Dear Dr. Singh:

Thank you very much for submitting your manuscript "Identification of anisomycin, prodigiosin and obatoclax as compounds with broad-spectrum anti-parasitic activity" (PNTD-D-19-01940) 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

Alvaro Acosta-Serrano

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: Yes to all. The goal here was to test four repurposed drugs that are effective versus Entamoeba in vitro versus other pathogenic protists. In each case the investigators, which are experts in each organism tested and from numerous universities, use assays that allow them to determine the EC50 for each compound.

Reviewer #2: A previous screen of the ReFrame compound library against Entamoeba histolytica led to the discovery of 4 compounds, Prodigiosin, Obatoclax, Anisomycin, and Nithiamide, that have previously been in human trials or use. The authors report on advanced testing of these 4 compounds on advanced tests for E. histolytica bioactivity and to investigate a panel of other parasites including pathogenic free-living amoeba, cryptosporidium, plasmodium, and Schistosoma mansoni. The study design is appropriate to shed light on the broad activity of some of these compounds. I had no concerns about the methods

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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: Yes to all. The results are clearly presented in tables, figures, and text. While the results vary from drug to drug and organism to organism, enough of them are promising to make the study of interest and enough results are negative to assure that investigators were honest.

Reviewer #2: The results are clearly presented and well written. The results show they have much promise, in vitro, for rapid action against E. histolytica, though somewhat disappointing in the cidal washout experiment. Some of the drugs have activity against other of the panel of parasites tested, though the only in vivo study was a negative study with Prodigiosin in C. parvum infection of NOD/SCID/gammaKO mice. Despite this negative study, there is abundant information to justify rapid publication of the results.

The figures are of good quality, except in the figure legend 4, it states we should see dots representing the values of the 4 animals tested but I can only see a very few dots.

On Page 22 of the PDF, line 309, there is a reference to Table S1, but the Table they are referring to in line 309 should actually be Table 1. Furthermore Table S1 should be moved down towards the end of the paper where it is discussed.

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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: yes to all. Their general hypothesis that there are repurposable drugs out there that might be tested versus pathogenic protists was confirmed. With the exception of one negative mouse study of Cryptosporidium, none of the drugs were tested in animal models, and no mechanism was investigated. Despite these limitations, the study has impact based upon the breadth of organisms studied.

Reviewer #2: The conclusions are reasonably well supported by the data but could be improved by my suggestions below. The relevance to advancing the field and therapies are discussed as well as (indirectly) the public health relevance.

1) I feel the paper's findings' applicability and promise would be way easier to evaluate if the authors made a comparative table of the 4 drugs, especially including their pharmacokinetic (PK) parameters. The PK should include, where ever available: plasma peak (Cmax) and exposure (AUC) information after various doses when give by oral (and IV) forms. This could be only the human data or better yet, include major efficacy species, such as mice. The plasma protein binding of each drug would be useful information as well as oral bioavailability and CNS penetration. Some of this data is (nonquantitatively) mentioned in passing, and is probably available in the public domain. Then the discussion could be expanded into whether higher or lower doses that have previously been given to humans would likely be necessary, or even whether intrathecal delivery for the amoebic encephalitis cases might be necessary.

2) The above data might further inform the negative study on C. parvum and expand the discussion here. For instance is prodigiosin so well absorbed that it might not be delivered to the terminal ileum/proximal colon where the C. parvum infection is highest.

3) The host cell toxicity data is only shown for one mammalian cell line, HepG2, and is certainly alarming for Anisomycin, Prodigiosin, and Obatoclax where the CC50 values for HepG2 are well below what is considered efficacious for many parasites. The authors don't discuss this data, but I feel they should. Perhaps that data is mitigated by the sheer fact that these therapies have been in humans already, but it's all relative to the levels achieved in vivo and the serum protein binding of the compounds (see #1 above).

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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: in the abstract line 54 and discussion line 424 they should be careful to say that there are no treatments for brain infection with Acanthamoeba, although there are available topical treatments for eye infections.

Reviewer #2: See above, minor revisions

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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: well done study by a large group of experts.

Reviewer #2: Outstanding paper and only minor revisions are needed

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Reviewer #1: Yes: John Samuelson

Reviewer #2: Yes: Wes Van Voorhis

Dear Dr. Singh,

We are pleased to inform you that your manuscript 'Identification of anisomycin, prodigiosin and obatoclax as compounds with broad-spectrum anti-parasitic activity' 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 within two working days 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.

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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,

Alvaro Acosta-Serrano

Deputy Editor

PLOS Neglected Tropical Diseases

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