Dear Prof. Hoffmann:

Thank you very much for submitting your manuscript "The repositioning of epigenetic probes/inhibitors identifies new anti-schistosomal lead compounds and chemotherapeutic targets" (#PNTD-D-19-01335) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised a substantial concerns about the manuscript as it currently stands. This issue must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

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Guest Editor

PLOS Neglected Tropical Diseases

Jennifer Keiser

Deputy Editor

PLOS Neglected Tropical Diseases

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Your manuscript, indicated above, has been evaluated by three referees whose comments are attached. In addition, I have reviewed both the referee’s comments and the manuscript itself. The referees provided a number of comments and suggestions for improving the manuscript. There was one major comment that is worth addressing that is "why [the] authors used such a small incubation time (20h) with cytotoxicity assays on the human cell line for calculating selectivity indexes. It's an issue since parasite assays were all performed with 72h incubation time. The authors should generate new data with 72h incubation or make a strong case justifying their choice for 20h since keeping the later should overestimated the selectivity/safety of compounds"

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

Yes

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

Yes

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

NA

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

NA

-Were correct statistical analysis used to support conclusions?

Yes

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

NA

Reviewer #2: Several minor details in the methods text could be clarified.

- Line 149. What strain of Mus musculus?

- Line 188. Schistosomules were cultured with compound at 37 degrees – were they also imaged at 37 or at room temperature?

- Line 189. Reference 50 (Paveley et al., 2012) is mentioned regarding phenotype and motility. However, some details on this package could be clarified. Is this package publically available? What was the rational for the hit thresholds (dotted line figure 1A and 1B)?

- For RNAi, what concetration of siRNA duplexes were used?

- Line 263. How many worms sample input are used in this assay?

Reviewer #3: Methods are appropriate for the study

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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: -Does the analysis presented match the analysis plan?

Yes

-Are the results clearly and completely presented?

Mostly.

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

Yes.

Reviewer #2: The results are clearly presented. As mentioned above in the methods, a description of the hit threshold scores shown in Figure 1A and 1B could be elaborated upon. Either on the legend, or in the text around line 361-364 when these cutoffs are mentioned.

For table 2, add in either the table or the legend a description of the assay used to generate the IC50 data for adult males and females. Also, in the text around line 409 where these results are discussed.

The structures of the chemicals shown in table 2 should be included. It may not be convenient to include them in the actual table 2, but perhaps a supplemental table or in text form (for example, in SMILES format).

While this is not essential, I am interested in whether pharmacological or genetic inhibition of histone methylation effects mitotic activity of somatic neoblasts or germ tissues (testies, ovaries, vitellaria)? Is this the mechanism by which egg laying is arrested?

Reviewer #3: Results match the analysis plan.

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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: -Are the conclusions supported by the data presented?

Generally, yes.

-Are the limitations of analysis clearly described?

Mostly.

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

Yes.

-Is public health relevance addressed?

Yes.

Reviewer #2: The conclusions are well supported by the data. Some discussion could elaborate the repurposing of epigenetic probes / inhibitor compounds. As mentioned in the introduction, praziquantel is not effective against juvenile life stages (~4 week, liver stage immature worms). Granted, ex vivo assays on these worms are more challenging – but is anything known about the expression of targets such as Smp_007000 across the schistosome life cycle? Given the effects on egg laying, it would be interesting whether these are differentially expressed in males and females or in the gonads (PMID: 27499125).

Reviewer #3: Conclusions are appropriate.

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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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: minor revision

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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: The authors describe the anti-schistosomal activities of compounds that are known modulators/inhibitors of human molecular targets involved in epigenetic processes. The deck of 37 compounds was obtained from the Structural Genomics Consortium and tested against schistosomula and adult schistosome lifecycle stages. The study combines several approaches to support/validate the main hits from the initial screenings: in silico modelling, RNAi-mediated functional genomics, enzyme assays and confocal microscopy.

This is an important work with interesting findings to schistosomal drug discovery targeting epigenetic processes. Hence, it should be highly appreciated by the journal readership. I list below several minor points to be corrected but the only major point to be addressed is why authors used such a small incubation time (20h) with cytotoxicity assays on the human cell line for calculating selectivity indexes. It's an issue since parasite assays were all performed with 72h incubation time. The authors should generate new data with 72h incubation or make a strong case justifying their choice for 20h since keeping the later should overestimated the selectivity/safety of compounds.

Minor points:

- Methods:

1- Compound acquisition, storage and handling: Please indicate where appropriate that compounds structures can be found at Table S1

2- Homology modelling and epi-drug docking:

2a - Provide (as supplementary material) evidence for model validation and quality, having special attention to any issues involved in the proposed docking site.

2b - Are all the EP/Eis know to be competitive with the substrates? if so, authors should state it earlier.

- Results:

Lines 311-313: Kd, Ki or IC50? the term potency may be misleading since it's usually expressed as an inverse proportional term. Then, higher potency may actually mean < 100nm.="" lines="" _348-3493a_="" figure="" 1="" _legend3a_="" please="" indicate="" if="" dashes="" crossing="" vertical="" connecting="" each="" replicate="" mean="" averages="" or="" medians.="" line="" _3853a_="" _please2c_="" complement="" s1="" by="" including="" chemical="" structures="" for="" the="" compounds.="" _386-3873a_="" authors="" contend="" fact="" that="" none="" of="" 386="" these="" chemotype="" controls="" demonstrated="" anti-schistosomula="" activity="" suggests="" specificity="" hits="" s.="" mansoni="" homologue="" is="" similar="" to="" found="" original="" human="" target="" but="" other="" interpretation="" would="" be="" they="" lack="" proper="" sar="" targets.="" it="" should="" discussed.="" table="" _23a_="" all="" ec50="" and="" cc50="" accompanied="" standard="" errors="" _9525_="" confidence="" intervals.="" also="" incubation="" time="" below="" headings.="" in="" _footnote2c_="" define="" _22_accurate22_.="" _413-4153a_="" are="" there="" any="" discussed="" compounds="" lly-507="" _bay-5983f_="" _4443a_="" _22_strong22_="" _22_close22_="" _463-4783a_="" make="" their="" conclusions="" lighter="" using="" expressions="" like="" _22_is="" predicted="" _bind...22_="" _22_predicted="" interaction="" may="" _prevent...22_="" _5233a_="" clarify="" why="" gsk-j4="" a="" prodrug.="" how="" drug="" _processed3f_="" _typos3a_="" _713a_="" theat="" threat="" _1273a_="" _22_cultured="" _this22_="" with="" _4523a_="" _22_ceb2_="" _stands22_="" _strands22_="" reviewer="" _23_23a_="" this="" study="" screened="" panel="" known="" epigenetic="" against="" schistosomules="" then="" proceeded="" active="" more="" detail="" on="" adult="" parasites="" _e28093_="" profiling="" phenotypic="" outcomes="" such="" as="" motility="" egg="" _laying2c_="" well="" assaying="" histone="" methylation.="" pharmacological="" experiments="" were="" controlled="" negative="" further="" validated="" rnai="" data="" showing="" knockdown="" putative="" methyltransferase="" phenocopies="" treatment.="" convincing="" controlled.="" laying="" phenotype="" particular="" _promising2c_="" since="" pathology="" associated="" disease="" driven="" parasite="" eggs.="" several="" suggestions="" clarification="" methods="" results="" have="" been="" outlined.="" _23_33a_="" manuscript="" written="" will="" interest="" pntd="" readers.="" minor="" revisions="" suggested.="" abstract="" author="" summary="" _numerous2c_="" undefined="" abbreviations.="" distracting="" _confusing3a_="" _482c_="" _532c_="" 76.="" _general2c_="" has="" large="" number="" _abbreviations2c_="" making="" tedious="" _read2c_="" do="" not="" addressed.="" 54="" _1525_="" something="" indicated.="" define.="" at="" places="" _e2809c_cell="" permeable="" _prodruge2809d_="" described.="" _however2c_="" _e2809c_worm="" _permeablee2809d_="" another="" matter="" general="" poorly="" understood.="" from="" overstated="" absence="" direct="" enzymatic="" evidence="" inhibition.="" was="" done="" indirectly="" h3k36me2="" levels="" _studies2c_="" which="" better="" support="" inhibitor="" _link2f_mechanism="" action.="" 311="" 313="" /> signs should be reversed.

A general comment: the reality of therapy for schistosomiasis is that compounds need to be active after a single dose, where active means worm killing. Blocking motility or egg production after extended exposure to a compound will not be adequate for novel therapies. Did the compounds tested actually _kill_ the worms?

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Reviewer #1: Yes: Floriano Paes Silva-Jr

Reviewer #2: No

Reviewer #3: No

Dear Prof. Hoffmann,

We are pleased to inform you that your manuscript, "The repositioning of epigenetic probes/inhibitors identifies new anti-schistosomal lead compounds and chemotherapeutic targets", has been editorially accepted for publication at PLOS Neglected Tropical Diseases.

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Best regards,

Philip T. LoVerde

Guest Editor

PLOS Neglected Tropical Diseases

Jennifer Keiser

Deputy Editor

PLOS Neglected Tropical Diseases

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

The authors have satisfactorily addressed the concerns of the reviewers.