Dear Dr. Bresciani_Alberto,

Thank you very much for submitting your manuscript "Spiro-containing derivatives show antiparasitic activity against Trypanosoma brucei through inhibition of the trypanothione reductase enzyme" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.  

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. 

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Grace Adira Murilla, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Ana Rodriguez

Deputy Editor

PLOS Neglected Tropical Diseases

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: I have no concerns regarding the methods - all appropriate to the questions addressed

Reviewer #2: the method is clearly stated and adequate

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Results are clearly presented using appropriate figures. All data is accessible and accurately summarised in the narrative text. Supplementary material is appropriate and necessary.

Reviewer #2: Results are clear

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Conclusions are consistent with the data and relevant implications discussed.

Reviewer #2: conclusions are straight forward

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Typos, clarifications

Lines 94/5 – suggested alternative wording: ‘The X-ray structure of compound 1-bound TbTR revealed that compound 1 binds a TR-specific hydrophobic pocket in the TS2 binding site.’

Line 138/9 – suggested alternative wording: ‘Human glutathione reductase (hGR) activity was assayed as described by Turcano et al [21]’

Line 347 – complementarity

Line 394 – replace ‘added’ with ‘supplemented’

Micromolar IC50 values are reported for compound 1, however the x-axes in Fig 7 are labelled nM.

Line 396 – suggested alternative wording: ‘Fig. 7A shows that compound 1 inhibits TbTR activity in a dose-dependent manner…’

Line 402 – consistent use of italics and abbreviated species format (T. brucei)

Reviewer #2: there are some typographical errors

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Turcano and colleagues describe a new lead compound with activity against trypanothione reductase (TR) and in vitro cultured Trypanosoma brucei at low micromolar concentrations. They define its binding to TR, demonstrate its in vitro selectivity for TR versus human glutathione reductase (hGR), and present data indicating enzyme inhibition via an interaction with the trypanothione binding site. The results are interesting and clearly presented. I only have a couple of minor comments.

The authors discuss the toxicity of the current anti-trypanosomal drugs (highlighting pentamidine and melarsoprol), the need for new treatment development (lines 70-4) and the identification of new biochemical pathways for targeting (lines 74-6). They should also mention recent successes in drug development, including the oxaboroles and the approval of fexnidazole. The available drugs are still limited and other challenges remain, such as the need for drugs to cross the blood brain barrier in late stage HAT, which aren’t mentioned in the introduction – interestingly, the brain penetrance of other spiro core-containing compounds is highlighted in the discussion (lines 441-3).

The authors describe the conservation of TR within the kinetoplastid parasites and suggest their lead compound may be applicable to Leishmania and T. cruzi (lines 432-4). Although not essential, this conclusion could be strengthened by the inclusion of data on compound activity against Leishmania promastigotes (and recombinant LiTR?), as per the authors’ 2018 paper (PMID 30475811).

Reviewer #2: clear

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Reviewer #1: No

Reviewer #2: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

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Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-materials-and-methods

Dear Dr Bresciani,

Thank you very much for submitting your manuscript "Spiro-containing derivatives show antiparasitic activity against Trypanosoma brucei through inhibition of the trypanothione reductase enzyme" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please respond further to this comment from the first round of review:

‘Lines 72-73: More recently, oral compounds like fexinidazole or oxaboroles have come to fruition’.

Fexinidazole is the first all-oral treatment for both early and late stages of Trypanosoma brucei gambiense sleeping sickness; distribution in endemic countries to start in 2019.

This is significant achievement in the treatment of sleeping sickness, and should be included in the response

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.  

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript. 

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Grace Adira Murilla, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Ana Rodriguez

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Please respond further to this comment from the first round of review:

‘Lines 72-73: More recently, oral compounds like fexinidazole or oxaboroles have come to fruition’.

Fexinidazole is the first all-oral treatment for both early and late stages of Trypanosoma brucei gambiense sleeping sickness; distribution in endemic countries to start in 2019.

This is significant achievement in the treatment of sleeping sickness, and should be included in the response

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-materials-and-methods

Dear Dr Bresciani,

We are pleased to inform you that your manuscript 'Spiro-containing derivatives show antiparasitic activity against Trypanosoma brucei through inhibition of the trypanothione reductase enzyme' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Grace Adira Murilla, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Ana Rodriguez

Deputy Editor

PLOS Neglected Tropical Diseases

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