Round 1

Reviewer 1 Report

 

Reviewer’s comments on the article entitled “Structural parameters of the interaction between ciprofloxacin 2 and human topoisomerase-II β enzyme: toward new NMR 3 probes” by Sales, T. A., et al submitted to Magnetochemistry

 

The authors noted that topoisomerase-II enzyme overexpression is correlated with the onset of cancer in human cells. With the hypothesis that CPX, which targets DNA gyrase and topoisomerase-II enzyme, can be used as a 19F NMR probe in detecting overexpression of topoisomerase-II, the authors selected a set of well-studied theoretical tools in simulating the 19F NMR spectra of CPX. The authors validated the accuracy of the theoretical method by demonstrating high similarity between the experimental 19F NMR shift and the calculated shift for the CPX:Explicit water model. In addition, the importance of intermolecular interaction, especially hydrogen bonding, in calculating NMR shifts has been emphasized by comparison of the experimental result with both the CPX:Explicit water and CPX:Implicit water models. However, a series of aspects require more information or clarification to justify the authors’ claim of using CPX to be a potential NMR probe in cancer diagnosis.

1.     “… Toward new NMR probes” in the title of the journal is confusing as it doesn’t describe what type of probe it is going to be and / or what the impact of this new probe would be.

2.  The abstract explains the study in a concise fashion. However, it remains unclear to the audience what is the connection between this study and cancer diagnosis. The authors must clearly state the connection between overexpression of topoisomerase-IIβ and diagnosis of specific cancerous activities in establishing the relevance of their work.

3.   One of the introductory statements (line-32) says, “… one factor that contributes for many deaths is the difficulty in diagnosing [14–16].” While the value of improved prognosis by early detection of cancer is well established, the current statement is vague. The authors can specify what type of cancers they are talking about and what diagnostic difficulties exist. In addition, the relevance of the cited references to the statement is unclear. References 14 – 16 are related to the development of rapid diagnosis of cancer, a new spectroscopic method development for cancer diagnosis, and studying a potential fluorescence probe for cancer detection. None of the articles are directly related to research on “diagnostic challenges in cancer”.

4.   Frequent switching between nuclear magnetic resonance (NMR) and magnetic resonance imaging have been confusing as those are fundamentally different techniques (line 35 – line 39).

5.  The total average energy for CPX:hTOPO-II was given to be -94.27 KJ/mol (line-129), is this correct? The energy variation given in Figure.2 doesn’t correspond to this value. Either this should be corrected or a better interpretation should be given for the audience to understand how’s that value obtained.  

6.     The hydrogen bonding and π-π interactions can be seen from the MD simulation. Are those interactions sufficiently strong to make a 19F NMR shift of 6 ppm? It would have been better to provide a discussion with a set analogues or a discussion on electron density distribution of CPX in CPX:hTOPO-II.

7.    The authors noted that CPX “… exert antibacterial activity due to inhibition of two bacterial enzymes, DNA gyrase and topoisomerase II enzymes.” While the authors studied CPX : topoisomerase-II enzyme interaction, no discussion was presented on CPX : DNA gyrase interaction and what kind of NMR shift would be resulted. Without this study, it is unclear how an exclusive interaction between CPX and topoisomerase-II can be detected from 19F NMR shift alone.      

 Please check for typographic errors:

1.      Should be “GAUSSIAN 09” and “Dunning” (line-82)  

2.      Should be “Figure (4-B)” (line-149)

 

 

Author Response

1. English language and style: (x) Moderate English changes required

A: We perform a careful proofreading to eliminate errors and improve the writing of the text.

 

 

The authors noted that topoisomerase-II enzyme overexpression is correlated with the onset of cancer in human cells. With the hypothesis that CPX, which targets DNA gyrase and topoisomerase-II enzyme, can be used as a 19F NMR probe in detecting overexpression of topoisomerase-II, the authors selected a set of well-studied theoretical tools in simulating the 19F NMR spectra of CPX. The authors validated the accuracy of the theoretical method by demonstrating a high similarity between the experimental 19F NMR shift and the calculated shift for the CPX:Explicit water model. In addition, the importance of intermolecular interaction, especially hydrogen bonding, in calculating NMR shifts has been emphasized by a comparison of the experimental result with both the CPX:Explicit water and CPX:Implicit water models. However, a series of aspects require more information or clarification to justify the authors’ claim of using CPX to be a potential NMR probe into cancer diagnosis.

2. “… Toward new NMR probes” in the title of the journal is confusing as it doesn’t describe what type of probe it is going to be and / or what the impact of this new probe would be.

A: Thank you for this suggestion. The authors agree with the reviewer. In this new version, we detail more about the type of probe that is being investigated. Now, the new title is:

 “Structural parameters of the interaction between ciprofloxacin and human topoisomerase-II β enzyme: toward new F19 NMR Chemical Shift Probes”

3. The abstract explains the study in a concise fashion. However, it remains unclear to the audience what is the connection between this study and cancer diagnosis. The authors must clearly state the connection between overexpression of topoisomerase-IIβ and diagnosis of specific cancerous activities in establishing the relevance of their work.

A: The authors thank you for the relevant comment. In fact, the abstract needs to contain a better link between the objective of the research, which is to show a possibility for the development of new tools for cancer diagnosis using the repositioning of the drug ciprofloxacin; and the enzyme chosen for analysis, due to its overexpression in tumor cells. To improve this presentation, we have inserted the following sentence into the abstract:

“Increased topoisomerase-II expression has been associated with cancer occurrence, mainly with aggressive forms of breast cancer, thus constituting a promising molecular target for new tumor cell identifiers.”

4. One of the introductory statements (line-32) says, “… one factor that contributes for many deaths is the difficulty in diagnosing [14–16].” While the value of improved prognosis by early detection of cancer is well established, the current statement is vague. The authors can specify what type of cancers they are talking about and what diagnostic difficulties exist. In addition, the relevance of the cited references to the statement is unclear. References 14 – 16 are related to the development of rapid diagnosis of cancer, a new spectroscopic method development for cancer diagnosis, and studying a potential fluorescence probe for cancer detection. None of the articles are directly related to research on “diagnostic challenges in cancer”.

  A: In the revised version, we have added more details about the types of cancers we are considering, the main diagnostic difficulties that can occur, and finally, the references have also been inserted about diagnostic challenges for cancer disease. The entire sentence has been redone, and the corrections are highlighted in the text (lines 33-51 of the introduction section). Thank you for the comment.

5. Frequent switching between nuclear magnetic resonance (NMR) and magnetic resonance imaging have been confusing, as those are fundamentally different techniques (line 35 – line 39).

A: The reviewer is right. Now, these misleading terms have been corrected. Thank you for your observation.

6. The total average energy for CPX:hTOPO-II was given to be -94.27 KJ/mol (line-129), is this correct? The energy variation given in Figure.2 doesn’t correspond to this value. Either this should be corrected or a better interpretation should be given for the audience to understand how that value is obtained.  

A: The authors thank you for your valuable observation. In fact, the sentence could lead this interpretation and that value could be the total energy value. However, it corresponds to the value of the interaction energy and not the total energy of the system. So, in line with the reviewer´s comment, the sentence was rewritten to clarify this discussion (lines 147-148). Additionally, the graph corresponding to the obtained interaction energies in all simulation times was inserted in figure 2 (Fig 2-C).

 

7. The hydrogen bonding and π-π interactions can be seen from the MD simulation. Are those interactions sufficiently strong to make a 19F NMR shift of 6 ppm? It would have been better to provide a discussion with a set analogues or a discussion on electron density distribution of CPX in CPX:hTOPO-II.

A: Thank you for your comment. In fact, the NMR technique is very sensitive to the environment, and variations in NMR parameters of a molecule are a consequence mainly of chemical environment modifications (in this case, inside and outside the enzyme) [1]. As a result, the modification in the environment brings the modification of the molecule electronic structure and conformation because of the occurrence of possible intermolecular interactions [2]. In this line, we believe that in this case, the hydrogen bonds and π-π interactions that occur between ciprofloxacin and topoisomerase eventually influence the alteration of the chemical shift, since they can change the conformation of the ligand, as well as shift its electron density, as the reviewer well observed. Thus, to enrich this discussion in the paper, we inserted 2 more figures containing these suggested data (figures 6 and 7). 

 

8. The authors noted that CPX “… exert antibacterial activity due to inhibition of two bacterial enzymes, DNA gyrase and topoisomerase II enzymes.” While the authors studied CPX: topoisomerase-II enzyme interaction, no discussion was presented on CPX: DNA gyrase interaction and what kind of NMR shift would be resulted. Without this study, it is unclear how an exclusive interaction between CPX and topoisomerase-II can be detected from 19F NMR shift alone.      

A: Thank you for pointing out this important question. Indeed, as mentioned in the text, the mechanisms of the action of ciprofloxacin related to its antibacterial activity are already well elucidated in the literature and include the inhibition of these two enzymes of the bacteria, DNA gyrase and topoisomerase. However, considering that in this work the proposal is to use this drug as a NMR probe to be used in humans, and that the enzyme DNA gyrase is apparently present in bacteria, but absent in large eukaryotes [3–5], the authors understand that there is no need for a study considering the interaction of ciprofloxacin with the enzyme DNA gyrase, in this case. But, considering this important comment, we have added in the text a brief sentence about this discussion (lines 79-84).

 

9. Please check for typographic errors:
10.     Should be “GAUSSIAN 09” and “Dunning” (line-82)  
11. Should be “Figure (4-B)” (line-149)

A: We acknowledge this observation. We have corrected the wrong terms in the updated version.  We carried out a careful revision of the manuscript to eliminate grammatical errors.

Reviewer 2 Report

The authors presented the paper "Structural parameters of the interaction between ciprofloxacin and human topoisomerase-II β enzyme: toward new NMR probes"

1) The novelty of the work should be clearly mentioned in The Conclusion section. It is clearly known that ciprofloxacin inhibit this enzyme. Moveover, the docking studies was previously done, for example: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5493602/

You don't compare this results with the previous works.

2) Have you done RMSF cacuation swith aminoacids?

3) After binding the shift will be changed of course. There is many problems

3a) the peak width will be much higher than in the Fig. 5

3b) the concentration of enzyme will be low.

3c)  the probe have to go efficeciently to the cancer tissue, into the cell

3d) is there any interfering proteins that can bind this compound? May be human serum albuminin plasma?

It may be not seen anything under organism conditions. Much more discussion, proof of the design should be presented. It is not so simple to do good in vivo probe. 

4) CPX in water calculation is good. However, the organism is not water. What about salts, viscosity, etc.? Is the influence on the fluorine shift will be high.

 

 

Author Response

Reviewer 2:

 

1. English language and style

 ( ) English very difficult to understand/incomprehensible
( ) Extensive editing of English language and style required
(x) Moderate English changes required
( ) English language and style are fine/minor spell check required

( ) I don't feel qualified to judge about the English language and style

 

A: We perform a careful proofreading to eliminate errors and improve the writing of the text.

 

 

Comments and Suggestions for Authors

The authors presented the paper "Structural parameters of the interaction between ciprofloxacin and human topoisomerase-II β enzyme: toward new NMR probes"

1) The novelty of the work should be clearly mentioned in The Conclusion section. It is clearly known that ciprofloxacin inhibits this enzyme. Moreover, the docking studies was previously done, for example: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5493602/. You don't compare this result with the previous works.

A: We acknowledge for pointing out this important reference. In the current version, this reference has been explored in the discussion section, along with other missing theoretical works (lines 128-156). Regarding conclusion, the text has been improved to contemplate more objectively the novelty of the work.

2) Have you done RMSF calculations with aminoacids?

A: In fact, the RMSF analysis is an enhancement of the stability of the enzyme in simulation. We insert the RMSF graph together with the RMSD in Figure 1.

3) After binding, the shift will be changed of course. There is many problems

1. a) the peak width will be much higher than in the Fig. 5

A: The authors thank you for your comments. The figure 5 is only a graphic representation of the difference between the chemical shifts obtained. In fact, it would be more interesting to represent the overlap of the spectra obtained for the molecule in the different environments, but this chemical shift indicated is an average got from the actual calculation of all the frames got in the simulation.  

1. b) the concentration of enzyme will be low. c)  the probe have to go efficiently to the cancer tissue, into the cell d) is there any interfering proteins that can bind this compound? May be human serum albuminin plasma? It may be not seen anything under organism conditions. Much more discussion, proof of the design should be presented. It is not so simple to do a good in vivo probe. 

A: Thank you for your relevant and expert opinion on the subject. We agree that the development of a probe is much more complex, and in fact, a lot of information and experimental work are needed to be able to really affirm that the ciprofloxacin can really act as an efficient probe. However, as mentioned in the text (lines 322-330), our theoretical study is a first attempt to explore this interesting property of an existing drug, based on previous evidences, such the effectiveness of this molecule to bind in human topoisomerases and consequently induces the cell apoptosis and also the overexpression of this enzyme in tumor cells. Furthermore, this initial study may also serve as a parameter for possible new probes that will be derived from the CPX molecule.

 Whilst the obtained theoretical model provides insightful information on the relationship between structure and biological activity and the clear practical utility lies in its ability to predict the performance of unique substrate classes, thereby directing future synthetic efforts. Then, if effective internal and external validation tests were possible, the model could estimate with a good accuracy of prediction. We agree that the limitation of our study is the absence of wet lab studies and in vitro, in vivo experiments or clinical trials for the proposed compounds. On the other hand, we believe that the strong point of the present study is to rationalize a predictive model for developing new NMR probes, which can be synthetized and biologically evaluated in the future. So, we believe that such information will be essential in any future effort towards design and discovery of new spectroscopic probes for cancer diagnosis. In this context, theoretical tools can be employed as filters to flag and de-select the potentially harmful compounds at the preclinical stage of drug development, thereby potentially avoiding significant economic and human health consequences incurred at later stages of drug discovery.

In line with that, we believe to report our current theoretical findings in the literature can stimulate new experiments and other theoretical investigations that could assess the validity of this assumption.

 

4) CPX in water calculation is good. However, the organism is not water. What about salts, viscosity, etc.? Is the influence on the fluorine shift will be high.

A: This is a very important point to address. Thank you for considering that. During the MD simulation, although we built the box with water, we inserted some ions to neutralize the system. In this case Na⁺ cations were inserted in a concentration range of 74 μM, which was sufficient for the neutralization. Although physiological sodium cation concentrations are around 140 mM [6], within the enzyme active site, which is generally a more hydrophobic region [7,8], this concentration is much lower. Regarding viscosity, in general, it is not considered in MD simulations. However, in most mostly aqueous media with very low ion contentions, we believe that the viscosity of water is a good approximation.

 

1. Gonçalves, M.A.; Gonçalves, A.S.; Franca, T.C.C.; Santana, M.S.; da Cunha, E.F.F.; Ramalho, T.C. Improved Protocol for the Selection of Structures from Molecular Dynamics of Organic Systems in Solution: The Value of Investigating Different Wavelet Families. J. Chem. Theory Comput. 2022.
2. Evanics, F.; Kitevski, J.L.; Bezsonova, I.; Forman-Kay, J.; Prosser, R.S. 19F NMR studies of solvent exposure and peptide binding to an SH3 domain. Biochim. Biophys. Acta - Gen. Subj. 2007, 1770, 221–230.
3. Pudipeddi, A.; Vasudevan, S.; Shanmugam, K.; Mohan S, S.; Vairaprakash, P.; Neelakantan, P.; Balraj, A.S.; Solomon, A.P. Design, dynamic docking, synthesis, and in vitro validation of a novel DNA gyrase B inhibitor. J. Biomol. Struct. Dyn. 2022, 4, 1–14.
4. Khan, T.; Sankhe, K.; Suvarna, V.; Sherje, A.; Patel, K.; Dravyakar, B. DNA gyrase inhibitors: Progress and synthesis of potent compounds as antibacterial agents. Biomed. Pharmacother. 2018, 103, 923–938.
5. Idowu, T.; Schweizer, F. Ubiquitous nature of fluoroquinolones: The oscillation between antibacterial and anticancer activities. Antibiotics 2017, 6.
6. Ye, L.; Neale, C.; Sljoka, A.; Lyda, B.; Pichugin, D.; Tsuchimura, N.; Larda, S.T.; Pomès, R.; García, A.E.; Ernst, O.P.; et al. Mechanistic insights into allosteric regulation of the A2A adenosine G protein-coupled receptor by physiological cations. Nat. Commun. 2018 91 2018, 9, 1–13.
7. March, D.; Bianco, V.; Franzese, G. Protein Unfolding and Aggregation near a Hydrophobic Interface. Polym. 2021, Vol. 13, Page 156 2021, 13, 156.
8. Abdelgawad, M.A.; Mohamed, F.E.A.; Lamie, P.F.; Bukhari, S.N.A.; Al-Sanea, M.M.; Musa, A.; Elmowafy, M.; Nayl, A.A.; Karam Farag, A.; Ali, S.M.; et al. Design, synthesis, and biological evaluation of novel pyrido-dipyrimidines as dual topoisomerase II/FLT3 inhibitors in leukemia cells. Bioorg. Chem. 2022, 122, 105752.

 

 

We acknowledge again the referee’s comments, which have enabled us to significantly improve our paper. Furthermore, we thank the editorial assistance and hope with the changes and clarifications implemented, the manuscript would be now acceptable for publication in the Magnetochemistry Journal. Finally, we also remain at your disposal for any further inquiries.

 

With best regards,

Round 2

Reviewer 1 Report

The motivation behind the work is much well demonstrated in this revised version of the manuscript, and the additional justifications provided for the study design, and various observations have improved the quality of the presentation significantly.

The authors should review the language and fix some grammatical errors to further improve the manuscript. Following are two such instances:

Line 42: Please correct the sentence- "In general, it can be considered three main aspects that influence the cancer early diagnosis, ...".

Line 135: "Recently, in order to ... the action mechanisms ..." - please change to "... mechanism of action ...".

Overall, the authors have done a good job revising the manuscript and in my opinion, the manuscript should be accepted for publication after minor text editing by the authors.

Author Response

Editorial Office

                    Magnetochemistry    

                                                                                                     25^th November, 2022

 

            Dear Editor,

            Please find enclosed the minor revised version of the manuscript entitled “Structural parameters of the interaction between ciprofloxacin and human topoisomerase-II β enzyme: toward new 19F NMR Chemical Shift probes” by Sales, T. A., et al submitted to Magnetochemistry journal.

            Once again, we would like to thank you for your new observations and suggestions. We have made a new revision of the manuscript in order to further refine the article, following the reviewers suggestions. The changes are highlighted throughout the text, and the list of changes is listed below. 

 

Reviewer 1

The motivation behind the work is much well demonstrated in this revised version of the manuscript, and the additional justifications provided for the study design, and various observations have improved the quality of the presentation significantly.

The authors should review the language and fix some grammatical errors to further improve the manuscript. Following are two such instances:

Line 42: Please correct the sentence- "In general, it can be considered three main aspects that influence the cancer early diagnosis, ...".

Line 135: "Recently, in order to ... the action mechanisms ..." - please change to "... mechanism of action ...".

Overall, the authors have done a good job revising the manuscript and in my opinion, the manuscript should be accepted for publication after minor text editing by the authors.

 

A: Thank you for the observations. The entire manuscript has been revised in order to correct or improve these and other clerical errors. The modifications are highlighted in the text.

 

 

Finally, we acknowledge once again for the editorial assistance and hope that with the implemented changes and clarifications, the manuscript is now fit for publication in the Magnetochemistry Journal. We also appreciate the referee's comments, which allowed us to significantly improve our paper in the two requested revisions. In addition, we also remain at your disposal for any further questions.

 

With best regards,

 

Teodorico C. Ramalho

Author Response File: Author Response.docx

Reviewer 2 Report

Thank you for the revised paper.

Author Response

            Once again, we would like to thank you for your new observations and suggestions. Please find enclosed the minor revised version of the manuscript entitled “Structural parameters of the interaction between ciprofloxacin and human topoisomerase-II β enzyme: toward new 19F NMR Chemical Shift probes” by Sales, T. A., et al submitted to Magnetochemistry journal. 
In addition, we also remain at your disposal for any further questions. 
With best regards,
 
Teodorico C. Ramalho

Author Response File: Author Response.docx