Dear Dr. Chan:

Thank you very much for submitting your manuscript "Non-sedating benzodiazepines cause paralysis and tissue damage in the parasitic blood fluke Schistosoma mansoni " (PNTD-D-19-01242) 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 topic but identified some aspects of the manuscript that should be improved.

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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: The methods are appropriate and are clearly presented. My only minor concern is that previous work has been done on BZ binding to membranes; were these methods designed de novo or were they based on earlier, similar work? If the latter is the case, that earlier work should be cited.

Reviewer #2: - Bioinformatics of LGICs: were homology-based searches also used to confirm missing channels? This would help in case fragmented gene models (or missing gene models) did not meet the transmembrane count filter.

- L126 typo (missing 'washed')

Reviewer #3: (No Response)

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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: The results are clearly and concisely presented and the data are convincing. The authors might note that previous bioinformatics searches had already shown that S. mansoni lacks GABAr sequences.

Reviewer #2: - Throughout the manuscript, 'selectivity' is profiled by measuring in vitro effects on schistosomes and specific interactions with mammalian GABAAR channels. There is the real possibility that selecting for chemicals that maintain 'parasite-selectivity' and diminished affinity for GABAAR channels is akin to selecting for activity against a different homologous parasite and host target set. Since the mode of action that drives this selectivity is unknown, this possibility cannot yet be ruled out. While not in the scope of this manuscript, I think it would be valuable to devote some text to discussing this possibility.

- Differences in recovery among imidazobenzodiazepines (Fig 4) are not well-explained. I would be curious to know whether higher concentrations of XliHe-II-048 and SH-I-055 imidazobenzodiazepines would have prevented recovery - suggesting this is a threshold effect.

Reviewer #3: (No Response)

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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: The conclusions are reasonable and well-supported by the data. The sentence beginning on line 286 is unclear; it seems to indicate that it is expected that the sedative dose of MCLZ is the same as the therapeutic dose for schistosomiasis. Please clarify.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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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: Minor concerns only:

1.Line 55: current estimates of death due to schistosomiasis are much lower than 300,000; the cited refreence is 16 years old and should be updated.

2. Line 197: replace 'at' with 'for'

3. Use 'concentration' instead of 'dose' when referring to units of mass/units of volume

4. Line 264: delete 'exhibit'

5. Line 352: 'pose' is an unusual term in this context; replace with 'pocket'?

6. Line 375: delete first use of 'the'

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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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: This is an interesting and important paper, helping to clear up a long-standing mystery in this field. I congratulate the authors.

Reviewer #2: This is a very interesting manuscript that seeks to re-explore the utility of benzodiazepines as agents of schistosome control. The authors make a compelling case that the dose-limiting sedatory effects of meclonazepam (MCLZ) are not necessarily coupled to MCLZ schistocidal activity. They make this case in two primary ways. First, bioinformatics analyses are used to show loss of GABAAR-like channels in this flatworm lineage and to suggest that different (yet unknown) target(s) exists for MCLZ in schistosomes. Second, various non-sedating benzodiazepines are shown to have strong effects on worm motility and tegument integrity. This study is a starting point to identifying benzodiazopines that fit this selectivity profile and identifying the likely novel mediators of benzodiazopine activity against schistosome parasites. I suspect that target identification will allow for broader screening efforts to identify other classes of chemicals that phenocopy these effects. The experiments were well-designed and presented. My comments are minor.

Reviewer #3: I enjoyed reading this paper and consider it to be of high interest to the field of parasitology. I think it is an excellent idea to reconsider the benzodiazepines as potential antischistosomal drugs, and exciting that the authors have found novel benzodiazepine compounds that appear to lack sedative side effects.

I am not an expert in the area of protein-ligand docking so do not consider myself qualified to assess that section.

I recommend that this paper be accepted for publication, as long as the authors address several points:

- Figure 1: in figure 1 the authors show the GABA_A_R class is absent from Schmidtea, Echinococcus and Schistosoma. Given that there are many more flatworm species available in WormBase ParaSite, would it be possible for the authors to screen those for homologs too, to be absolutely sure these genes are not present in some trematodes?

- Figure 2: In figure 2 the authors show 180 benzodiazepines that they screened. It's not clear to me how these compounds were chosen, are they just all benzodiazepines that the authors could get their hands on, or was there some criteria for selecting the compounds? Please could the authors clarify this.

- Figure 4: the authors show what happens with MCLZ at 5 microM but Xhe-II-048 at 10 microM. I may have missed it, but is there no effect with Xhe-II-048 at 5 microM? Also, I am wondering have the authors tested Xhe-II-048 on other species such as S. haematobium and tapeworms to see if there is an effect on tegument of other flatworm species, which might suggest it would act as a drug for those species too?

- Discussion: It would be of great interest to find a drug that can be used for all the different species of schistosome, for example, for Schistosoma haematobium and S. japonicum also. Have the authors tested or considered testing the imidazobenzodiazepines against other schistosome species (and even other flatworm parasites like Fasciola and Echinococcus)? I presume that this will be a next step for the authors, in addition to tests on different stages of schistosomes (somules, juveniles), and in vivo studies with these drugs in mice? I would like the authors to add some discussion on what they consider the most important next step to develop these compounds as new drugs.

--- Avril Coghlan

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Avril Coghlan

Dear Dr. Chan,

We are pleased to inform you that your manuscript, "Non-sedating benzodiazepines cause paralysis and tissue damage in the parasitic blood fluke Schistosoma mansoni", has been editorially accepted for publication at PLOS Neglected Tropical Diseases.

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Best regards,

Maria Victoria Periago

Deputy Editor

PLOS Neglected Tropical Diseases

Aaron Jex

Deputy Editor

PLOS Neglected Tropical Diseases

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