Fiscalin Derivatives as Potential Neuroprotective Agents
, Bárbara Silva 1,2, Solida Long 3,4, Madalena Pinto 4,5, Fernando Remião 1,2, Emília Sousa 4,5 and Renata Silva 1,2,*Round 1
Reviewer 1 Report
The manuscript entitled “New marine-derived fiscalins as potential neuroprotective agents” describes the results of the in vitro studies performed to assess the toxicity of some fiscalins derivatives in the SH-SY5Y neuroblastoma cell line. Fiscalins are a group of marine-derived compounds isolated from the fungus Neosartorya fischeri. The rational of this study is based on the already described properties of fiscalins derivatives as anticancer, antimicrobial and in particular as neuroprotective in neurodegenerative diseases.
The SH-SY5Y neuroblastoma cell line has been already used as a neuronal in vitro model since these cells, under specific protocol treatments, can be induced to differentiate into neuronal-like cells that express mature neuronal markers. Thus, this cell line has been extensively used for inducing neurodegenerative diseases (ND) such as Parkinson disease (PD) and Alzheimer disease (AD).
Overall the study is well designed and the presentation of the results is quite clear and well organized, however the manuscript should be improved.
Some criticisms are described below.
- Abbreviations should be described the first time they appear in the text, also in the abstract. This has not been the case of MPP+, FeNTA, etc .
- Through all the text, the sentence “(Error! Reference source not found”) appears instead of the Figure author refers to. This should be corrected.
- When describing statistical results, the p value from the statistical studies performed should be always specified.
- Regarding the statistics, when performing 2-way ANOVA, statistical results including interaction between the variables studied, F values, etc…should be described in the results section or figure captions.
- In the text, results are sometimes expressed as Mean ± SD, sometimes as Mean ± SEM. Is there any reason why authors have not used always the same statistical parameters?
- Typos and other mistakes are present in the text. For instance, in pg 2, line 82 “..we have been studding…” “…have been studying…”; pg 2, line 92, “…have showed..”, “…have shown…”, etc.
- When describing methods (pg 5, line 203), authors state that the NR uptake assay provides a “quantitative estimation” of the number of viable cells in a culture. Using such quantitative methods, if performed properly (including biological and technical replicates), the obtained results are not an estimation but a reliable data regarding cell viability.
- In all the procedures used to study the cytotoxicity, cells are first treated with fiscalins, then medium is removed and 50 μg/mL NR, or 10 μg/mL REZ is added, without washing cells first? This could be a problem because the molecules included in the medium treatment could interfere with the biochemical assay.
- Pg 6, line 245, when authors write “…The percentage of SRB binding relatively to that of the control cells (0 μM) was used as the cytotoxicity measure”, does it mean cytotoxicity VALUE?
- Pg 16, line 557, there is an error, this is not Figure 1 but Figure 5. The same at pg 22, line 814, this is not Figure 2 but Figure 6.
- False Figure 2 at pg 21, line 807 “, is not clear at all. Same colors from the “heat map” have different results. This should be changed.
- In the description of Supplementary material, pg 22, line 848, the numbers of supplementary figures from S1 till S8 are wrong, figure S1 has been used twice.
- In the Discussion and in the Conclusions sections, authors have summarized all the obtained results with all the 18 fiscalins derivatives. It would be of help for the reader to have all these results included in a table comparing the effect of all the fiscalins derivatives in terms of the studied parameters, so cytotoxicity through the different assays performed, P-glycoprotein activation/inhibition, MPP+ , FeNTA pro- or anti- toxicity effects, etc.
- Lastly, since in this research no mechanisms have been studied, authors should include this statement in the conclusions, also as a future direction of these studies.
Author Response
The manuscript entitled “New marine-derived fiscalins as potential neuroprotective agents” describes the results of the in vitro studies performed to assess the toxicity of some fiscalins derivatives in the SH-SY5Y neuroblastoma cell line. Fiscalins are a group of marine-derived compounds isolated from the fungus Neosartorya fischeri. The rational of this study is based on the already described properties of fiscalins derivatives as anticancer, antimicrobial and in particular as neuroprotective in neurodegenerative diseases.
The SH-SY5Y neuroblastoma cell line has been already used as a neuronal in vitro model since these cells, under specific protocol treatments, can be induced to differentiate into neuronal-like cells that express mature neuronal markers. Thus, this cell line has been extensively used for inducing neurodegenerative diseases (ND) such as Parkinson disease (PD) and Alzheimer disease (AD).
Overall the study is well designed and the presentation of the results is quite clear and well organized, however the manuscript should be improved.
Some criticisms are described below.
Point 1: Abbreviations should be described the first time they appear in the text, also in the abstract. This has not been the case of MPP+, FeNTA, etc .
Response 1: We acknowledge the Reviewer’s concern and the abbreviations were altered accordingly.
Point 2: Through all the text, the sentence “(Error! Reference source not found”) appears instead of the Figure author refers to. This should be corrected.
Response 2: We acknowledge the Reviewer’s concern and the errors have been corrected in the revised version of the manuscript.
Point 3: When describing statistical results, the p value from the statistical studies performed should be always specified.
Response 3: We acknowledge the Reviewer’s comment and the p value was added to the results section, accordingly to the Reviewer’s suggestion.
Point 4: Regarding the statistics, when performing 2-way ANOVA, statistical results including interaction between the variables studied, F values, etc…should be described in the results section or figure captions.
Response 4: We acknowledge the Reviewer’s concern and these statistical results were added in the figure captions of the revised manuscript.
Point 5: In the text, results are sometimes expressed as Mean ± SD, sometimes as Mean ± SEM. Is there any reason why authors have not used always the same statistical parameters?
Response 5: We acknowledge the Reviewer’s concern and, in fact, it was always used the Mean ± SD parameter, and it was just wrongly written. Therefore, it was rectified in the revised version of the manuscript.
Point 6: Typos and other mistakes are present in the text. For instance, in pg 2, line 82 “..we have been studding…” “…have been studying…”; pg 2, line 92, “…have showed..”, “…have shown…”, etc.
Response 6: We acknowledge the Reviewer’s concern and an english check was performed troughout the revised manuscript.
Point 7: When describing methods (pg 5, line 203), authors state that the NR uptake assay provides a “quantitative estimation” of the number of viable cells in a culture. Using such quantitative methods, if performed properly (including biological and technical replicates), the obtained results are not an estimation but a reliable data regarding cell viability.
Response 7: We acknowledge the Reviewer’s concern and, definitely “quantitative estimation” gives a contradictory idea of what we intend to say. It has been rectified accordingly to “quantitative analysis”.
Point 8: In all the procedures used to study the cytotoxicity, cells are first treated with fiscalins, then medium is removed and 50 μg/mL NR, or 10 μg/mL REZ is added, without washing cells first? This could be a problem because the molecules included in the medium treatment could interfere with the biochemical assay.
Response 8: We acknowledge the Reviewer’s concern and, for sure it could interfere with the obtained results. However, in fact, we washed the cells with HBSS prior to the adiction of the NR and REZ solutions, even though it is not specified in the materials section. Therefore, it was added to the method explanation in the revised version of the manuscript.
Point 9: Pg 6, line 245, when authors write “…The percentage of SRB binding relatively to that of the control cells (0 μM) was used as the cytotoxicity measure”, does it mean cytotoxicity VALUE?
Response 9: We acknowledge the Reviewer’s concern and the sentence was changed to: “The results were expressed as percentage of SRB binding relatively to that of the control cells (0 µM)”.
Point 10: Pg 16, line 557, there is an error, this is not Figure 1 but Figure 5. The same at pg 22, line 814, this is not Figure 2 but Figure 6.
Response 10: We acknowledge the Reviewer’s concern and the mentioned errors were corrected accordingly.
Point 11: False Figure 2 at pg 21, line 807 “, is not clear at all. Same colors from the “heat map” have different results. This should be changed.
Response 11: We acknowledge the Reviewer’s comment and it has been rectified accordingly, and the colours altered to facilitate the interpretation of the heatmap.
Point 12: In the description of Supplementary material, pg 22, line 848, the numbers of supplementary figures from S1 till S8 are wrong, figure S1 has been used twice.
Response 1: We apologize for the lapses and the identified errors were corrected accordingly.
Point 13: In the Discussion and in the Conclusions sections, authors have summarized all the obtained results with all the 18 fiscalins derivatives. It would be of help for the reader to have all these results included in a table comparing the effect of all the fiscalins derivatives in terms of the studied parameters, so cytotoxicity through the different assays performed, P-glycoprotein activation/inhibition, MPP+ , FeNTA pro- or anti- toxicity effects, etc.
Response 13: We aknowledge the reviewer’s sugestion and we add a schematic structure-activity relashionship (now Figure 6), along with the heatmap (now figure 7) in which the obtained data was combined. By putting the results altogether in these figures, we expect to provide the reader an overall analysis of all the parameters studied for each fiscalin derivative.
Point 14: Lastly, since in this research no mechanisms have been studied, authors should include this statement in the conclusions, also as a future direction of these studies.
Response 14: We acknowledge the Reviewer’s concern and the following sentence was added to the conclusions section of the revised manuscript: “Despite the important biological properties presented in this work, the mechanisms underlying the observed neuroprotective effects are yet unknown, persisting the need for more mechanistic and in vivo studies to further elucidate their mechanisms of action and their potential as new disease-modifying drugs. Indeed, future perspectives of the present work aim at the evaluation, in vivo, in a MPTP model of PD, of the pharmacokinetics and pharmacodynamics profiles of the most promising fiscalin derivatives, as well as the assessment of potential side effects. Furthermore, and knowing that ferroptosis is involved in the etiology of several ND apart from PD and AD (such as Amyotrophic Lateral Sclerosis or Huntington disease), these derivatives may also, in the future, represent new therapeutic strategies to be tested in the scope of the treatment and/or prevention of such diseases.”
Author Response File: 
Author Response.docx
Reviewer 2 Report
Manuscript #pharmaceutics-1744625 addresses an interesting topic of research. Development of anti-neurodegenerative chemical entities is yet unmet clinical need. However, the current manuscript reports synthetic analogs of fiscalins which are quinazolinone derivatives that are previously reported to elicit neuroprotective activities (reference #30 which is duplicated also as reference #39!!!!). Accordingly, the chemistry and the neuroprotective biological activity are literature-reported and known. Measuring cytotoxicity by three methods does not add much to the biology! In short, the current manuscript does not provided significant new data or insights of biological activity such as molecular target or mechanistic study. In addition, many errors exist in references (“Error! Reference source not found” is encountered many times in this manuscript). A minor issue is the lengthy introduction. In my humble opinion, the current manuscript might NOT be suitable for publication. I suggest authors to conduct additional studies to get insights of the neuroprotective activity of these compounds and to correct text and references errors and then resubmit again.
Author Response
Manuscript #pharmaceutics-1744625 addresses an interesting topic of research. Development of anti-neurodegenerative chemical entities is yet unmet clinical need. However, the current manuscript reports synthetic analogs of fiscalins which are quinazolinone derivatives that are previously reported to elicit neuroprotective activities (reference #30 which is duplicated also as reference #39!!!!). Accordingly, the chemistry and the neuroprotective biological activity are literature-reported and known. Measuring cytotoxicity by three methods does not add much to the biology! In short, the current manuscript does not provided significant new data or insights of biological activity such as molecular target or mechanistic study. In addition, many errors exist in references (“Error! Reference source not found” is encountered many times in this manuscript). A minor issue is the lengthy introduction. In my humble opinion, the current manuscript might NOT be suitable for publication. I suggest authors to conduct additional studies to get insights of the neuroprotective activity of these compounds and to correct text and references errors and then resubmit again.
Response 1: This study is a follow-up work after a first disclosure of neuroprotective activity by fiscalins. Herein, a deeper analysis was perform not only in the derivatives investigated but also in the in vitro studies which allowed to propose a structure-activity relationship study and gave further insights into their neuroprotection action with modulating effects on the P-gp and protective effects towards MPP+ and Iron (III) induced cytotoxicity. These results can guide readers for the future design of new neuroprotective quinazoline-3,6-diones. We also acknowledge the Reviewer’s concern and the references and existing errorrs were all rectified in the revised version of the manuscript.
Author Response File: Author Response.docx
Reviewer 3 Report
This study investigates the in vitro neuroprotective efficacy of marine-derived fiscalins.
a few remarks and suggestions are as follows:
1. check whole manuscript for "Error! Reference source not found" issue and update accordingly
2. The most of of the derivatives were previously synthesized, so the word "New" does not pertain in the title of the manuscript.
3. I could not find the description of figures in result and discussion section, only supplementary figures are explained.
4. figure numbering and their arrangement is not clear to me.
Author Response
This study investigates the in vitro neuroprotective efficacy of marine-derived fiscalins.
a few remarks and suggestions are as follows:
Point 1: check whole manuscript for "Error! Reference source not found" issue and update accordingly
Response 1: We acknowledge the Reviewer for identifying these lapses and the errors have been rectified accordingly.
Point 2: The most of of the derivatives were previously synthesized, so the word "New" does not pertain in the title of the manuscript.
Response 2: We acknowledge the Reviewer’s concern and the word “new” was removed from the title, which was also simplified and changed to “Fiscalin derivatives as potential neuroprotective agents”.
Point 3: I could not find the description of figures in result and discussion section, only supplementary figures are explained.
Response 3: We acknowledge the Reviewer’s concern and a more detailed description of figures was made in the revised version of the manuscript.
Point 4: figure numbering and their arrangement is not clear to me.
Response 4: We acknowledge the Reviewer’s concern and, indeed, there were some errors in the figures captions, which were rearranged and rectified in the revised version of manuscript.
Author Response File: Author Response.docx
Reviewer 4 Report
The current manuscript examined the neuroprotective effect of marine-derived fiscalins in invitro model of neurodegenerative disorders. they concluded based on the results that fiscalins are promising neuroprotective agents and therefore possible therapeutic agent for neurodegenrative disorders. The topic is interesting and the study is well designed, statistical analysis was performed accurately. there is just small thing in discussion which some of the references are missing and just shows error.
Author Response
The current manuscript examined the neuroprotective effect of marine-derived fiscalins in invitro model of neurodegenerative disorders. they concluded based on the results that fiscalins are promising neuroprotective agents and therefore possible therapeutic agent for neurodegenrative disorders. The topic is interesting and the study is well designed, statistical analysis was performed accurately. there is just small thing in discussion which some of the references are missing and just shows error.
Response 1: We acknowledge the Reviewer’s comments and, in fact, there were some errors alongside the text and in the figures captions, which were rearranged and rectified in the revised version of manuscript.
Author Response File: Author Response.docx
Reviewer 5 Report
It is a very well written manuscript where authors explored the potential of fiscalins derived compounds. They report promising compounds with modulating effects on the P-gp and protective effects towards MPP+ and Iron (III) induced cytotoxicity. Manuscript can benefit from some modifications.
The title seems misleading by saying ‘marine-derived’ while the compounds are synthetic and are derivatives of fiscalins. Authors have discussed the structure activity relationship of the compounds. It would be really interesting and appealing to the readers if a schematic explanation can be put together. At this point, the authors have only performed cell culture assay and have not commented on the future directions of this work. What could be the possible explanation about the mechanism of action of the compounds. It would be important to explore in order to determine if the compounds are suitable to be pursued further to target ND and what could possibly be the side effects of the compounds.
Author Response
It is a very well written manuscript where authors explored the potential of fiscalins derived compounds. They report promising compounds with modulating effects on the P-gp and protective effects towards MPP+ and Iron (III) induced cytotoxicity. Manuscript can benefit from some modifications.
The title seems misleading by saying ‘marine-derived’ while the compounds are synthetic and are derivatives of fiscalins. Authors have discussed the structure activity relationship of the compounds. It would be really interesting and appealing to the readers if a schematic explanation can be put together. At this point, the authors have only performed cell culture assay and have not commented on the future directions of this work. What could be the possible explanation about the mechanism of action of the compounds. It would be important to explore in order to determine if the compounds are suitable to be pursued further to target ND and what could possibly be the side effects of the compounds.
Response 1: We acknowledge the Reviewer’s concern and, in fact, the title is misleading and, therefore it was changed and simplifyed to “Fiscalin derivatives as potential neuroprotective agents”.
Apart from the heatmap, and according to the Reviwer’s suggestion, we added a schematic structure-activity relashionship (now Figure 6), to highlight the structural modifications and their consequences in the biological activities of these compounds.
Also, to further clarify the future perspectives of the present work, namely the in vivo evaluation of the most promising fiscalins in a PD model to highlight their Pharmakokinetics/Pharmacodinamycs and potencial side effects, were clarified and the following sentence added to the conclusion section of the revised manuscript: : “Despite the important biological properties presented in this work, the mechanisms underlying the observed neuroprotective effects is yet unknown, persisting the need for more mechanistic and in vivo studies to further elucidate their mechanisms of action and their potential as new disease-modifying drugs. Indeed, future perspectives of the present work aim at the evaluation, in vivo, in a MPTP model of PD, of the pharmacokinetics and pharmacodynamics profiles of the most promising fiscalin derivatives, as well as the assessment of potential side effects. Furthermore, and knowing that ferroptosis is involved in the etiology of several ND apart from PD and AD (such as Amyotrophic Lateral Sclerosis or Huntington disease), these derivatives may also, in the future, represent new therapeutic strategies to be tested in the scope of the treatment and/or prevention of such diseases.”
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
The authors have revised the manuscript and replied to most of the raised concerns
