Probing Redox Responses and DNA Interactions in Drug Discovery

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This work is devoted to probing redox responses and DNA interactions in drug discovery. Here the electrochemical interactions of two thiazolopyrimidine-derived molecules, TP-NB and TP-PC, with DNA as potential drug candidate molecules were electrochemically investigated for the first time. Key parameters, including pH, scan rate, immobilization time, and drug concentration, were optimized to achieve highly sensitive detection of these molecules under ideal conditions. It was shown that both synthesized molecules TP-NB and TP-PC can be used as a potential anticancer agent with further studies. I think that this article may be published after minor revision.

Notes:

1. From what point of view, the fragment of piperidine-4-carboxamide was introduced to the structure of TP-NB? What were the authors' goals for this fragment? Short comment about it should be added in the Introduction.
2. In the Introduction authors reported that in intercalation, molecules insert between DNA base pairs without forming covalent bonds or disrupting hydrogen bonds, causing stabilization, elongation, stiffening, and unwinding of the double helix. If disrupting hydrogen bonds is not carried out so how unwinding of the double helix is happen? This is not entirely clear. Could the authors describe in more detail what happens when a molecule intercalates between DNA base pairs?
3. The meaning of ACB abbreviation should be written in the text at the first mentioning.
4. After section 3. Materials and Methods the numeration should be 3.1, 3.2, 3.3 but not 2.1, 2.2, 2.3. It should be checked and corrected.
5. Conclusions should be shortened and focused on the main findings of this work.

 

Author Response

This work is devoted to probing redox responses and DNA interactions in drug discovery. Here the electrochemical interactions of two thiazolopyrimidine-derived molecules, TP-NB and TP-PC, with DNA as potential drug candidate molecules were electrochemically investigated for the first time. Key parameters, including pH, scan rate, immobilization time, and drug concentration, were optimized to achieve highly sensitive detection of these molecules under ideal conditions. It was shown that both synthesized molecules TP-NB and TP-PC can be used as a potential anticancer agent with further studies. I think that this article may be published after minor revision.

1. From what point of view, the fragment of piperidine-4-carboxamide was introduced to the structure of TP-NB? What were the authors' goals for this fragment? Short comment about it should be added in the Introduction.

Answer 1: We thank the reviewer for this insightful comment. The necessary part is added to Introduction section.

‘The piperidine-4-carboxamide substituent, known for its activity against various biological targets, was introduced into the TP-NB structure to improve binding affinity and pharmacokinetic properties, such as solubility and metabolic stability. Its functional groups, capable of acting as hydrogen bond donors and acceptors, were expected to enhance interactions with key residues at the target site (doi.org/10.3390/ijms24108633).’

 

1. In the Introduction authors reported that in intercalation, molecules insert between DNA base pairs without forming covalent bonds or disrupting hydrogen bonds, causing stabilization, elongation, stiffening, and unwinding of the double helix. If disrupting hydrogen bonds is not carried out so how unwinding of the double helix is happen? This is not entirely clear. Could the authors describe in more detail what happens when a molecule intercalates between DNA base pairs?

Answer 2: We thank the reviewer for this insightful comment. Indeed, intercalating agents insert themselves between adjacent base pairs of the DNA double helix, which leads to a local unwinding and elongation of the helix. Although hydrogen bonds between complementary bases remain largely intact during intercalation, the stacking interactions between base pairs are perturbed. The insertion of a planar molecule between base pairs increases the distance between them, leading to helical unwinding due to a change in the torsional strain and overall geometry of the DNA. These changes result in a more rigid and elongated structure.

We have now revised the corresponding sentence in the Introduction to clarify that the unwinding results primarily from the disruption of base stacking rather than hydrogen bonding. Intercalating agents insert between adjacent DNA base pairs, causing local unwinding and elongation of the helix. While hydrogen bonds remain mostly intact, base stacking interactions are disrupted. The insertion increases the distance between base pairs, alters torsional strain, and results in a more rigid, extended DNA structure.

1. The meaning of ACB abbreviation should be written in the text at the first mentioning.

Answer 3: We appreciate the reviewer for his/her suggestions, which significantly improved our manuscript. In the modified version, we implemented necessary changes based on the reviewer’s questions throughout the manuscript. In the new version of manuscript, we added the meaning of ACB abbreviation in the text at the first mentioning.

1. After section 3. Materials and Methods the numeration should be 3.1, 3.2, 3.3 but not 2.1, 2.2, 2.3. It should be checked and corrected.

Answer 4: We appreciate your suggestion which will improve our manuscript. In the modified version, we implemented necessary changes based on the reviewer’s suggestion throughout the manuscript.

1. Conclusions should be shortened and focused on the main findings of this work.

Answer 5: We appreciate your suggestion which will improve our manuscript. In the revised version of the manuscript, the conclusion section was shortened and focused on the main findings. 

 

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript reports the electrochemical data from the interaction of pyrimidine-based scaffolds with double-stranded DNA using immobilisation of the scaffolds onto the electrode surface. Using voltammetry to explore such DNA interactions is clearly relevant to the scope of the journal and the data presented offers some insight into the nature and extent of these interactions and the impacts of pH. There are however a number of points that the authors need to address relating to the presentation and interpretation of the data and these are detailed below. All points require changes to the manuscript.

1. Page 1 line 32 “As both molecules . . . on DNA . . .” ‘High effectiveness’ is too vague a term. Be more specific about the results and their implications for drug development.
2. Page 4 lines 129-138 “Fires, the . . . in Fig 2.” Most of this content is experimental description and should be part of the Materials and Methods section.
3. Throughout the manuscript the authors use TP-NB and TP-NBe interchangeably. Please choose one abbreviation and use it consistently throughout.
4. Page 5 line 160 “. . . ACB . . .” All abbreviations should be defined at first use in the text.
5. Figure 3 legend. For the figure to be comprehensible independently from the main text, the identity of the supporting electrolytes should be mentioned here.
6. Page 5 line 181 “. . . around -0.35 V increased . . .” Why has the voltage for the reduction peak for TP-NB changed from -0.5 V in Figure 2 to -0.35 V in Figure 3? Some more explanation and interpretation is needed here.
7. Page 6 lines 200-201 “The slope of . . . 59 mV/pH.” This statement needs a supporting reference.
8. For Figures 3-6 provide error bars for the linear plots and give information on the number of replicates in the figure legends.
9. Page 9 line 276 “ . . . activated electrodes . . .” Provide succinct details on the process by which the electrodes are activated.
10. Figure 6B. A histogram is not the appropriate type of graph to use here as the x-axis concentrations are continuous rather than being binned categories. As such, a line chart is more appropriate.
11. Page 11 line 342 “. . . were calculated as . . . respectively.” The problem with this assessment is that both of these values are less than the lowest standard in your standard curve plot (Figure 6C). As such, you do not have knowledge of the shape and definition of the plot in this concentration region. Add lower concentration points to the standard curve to encompass these values.
12. Page 12 lines 394-396 “. . . shifted towards. . . intercalation [44].” This is not a very large change in voltage, yet it is critical to your interpretation of intercalation taking place. What is the degree of resolution of voltage available and what are the experimental errors? In other words, can you demonstrate that this difference in voltage is statistically distinguishable?
13. Page 12 line 402 This should read “Percentage of the guanine peak current change”
14. Page 13 lines 409-411 “Generally, a . . . classified as toxic.” This classification should be backed by a supporting literature reference.
15. Page 13 lines 442-444 “ . . . towards a more . . . unlike TP-NB.” See comment 12 above for this comparison also.
16. Page 14 lines 457-462 “As seen in . . . its adverse effects.” So why did you not try lower concentrations to determine over what concentration range you would get a proportional decrease relationship?

Comments on the Quality of English Language

The manuscript should be subject to an editorial check to correct minor errors.

Author Response

This manuscript reports the electrochemical data from the interaction of pyrimidine-based scaffolds with double-stranded DNA using immobilisation of the scaffolds onto the electrode surface. Using voltammetry to explore such DNA interactions is clearly relevant to the scope of the journal and the data presented offers some insight into the nature and extent of these interactions and the impacts of pH. There are however a number of points that the authors need to address relating to the presentation and interpretation of the data and these are detailed below. All points require changes to the manuscript.

1. Page 1 line 32 “As both molecules . . . on DNA . . .” ‘High effectiveness’ is too vague a term. Be more specific about the results and their implications for drug development.

Answer 1: We appreciate the reviewer for his/her suggestions, which significantly improved our manuscript. In the modified version of the manuscript, we have indicated in the relevant section which properties of this drug candidate molecule may have potential uses based on analysis with the electrochemical method we developed and what advantages our method provides in these analyzes.

1. Page 4 lines 129-138 “Fires, the . . . in Fig 2.” Most of this content is experimental description and should be part of the Materials and Methods section.

Answer 2: We appreciate your suggestion which will improve our manuscript. In the revised version of the manuscript, the section highlighted by the reviewer has been relocated to the Materials and Methods section. Accordingly, it has been removed from the Results and Discussion section and replaced with a more concise summary.

1. Throughout the manuscript the authors use TP-NB and TP-NBe interchangeably. Please choose one abbreviation and use it consistently throughout.

Answer 3: We appreciate the reviewer for his/her suggestions, which significantly improved our manuscript. In the revised version of the manuscript, only the abbreviation TP-NB has been consistently employed.

 

1. Page 5 line 160 “. . . ACB . . .” All abbreviations should be defined at first use in the text.

Answer 4: We appreciate the reviewer for his/her suggestions, which significantly improved our manuscript. In the new version of manuscript, we added the meaning of ACB abbreviation in the text at the first mentioning.

1. Figure 3 legend. For the figure to be comprehensible independently from the main text, the identity of the supporting electrolytes should be mentioned here.

Answer 5: We appreciate your suggestion which will improve our manuscript. In the revised version of the manuscript, as indicated by the reviewer, the identity of the supporting electrolytes for Figure 3 has been specified in the figure legend.

1. Page 5 line 181 “. . . around -0.35 V increased . . .” Why has the voltage for the reduction peak for TP-NB changed from -0.5 V in Figure 2 to -0.35 V in Figure 3? Some more explanation and interpretation is needed here.

Answer 6: Thank you for your feedback. We would like to clarify our situation. To clarify the situation, in this section, the initial reduction signals for TP-NB and TP-NC in Figure 2 were recorded directly within their respective solutions, while in Figure 3 and subsequent measurements, both candidate molecules were passively adsorbed onto the electrode surface before measurements were taken within ACB. The observed discrepancy in the peak potentials of the substance can be attributed to the difference between the direct measurements and those obtained from the electrode surface. A comparison of the CV measurements from the direct measurements in Figure 4 reveals that the peak positions correspond to the potential values observed in Figure 2. In contrast, the measurements taken from the electrode surface in Figure 5 show the peaks at the potential values observed in Figure 3. This supports our hypothesis that a slight shift in the peak potentials occurs between direct measurements and those obtained from the electrode surface. In the revised version of the manuscript, this explanation has also been added to the relevant section.

1. Page 6 lines 200-201 “The slope of . . . 59 mV/pH.” This statement needs a supporting reference.

Answer 7: Thank you for your feedback. In the revised version of the manuscript, a supporting reference has been added to the relevant section.

1. For Figures 3-6 provide error bars for the linear plots and give information on the number of replicates in the figure legends.

Answer 8: We appreciate the reviewer for his/her suggestions, which significantly improved our manuscript. In the revised version of the manuscript, the changes requested by the reviewer for the figures have been made.

1. Page 9 line 276 “ . . . activated electrodes . . .” Provide succinct details on the process by which the electrodes are activated.

Answer 9: We appreciate your suggestion which will improve our manuscript. In the modified version, we implemented necessary changes based on the reviewer’s suggestion throughout the manuscript.

1. Figure 6B. A histogram is not the appropriate type of graph to use here as the x-axis concentrations are continuous rather than being binned categories. As such, a line chart is more appropriate.

Answer 10: Thank you for your feedback. We would like to clarify our presentation. In this context, the histogram graph was utilized to track the changes in the guanine oxidation current corresponding to varying dsDNA concentrations, with the objective of identifying the concentration at which saturation occurs on the electrode surface. The use of histogram graphs to illustrate concentration-dependent variations in oxidation currents and the saturation point is also commonly observed in the literature (https://doi.org/10.1016/j.talanta.2020.121647 , https://doi.org/10.1016/j.jelechem.2020.114011). As indicated by the reviewer, a line chart could certainly be used; however, a histogram was selected and employed in our study, as it provides a clearer representation for determining the saturation point.

1. Page 11 line 342 “. . . were calculated as . . . respectively.” The problem with this assessment is that both of these values are less than the lowest standard in your standard curve plot (Figure 6C). As such, you do not have knowledge of the shape and definition of the plot in this concentration region. Add lower concentration points to the standard curve to encompass these values.

Answer 11: Thank you for your feedback. We would like to clarify our statement. In this section, the calibration curve was constructed using the range in which a linear relationship was observed between dsDNA concentration and the guanine oxidation current. Based on the equation derived from this calibration curve, the LOD value was calculated using the formula 3σ/m and the LOQ using the formula 10σ/m, where σ corresponds to the standard deviation of the intercept and m represents the slope of the calibration curve. Therefore, it is expected that these values fall below the lowest concentration point on the standard curve. A review of various studies in the literature reveals that, similarly to our findings, LOD and LOQ values have been calculated at concentrations lower than those included in the standard curve. For example, in a study conducted for the electrochemical detection of the drug balofloxacin, a calibration curve was established using concentrations ranging from 150 to 1000 nM, yet the LOD and LOQ values were calculated as 40.5 nM and 135 nM, respectively, using the same formulas (https://doi.org/10.1016/j.jpha.2020.03.013). In another study focused on the electrochemical detection of ciprofloxacin, a calibration curve was generated over the concentration range of 0.3 to 7 µM, while the LOD and LOQ were determined as 0.03 µM and 0.1 µM, respectively (https://doi.org/10.3390/ma15227872). These studies support the validity of obtaining LOD and LOQ values below the concentration range used for the calibration curve, as observed in our study.

1. Page 12 lines 394-396 “. . . shifted towards. . . intercalation [44].” This is not a very large change in voltage, yet it is critical to your interpretation of intercalation taking place. What is the degree of resolution of voltage available and what are the experimental errors? In other words, can you demonstrate that this difference in voltage is statistically distinguishable?

Answer 12: We appreciate the reviewer for his/her suggestions, which significantly improved our manuscript. We would like to clarify our statement. In the section where this interpretation was made, we also referred to a study investigating the interaction between palbociclib and dsDNA. In that study, although the difference between the guanine oxidation peak potentials of dsDNA before and after interaction was not very large, a shift toward more positive values—similar to what we observed—was reported following the interaction. It was suggested that this shift could be attributed to a potential intercalation-mediated interaction (https://doi.org/10.1016/j.bioelechem.2022.108264). Furthermore, in another study, the changes in the guanine oxidation peak potentials before and after interaction with various intercalating agents—namely epirubicin, idarubicin, and doxorubicin—were systematically evaluated. In particular, interactions with idarubicin and doxorubicin resulted in a slight shift of the guanine oxidation peak potential toward more positive values, closely resembling the trend observed in our study. These shifts were interpreted as indicative of potential intercalation-mediated interactions (https://doi.org/10.3390/mi12111337). Moreover, in our study, all measurements were conducted in five replicates, and in each case, the guanine peak potential consistently shifted to a similarly higher level following the interaction. This uniform and reproducible shift further supports the hypothesis of intercalation.

1. Page 12 line 402 This should read “Percentage of the guanine peak current change”

Answer 13: We appreciate your suggestion which will improve our manuscript. In the modified version, we implemented necessary changes based on the reviewer’s suggestion throughout the manuscript.

1. Page 13 lines 409-411 “Generally, a . . . classified as toxic.” This classification should be backed by a supporting literature reference.

Answer 14: Thank you for your feedback. In the revised version of the manuscript, a supporting reference has been added to the relevant section.

1. Page 13 lines 442-444 “ . . . towards a more . . . unlike TP-NB.” See comment 12 above for this comparison also.

Answer 15: We appreciate the reviewer for his/her suggestions, which significantly improved our manuscript. We would like to clarify our statement. In the section where this interpretation was made, we also referred to a study investigating the interaction between ibrutinib and dsDNA. In that study, although the difference between the guanine oxidation peak potentials of dsDNA before and after interaction was not very large, a slightly shift toward more negative values—similar to what we observed—was reported following the electrostatic interaction (https://doi.org/10.1016/j.microc.2022.107622). Another study investigates the interaction of nitrofurazone with DNA. In this study, it was reported that as a result of the interaction with DNA, the peak potential of nitrofurazone shifted from -0.616 V to -0.652 V. This shift is approximately of the same magnitude as the negative potential shift observed in our study, and it was suggested that this change indicates an electrostatic interaction (https://doi.org/10.1016/j.bios.2012.05.034). These similar examples in the literature provide evidence that an electrostatic interaction also occurs in our study. In addition, in our study, all measurements were conducted in five replicates, and in each case, the guanine peak potential consistently shifted to a similarly lower level. This uniform and reproducible shift further supports the hypothesis of electrostatic interaction.

1. Page 14 lines 457-462 “As seen in . . . its adverse effects.” So why did you not try lower concentrations to determine over what concentration range you would get a proportional decrease relationship?

Answer 16: The reviewer is correct. There is a possibility of obtaining a dose-dependent response at lower concentrations. However, in this section, concentration of 100 µg/mL, which are known to achieve surface saturation for TP-NB, and nearby values were used. For TP-PC, experiments were conducted at 250 µg/mL, concentration known to achieve surface saturation, as well as concentrations within ±100 µg/mL. By doing so, when a significant portion of the sensor surface is covered with the drug, the level of interaction with DNA and the binding kinetics were investigated.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The article is devoted to the Probing Redox Responses and DNA Interactions in Drug Discovery. It is written in fine language and sounds scientifically. Not very well structured, unfortunately. This article will be of interest to electrochemists and researchers in the field.

 

Reviewer comments:

 

Authors should put their affiliations in order. Authors should carefully restructure their article according to the comments provided below.

About blank experiments and their conditions. Authors should inform readers about what they use as a blank solution. Next, according to high current values in blank curves - was the blank subtracted or not during the present work? This and the next question are crucial to the entire work.

Why do the authors talk about potential anticancer activity? After all, these drug candidates may not penetrate the nucleus of eukaryotic cells at all. The statement is very bold and should be confirmed at least by in vitro experiments in living cells. Is this single electrochemical profile for such a statement enough? Can the authors provide analogies in which real anticancer drugs of similar structure behave in a similar electrochemical manner? Electrochemical experiments should be started when it is known about the penetration of the drug candidate into the nucleus of a living cell. Or at least this should be mentioned in the article.

At what potential value were the current measurements made for calibration graph in the concentration/calibration tests? Despite the large fluctuations in the peak maxima positions, the authors obtained good values of linearity and R². It looks dubious. Explanation is needed. Why were calibrations, LOQ and LOD done at all? And this constitutes a significant part of the study. The conclusion says nothing about this.

Paragraph 2.2 must be moved close to Figure 1, the last one should be renamed to Scheme 1, to be precise.

About ACB. As the reviewer realized, it is an acetate buffer. In the “Materials and methods” section provided three different by pH ACB. Authors should provide precise info in the whole document about what types of ACB were used in all arising cases.

 

Line 26, 27; 40; 132; 134; 304; 358 “TP-NB and TP-PC; cAMP, cGMP; ACB; PGEs; dsDNA; GC-rich” First appearance of the abbreviation/term must be followed by decipher. An unprepared reader will feel uncomfortable following the article. To partially prevent this inconvenience the “Materials and methods” paragraph should be transferred before the “Results and discussion” one.

Line 401 “S% = (Sa/Sb) x 100 (Equation 7)” Reformat all equation’s presentations as follows, please:

S = (S_a/S_b) × 100%                                                                                                                  (7)

where S - …

Line 55 “il-lustrated in Figure 1.” Typo. Please make uniform references to figures.

Line 56-58 Repeat. Compare the lines 51-53.

Line 153 “For this reason; reduction peak” “;” - typo?

Line 159 and below “TP-NBe” typo?

Line 192 “pH: 4.8” typo?

Line 199 “equation of Epc = -0.0501 pH - 0.1217 R² = 0,995” At this stage of the presentation, it is not entirely clear how the different pHs were created.

Figure 4 and Table 1. When using logarithms, the following forms are common and correct: decimal logarithms lg, natural logarithms ln, and logarithms to the base a - log_aN. What exactly did the authors use? What “ν” represents? Please do indicate that square roots are used in legends.

Line 292 “At higher concentrations (250 and 500 µg/mL), the reduction peak currents were lower” Please indicate this before Figure 5.

Figure 6, caption. There is no dsDNA solution under investigation directly, in experiments utilized pre-adsorbed on electrode dsDNA from mentioned solution, see text above. The same in line 414, please pay attention.

Line 450 “according to the Equation 9” The Equation 9 not found in the text as well as the Equation 8.

Line 508 “TP-PC synthesized for the first time with various substitutions to the main thiazolopyrimidine skeleton” The statement is not true. Only one compound has been synthesized.

 

Conclusion:

Accept after major revision.

 

Author Response

1. Authors should put their affiliations in order. Authors should carefully restructure their article according to the comments provided below.

Answer 1: We appreciate the reviewer for his/her suggestions, which significantly improved our manuscript. In the modified version, we implemented necessary changes based on the reviewer’s questions throughout the manuscript.

2. About blank experiments and their conditions. Authors should inform readers about what they use as a blank solution. Next, according to high current values in blank curves - was the blank subtracted or not during the present work? This and the next question are crucial to the entire work.

Answer 2: We appreciate the reviewer for his/her suggestions, which significantly improved our manuscript. The peak potentials obtained from the blank solutions were found to be very close to those recorded for TP-NB and TP-PC. However, the peak currents associated with TP-NB and TP-PC were significantly higher. Consequently, except for the voltammograms, all subsequent graphical representations in the study were constructed after subtracting the signals originating from the blank solutions. In the revised version of the manuscript, information regarding the composition and measurements of the blank solutions, as well as the subtraction of blank signals during the preparation of TP-NB and TP-PC graphs, has been incorporated into the relevant sections.

3. Why do the authors talk about potential anticancer activity? After all, these drug candidates may not penetrate the nucleus of eukaryotic cells at all. The statement is very bold and should be confirmed at least by in vitroexperiments in living cells. Is this single electrochemical profile for such a statement enough? Can the authors provide analogies in which real anticancer drugs of similar structure behave in a similar electrochemical manner? Electrochemical experiments should be started when it is known about the penetration of the drug candidate into the nucleus of a living cell. Or at least this should be mentioned in the article.

Answer 3: We thank the reviewer for this important comment. We agree that any claim regarding anticancer potential must be made with caution, particularly in the absence of in vitro or in vivo biological data. Our intention was not to make a definitive claim about anticancer activity, but rather to highlight the structural and electrochemical features of the investigated compounds that are commonly observed in molecules with known anticancer properties. We acknowledge that electrochemical behavior alone is not sufficient to establish biological activity.

To address this concern, we have revised the relevant section of the manuscript to clearly state that our hypothesis regarding potential anticancer activity is preliminary, based solely on structural analogy and electrochemical profile. We now explicitly note that further studies, including cellular uptake and nuclear localization assays, as well as in vitro cytotoxicity experiments, are necessary to confirm any such activity. Additionally, we have added a brief discussion referencing similar compounds reported in the literature (https://doi.org/10.1016/j.bioorg.2018.08.013 , doi.org/10.1016/j.ejmech.2017.04.056).

4. At what potential value were the current measurements made for calibration graph in the concentration/calibration tests? Despite the large fluctuations in the peak maxima positions, the authors obtained good values of linearity and R². It looks dubious. Explanation is needed. Why were calibrations, LOQ and LOD done at all? And this constitutes a significant part of the study. The conclusion says nothing about this.

Answer 4: Thank you for your feedback. We would like to clarify our statement. In the construction of the calibration graph, a potential of approximately -0.35 V was applied for TP-NB, and a potential of approximately -0.38 V was applied for TP-PC. But, at high concentrations, the components of the solution may interact more with the electrode surface, which could alter the reaction kinetics and lead to a shift in the peak potential. While a slight shift in the peak potentials was observed with increasing concentrations, the calibration graph was constructed based on the peak current intensities corresponding to the candidate drug concentrations. As a result, smooth curves with a high correlation coefficient were obtained. In addition to characterizing the electrochemical properties of these candidate drug molecules, a novel method for their detection is being developed. Determining the sensitivity of the method is crucial during its development. Consequently, a standard curve was constructed for the detection of TP-NB and TP-PC, and the LOD and LOQ values were established. Furthermore, determining the lowest detectable concentrations of these substances in the context of their potential use as pharmaceuticals will be essential for effective treatment monitoring. In the revised version of the manuscript, the importance of the LOD and LOQ values has been added to the conclusion section.

5. Paragraph 2.2 must be moved close to Figure 1, the last one should be renamed to Scheme 1, to be precise.

Answer 5: Thank you for your feedback. We would like to clarify our statement. To enhance the clarity with which readers can understand and visualize the chemical structures and syntheses of the relevant candidate drug molecules, we have incorporated this section into the introduction. This approach aims to provide readers with a comprehensive understanding of the candidate drug molecules throughout the remainder of the manuscript. Since the term 'figure' has been consistently used throughout the publication, we have designated it as Figure 1 to maintain clarity and avoid any potential confusion.

6. About ACB. As the reviewer realized, it is an acetate buffer. In the “Materials and methods” section provided three different by pH ACB. Authors should provide precise info in the whole document about what types of ACB were used in all arising cases.

Answer 6: We appreciate the reviewer for his/her suggestions, which significantly improved our manuscript. In the revised version of the manuscript, the specific pH values of the ACB used in the relevant instances have been clearly specified in the corresponding sections.  

7. Line 26, 27; 40; 132; 134; 304; 358 “TP-NB and TP-PC; cAMP, cGMP; ACB; PGEs; dsDNA; GC-rich” First appearance of the abbreviation/term must be followed by decipher. An unprepared reader will feel uncomfortable following the article. To partially prevent this inconvenience the “Materials and methods” paragraph should be transferred before the “Results and discussion” one.

Answer 7: Thank you for your feedback. We would like to clarify our situtaion. Since the journal's template places the Materials and Methods section after the Results and Discussion, we have indicated it in the same manner in our manuscript.  In the revised version of the manuscript, the explanations for the abbreviations were added where they first appear.

8. Line 401 “S% = (Sa/Sb) x 100 (Equation 7)” Reformat all equation’s presentations as follows, please:

S = (S_a/S_b) × 100%                                                                                                                  (7)

where S - …

Answer 8: Thank you for your feedback. We would like to clarify our representation. Equation 7 has been formatted as specified. However, for the other equations, we prefer to keep them as written for the sake of readability and ease of understanding for the readers.

9. Line 55 “il-lustrated in Figure 1.” Typo. Please make uniform references to figures.

Answer 9: We appreciate your suggestion which will improve our manuscript. In the modified version, we implemented necessary changes based on the reviewer’s suggestion throughout the manuscript.

10. Line 56-58 Repeat. Compare the lines 51-53.

Answer 10: We did not understand what the reviewer meant.

11. Line 153 “For this reason; reduction peak” “;” - typo?

Answer 11: We appreciate your suggestion which will improve our manuscript. In the revised version, we implemented necessary changes based on the reviewer’s suggestion throughout the manuscript.

12. Line 159 and below “TP-NBe” typo?

Answer 12: We appreciate your suggestion which will improve our manuscript. In the revised version, we implemented necessary changes based on the reviewer’s suggestion throughout the manuscript.

13. Line 192 “pH: 4.8” typo?

Answer 13: Thank you for your feedback. There is no typographical error in this section; however, the sentence was causing some ambiguity. The sentence has been restructured for clarity. In this section, TP-NB and TP-PC solutions were prepared using ACB at pH 4.8, and DPV measurements were conducted in electrolyte solutions with varying pH values.

14. Line 199 “equation of Epc = -0.0501 pH - 0.1217 R² = 0,995” At this stage of the presentation, it is not entirely clear how the different pHs were created.

Answer 14: We appreciate the reviewer for his/her suggestions, which significantly improved our manuscript. In the revised version of the manuscript, the method by which these pH values were obtained has been explained between lines 216 and 221 to clarify the ambiguity pointed out by the reviewer.

15. Figure 4 and Table 1. When using logarithms, the following forms are common and correct: decimal logarithms lg, natural logarithms ln, and logarithms to the base a - log_aN. What exactly did the authors use? What “ν” represents? Please do indicate that square roots are used in legends.

Answer 15: Thank you for your feedback. When various equations in the literature were examined, we observed that the term 'log' is mostly used for decimal logarithms. For this reason, we preferred to keep it as 'log' in our equations. As suggested by the reviewer, the definitions of the abbreviations have been provided in lines 267 and 268, following the caption of Table 1.

16. Line 292 “At higher concentrations (250 and 500 µg/mL), the reduction peak currents were lower” Please indicate this before Figure 5.

Answer 16: We appreciate the reviewer for his/her suggestions, which significantly improved our manuscript. In the section below Figure 5, where the results are interpreted, it was already stated that the current values decreased at concentrations of 250 and 500 µg/mL, following the saturation point. In order to maintain the logical flow and integrity of the interpretation, we preferred to retain the original structure.

17. Figure 6, caption. There is no dsDNA solution under investigation directly, in experiments utilized pre-adsorbed on electrode dsDNA from mentioned solution, see text above. The same in line 414, please pay attention.

Answer 17: We appreciate your suggestion which will improve our manuscript. In the revised version, we implemented necessary changes based on the reviewer’s suggestion throughout the manuscript.

18. Line 450 “according to the Equation 9” The Equation 9 not found in the text as well as the Equation 8.

Answer 18: We appreciate your suggestion which will improve our manuscript. In the revised version of the manuscript, we corrected it as Equation 7 instead of Equation 9.

19. Line 508 “TP-PC synthesized for the first time with various substitutions to the main thiazolopyrimidine skeleton” The statement is not true. Only one compound has been synthesized.

Answer 19: Thank you for your feedback. This article examines two newly synthesized candidate drug molecules.

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Whilst the authors have made changes to the manuscript in response to the more minor points raised in the earlier review, there are several points that the authors are unwilling or unable to address and these include some of the greatest deficiencies in the paper. As such, I cannot recommend this manuscript for publication. The points in question are as follows:

1. Page 5 line 155 “. . . ACB . . .” All abbreviations should be defined at first use in the text. The authors state this has been done but there is no sign of the corresponding change in the manuscript text.
2. Figure 6 does not have information on the number of replicates given in the accompanying figure caption.
3. Figure 6B. The authors are mistaken. Just because another publication/s may have used this approach does not make it right. A histogram, by definition, can only be applied to binned data on the x axis. The x axis is concentration, which is a continuous variable, as such a histogram cannot be used. The graph must be changed.
4. Page 14 line 385 “. . . were calculated as . . . respectively.” The problem with this assessment is that both of these values are less than the lowest standard in your standard curve plot (Figure 6C). As such, you do not have knowledge of the shape and definition of the plot in this concentration region. Add lower concentration points to the standard curve to encompass these values. Once again in their response, the authors are mistaken. Once again, just because another publication/s may do this does not make it right. There are a couple of different ways of computing the limit of detection, but the approach taken by the authors is critically dependent on the assumption that the calibration curve is linear right down to the limit of detection (LOD). When standards are included in the near vicinity of the LOD and linearity appears to have been maintained, then this assumption is supported. However, when standards stop at much higher concentration that the LOD then the assumption of continued linearity becomes questionable. Many calibration curves cease to be linear at low concentrations. The approach that you have taken therefore cannot be supported by the data given. The reader must be assured that the LOD is statistically distinguishable from the blank and you have not provided such evidence.
5. Page 15 lines 437-439 “. . . shifted towards. . . intercalation [46].” This is not a very large change in voltage, yet it is critical to your interpretation of intercalation taking place. What is the degree of resolution of voltage available and what are the experimental errors? In other words, can you demonstrate that this difference in voltage is statistically distinguishable? The authors’ response to this completely misses the point. The fact that similar shifts have been seen in other papers is really irrelevant. What you need to demonstrate is that these measured shifts are statistically significant, and you have still not done this.
6. Page 15 line 446 This should read “Percentage of the guanine peak current change”. This was not changed at this line in the manuscript; it still erroneously says ‘adenine peak current change’.
7. Page 16 lines 486-487 See comment 5 above.

Comments on the Quality of English Language

Whilst the standard of English is reasonable, an editorial check is advised to make any necessary minor corrections.

Author Response

Whilst the authors have made changes to the manuscript in response to the more minor points raised in the earlier review, there are several points that the authors are unwilling or unable to address and these include some of the greatest deficiencies in the paper. As such, I cannot recommend this manuscript for publication. The points in question are as follows:

1. Page 5 line 155 “. . . ACB . . .” All abbreviations should be defined at first use in the text. The authors state this has been done but there is no sign of the corresponding change in the manuscript text.

Answer 1: We thank the reviewer for the suggestion. In the revised version of the manuscript, the abbreviation 'ACB' has been defined at its first occurrence, and other abbreviations have also been rechecked, with some additional definitions included where necessary.

1. Figure 6 does not have information on the number of replicates given in the accompanying figure caption.

Answer 2: We thank the reviewer for the suggestion, which improved our manuscript. In the revised version, the number of replicates was added to the caption of Figure 6.

1. Figure 6B. The authors are mistaken. Just because another publication/s may have used this approach does not make it right. A histogram, by definition, can only be applied to binned data on the x axis. The x axis is concentration, which is a continuous variable, as such a histogram cannot be used. The graph must be changed.

Answer 3: We thank the reviewer for this insightful comment. In the revised version of the manuscript, we have followed the reviewer’s suggestion and replaced the histogram in Figure 6B with a line graph. 

1. Page 14 line 385 “. . . were calculated as . . . respectively.” The problem with this assessment is that both of these values are less than the lowest standard in your standard curve plot (Figure 6C). As such, you do not have knowledge of the shape and definition of the plot in this concentration region. Add lower concentration points to the standard curve to encompass these values. Once again in their response, the authors are mistaken. Once again, just because another publication/s may do this does not make it right. There are a couple of different ways of computing the limit of detection, but the approach taken by the authors is critically dependent on the assumption that the calibration curve is linear right down to the limit of detection (LOD). When standards are included in the near vicinity of the LOD and linearity appears to have been maintained, then this assumption is supported. However, when standards stop at much higher concentration that the LOD then the assumption of continued linearity becomes questionable. Many calibration curves cease to be linear at low concentrations. The approach that you have taken therefore cannot be supported by the data given. The reader must be assured that the LOD is statistically distinguishable from the blank and you have not provided such evidence.

 

Answer 4: Thank you for this important and constructive observation. We acknowledge that the two values reported fall below the lowest concentration point included in our standard curve, and that the accuracy of any interpolation or extrapolation in this range is indeed dependent on the assumption of linearity at low concentrations. We agree that including additional low-concentration standards would have strengthened our claim, and we acknowledge this as a limitation of our current study. However, our approach is based on widely used practices in the literature where the limit of detection (LOD) is calculated assuming a linear calibration curve, as long as the R² value remains high and the standard deviation of the blank is considered in the calculation. Although we did not include standard points in the LOD region, we based our estimation on the linear fit of the calibration curve (R² > 0.99), which remained consistent across replicates. We have now clarified this limitation in the revised manuscript and emphasized that future work should include lower concentration standards to fully validate the linearity assumption near the LOD. We appreciate the reviewer’s critical insight and have revised the discussion section to reflect this important point. We added the below paragraph into the manuscript in Figure 6C.

‘One limitation of our study is that the lowest analyte concentrations detected fell below the range of the standard curve shown in Figure 6C. While the calibration curve demonstrated high linearity (R² > 0.99) across the tested concentrations, we acknowledge that linearity at very low concentrations, particularly near the limit of detection (LOD), should ideally be confirmed by including additional lower concentration standards. Due to practical constraints, we were not able to include these points in the current study. Therefore, the reported LOD values should be interpreted with this limitation in mind’.

5. Page 15 lines 437-439 “. . . shifted towards. . . intercalation [46].” This is not a very large change in voltage, yet it is critical to your interpretation of intercalation taking place. What is the degree of resolution of voltage available and what are the experimental errors? In other words, can you demonstrate that this difference in voltage is statistically distinguishable? The authors’ response to this completely misses the point. The fact that similar shifts have been seen in other papers is really irrelevant. What you need to demonstrate is that these measured shifts are statistically significant, and you have still not done this.

Answer 5: We thank the reviewer for the insightful comment and would like to provide further clarification. In our previous revision, our aim was to emphasize that, in electrochemical studies of drug–DNA interactions, substantial shifts in peak potentials are typically not observed. Instead, only modest changes in the oxidation peak potentials of guanine and/or adenine bases are generally reported. These minor shifts are often used to infer possible modes of interaction. In this context, we suggested that the interaction between TP-NB and DNA might occur via intercalation, based on the observed changes in peak potential. However, we fully acknowledge that such electrochemical evidence alone is not sufficient to definitively confirm intercalation. As the reviewer correctly notes, additional analyses—such as rigorous statistical evaluation and molecular docking studies—are required to substantiate this hypothesis. Accordingly, we have revised the manuscript to avoid definitive language and now state that the interaction could have occurred through intercalation. The primary objective of this study is to determine whether an interaction exists between the candidate drug molecules and DNA, and if so, to provide preliminary insight into the potential mode of binding. Determining the exact nature of the interaction will be the focus of future studies involving more advanced techniques, including molecular docking.

In a recent study (Reference 1: https://doi.org/10.1038/s41598-024-52609-z), similar results were reported, and the authors stated:

"The decrease of the signals and the shift observed in PBS may be correlated with DNA double helix condensation and PNT intercalation. However, the observed shift in the peak potential is much smaller than shifts described by other authors (Reference 2) for intercalation, making it rather difficult to definitively identify the observed interaction type."

In Reference 2, the authors reported:

"When dsDNA is added to a solution of Dac, the peak current height decreases and peak potential shifts from −0.950 V to the more negative value of −1.09 V are observed." In that case, the shift was 0.14 V, whereas in our study, the observed shift was 0.03 V, which is more comparable to that of Reference 1. To prevent misunderstanding and clarify the limitations of our interpretation, we have added the following statement to the discussion:

"Although a shift in redox potential was observed and interpreted as evidence supporting intercalation, we acknowledge that the magnitude of this shift is relatively small. Statistical analysis to confirm that this shift is significantly different from baseline variability was not conducted in the current study. Therefore, the interpretation of intercalation based on this shift should be viewed with caution."

References

1. Smarzewska, S., Ignaczak, A. & Koszelska, K. Electrochemical and theoretical studies of the interaction between anticancer drug ponatinib and dsDNA. Sci Rep 14, 2278 (2024). https://doi.org/10.1038/s41598-024-52609-z
2. Temerk, Y. & Ibrahim, H. Binding mode and thermodynamic studies on the interaction of the anticancer drug dacarbazine and dacarbazine–Cu(II) complex with single and double stranded DNA. J. Pharm. Biomed. Anal. 95, 26–33),

1. Page 15 line 446 This should read “Percentage of the guanine peak current change”. This was not changed at this line in the manuscript; it still erroneously says ‘adenine peak current change’.

Answer 6: We thank the reviewer for this constructive comment, which has contributed to the refinement of our manuscript. As suggested, the specified correction regarding the peak current description has been implemented in the revised version at the indicated section to ensure accuracy and clarity.

1. Page 16 lines 486-487 See comment 5 above.

Answer 7: We thank the reviewer for the insightful comment. Consistent with our response to Comment 5, we recognize that conclusively determining the nature of the interaction requires supplementary approaches, such as molecular docking studies and comprehensive statistical analyses. Accordingly, to maintain scientific rigor and avoid overinterpretation of the electrochemical data, we have revised the manuscript to adopt a more cautious phrasing. Specifically, rather than stating that the interaction between TP-PC and DNA occurs via electrostatically, we now indicate that it may have occurred through electrostatic interactions. This revision ensures terminological precision and appropriately reflects the preliminary nature of the current findings.

Reviewer 3 Report

Comments and Suggestions for Authors

The article is devoted to the Probing Redox Responses and DNA Interactions in Drug Discovery. It is written in fine language and sounds scientifically. This article will be of interest to electrochemists and researchers in the field.

 

Reviewer comments:

 

Reviewer comment: “Authors should put their affiliations in order” - ignored by the authors. The reviewer leaves the decision on this issue to the editors.

Authors repeated TP-NB and TP-PC abbreviation decipher in triplicate. First appearance on page 60 then subsequent ones on pages 69 and 533. Why is it necessary to repeat this information?

Line 73 and below “AcA” First appearance of the abbreviation/term must be followed by decipher. An unprepared reader will feel uncomfortable following the article.

Figure 4 and Table 1. When using logarithms, the following forms are common and correct: decimal logarithms lg, natural logarithms ln, and logarithms to the base a - log_aN. Authors must indicate what type of logarithm was used in the article. Simple “log” is not enough. It should be as follows: “… where log corresponds to decimal logarithm”.

Line 506-510 Check font style and size please.

 

Conclusion:

 

The authors have worked well on the article. Some comments were ignored, but these comments are not of a principal nature. The article may be accepted after minor corrections.

Author Response

1. “Authors should put their affiliations in order” - ignored by the authors. The reviewer leaves the decision on this issue to the editors.

Answer 1: We appreciate the reviewer's comment. However, the authors' affiliations have already been properly listed in the correct order in the manuscript. We are sorry if this was not clear.

2. Authors repeated TP-NB and TP-PC abbreviation decipher in triplicate. First appearance on page 60 then subsequent ones on pages 69 and 533. Why is it necessary to repeat this information?

Answer 2: We thank the reviewer for pointing this out. We have removed the redundant abbreviations from lines 69 and 533, as the abbreviations were already defined on line 60.

3. Line 73 and below “AcA” First appearance of the abbreviation/term must be followed by decipher. An unprepared reader will feel uncomfortable following the article.

Answer 3: We appreciate the reviewer for his/her suggestions, which significantly improved our manuscript. We have provided the expansion of the abbreviation 'AcA' in the relevant section of the revised version of the manuscript.

4. Figure 4 and Table 1. When using logarithms, the following forms are common and correct: decimal logarithms lg, natural logarithms ln, and logarithms to the base a - log_aN. Authors must indicate what type of logarithm was used in the article. Simple “log” is not enough. It should be as follows: “… where log corresponds to decimal logarithm”.

Answer 4: We appreciate the reviewer's insightful suggestion. In accordance with the reviewer's suggestion, we have specified in the relevant section that 'log' refers to the decimal logarithm, in the revised manuscript.

Line 506-510 Check font style and size please.

Answer 5: We thank the reviewer for pointing this out. In the revised version of the manuscript, we have adjusted the font and size of this section to be consistent with the rest of the text.

 

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have more carefully considered the points raised in my earlier reviews of this manuscript. Certain errors that they had omitted to address have now been corrected and in areas where I felt their methodology or evidence was flawed, they have added cautionary statements in the manuscript. Considering these actions, I feel that the authors have now made minimally adequate changes to the manuscript and have now more meaningfully engaged with the earlier review comments.

Comments on the Quality of English Language

The manuscript would benefit from an editorial check on grammar.