indicated next to EC₅₀ and IC₅₀ represent.

Answer3: Thank you for your advice. These numbers represent the 95% confidence interval for EC₅₀ or IC₅₀, rather than single values. We have further clarified this detail in the manuscript. The confidence interval serves as a statistical measure of uncertainty in the estimation, allowing readers to gain a more comprehensive understanding of the reliability of the determined values.

Question4: The two-tailed Student’s t-test is not accurate for the data sets shown in Figure 3. ANOVA followed by Dunnett's test should be the better statistical approach.

Answer 4: Thank you for your suggestions and guidance. We have revised the statistical analysis method to ANOVA followed by Dunnett's test to more accurately analyze the datasets. Some of the test results, differing from the previous ones, have been updated in Figure 3.

Question 5: Minor editing of English language required

Answer 5: Thank you for your suggestion. We have enlisted a native English speaker to review our manuscript. They have identified and corrected some grammar and punctuation errors, as well as improved the expression of certain content. We believe these revisions will further enhance the quality of the manuscript.

Once again, thank you for your valuable time and professional guidance.

Sincerely,

Fanhao Meng

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I suggest to publish the manuscript in current form.

Author Response

Dear Reviewer,

I sincerely appreciate your thorough review of my manuscript and the valuable feedback provided. I am delighted to learn that you suggest publishing the manuscript in its current form. Your support and professional insights mean a lot to me. Once again, thank you for your patience and invaluable advice.

Best regards,

Fanhao, Meng

Reviewer 2 Report

Comments and Suggestions for Authors

In the revised manuscript, the authors added new data, where they showed gene and protein levels of cMYC expression in the cells treated by studied compounds as well as CX-3543 (Figures 3h, i, and j). These results support the authors’ points, clarifying that the newly synthesized compounds most likely work as DNA-G4 structure-mediated cMYC inhibitors, resulting in oxidative stress-mediated cell toxicity.

 

A small niggling point is that the authors mention ‘the compounds were initially dissolved in PBS to 10 m’ in the comments, whereas they describe ‘the compounds were initially dissolved by PFS to 10 mM’ in the revised manuscript. Please make sure they correctly describe the material of the vehicle and the initial concentration of compounds.

 

Overall, I recommend the revised manuscript for publication.

 

 

Comments on the Quality of English Language

 Minor editing of English language required

Author Response

Dear Reviewer,

    Thank you for thoroughly reviewing our manuscript and providing valuable feedback. We appreciate your attention to detail.

    We have taken note of the inconsistency you pointed out regarding the description of the vehicle and the initial concentration of compounds. We sincerely apologize for any confusion caused by this oversight. The discrepancy has been rectified, and the manuscript now accurately reflects the use of PBS to dissolve the compounds to 10 mM.

    In terms of English language issues, we conducted a comprehensive review and addressed some grammar and expression concerns.

    We are grateful for your overall positive recommendation for the manuscript. If you have any further specific suggestions or if there are points that require clarification, please feel free to let us know.

    Once again, thank you for your time and constructive feedback.

Best regards,

Fanhao, Meng