Dear Dr. Zhu,

Thank you very much for submitting your manuscript "On-target inhibition of Cryptosporidium parvum by nitazoxanide (NTZ) and paclitaxel (PTX) validated using a novel MDR1-transgenic host cell model and algorithms to quantify on/off-target rates" 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. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Reviewer's Comments:

1. The authors developed a novel anti-cryptosporidial drug model in vitro. How to evaluate the efficacy of these drugs anti-cryptosporidial effects (kill Cryptosporidium)? The methods in this manuscript was limited, which could not effectively evaluate the anti-cryptosporidial effects. So, these results and limited should be describe in discussion part.

2. In the cytotoxicity test, the biological function changes of HCT-8 cells should be added.

3. The function of MDR1 and it’s function in Cryptosporidium infection should be described in background.

4. According to the methods and the results in this study, the authors validated that NTZ killed C. parvum by solely acting on the parasite target. There was limited.

5. Line 3: Use of the terms on-target and off-target are confusing here, and not aligned with their standard use in pharmacology. “On-target” typically refers to action on the target of the therapeutic effect and “off-target” usually refers to a toxic effect. Instead, consider “parasite-target” and “host-target”.

6. Line 3: Use of the term “rates” in the title (“on/off-target rates”) is confusing, as this term typically refers to something happening over time. It also evokes an “off-rate” in a molecular binding, which is not at all what is being referred to here. I suspect that what the authors mean is “ratio”, but I suggest using the term “effect”, as in the Short Title.

7. Lines 95-100: I suggest an additional comment here about why it is important to understand MOA. For example, better understanding of MOA can enable optimization of drugs, reduction in toxic effects, and identification of new leads with the same or similar MOA.

8. Line 134: A critical hypothesis that is not at all addressed in this manuscript is that NTZ’s broad spectrum antimicrobial activity (reported against bacteria, viruses, and parasites) is due to stimulation of the host immune system, rather than a direct effect on pathogens (Jaseonsky et al. 2019 iScience DOI: 10.1016/j.isci.2019.07.003; Dang et al. 2018 Antimicrob Agents Chemother DOI: 10.1128/AAC.00707-18; Elazar et al. 2009 Gastroenterol DOI: 10.1053/j.gastro.2009.07.056; etc.). While this hypothesis does not exclude the possibility that NTZ may have direct effects on Cryptosporidium and/or direct effects on infected host cells that inhibit parasite growth, it should be acknowledged that this HCT-8/MDR1-transgenic model does not enable investigation of NTZ effects on immune response.

9. Line 155: drugs are “approved” or “registered” by the US FDA, only vaccines are “licensed”

10. Line 184: “…develop drug resistance more rapidly…”? According to Table S2, all lines were selected for the same duration. Instead consider, “…develop greater drug resistance…”

11. Lines 209-210: If there is no increase in MDR1 protein expression by IFA, what is the basis of increased PXT or NTZ tolerance in selected cells?

12. Line 360: Implying the ratio of TC50/EC50 is a major determinant of clinical drug safety could be considered miseading. Dose-limiting drug toxicity is usually not driven by cytotoxicity directly, but instead by toxic effects on other tissues (heart/QT, liver/metabolic pathways, etc.). Consider using the term “in vitro cytotoxicity index” rather than “safety interval”.

13. Lines 431-441: As mentioned previously above, there should be some discussion of the potential impact of NTZ on immune modulation.

14 Line 539: Method of quantitation of western blots (phosphoimager?) in Figs 2C and 2F should be described. The bands on the westerns in Figs 2B and 2E look more than 1.5x darker/bigger.

15. Line 44: “…newly hits/leads” should be modified to “new hits/leads” or “newly identified hits/leads”.

16. Lines 70-71: For “…big weight loss”, consider instead “significant” or “substantial”.

17. Lines 83-84: …a parasite target…a host cell target… (indefinite articles are missing for both).

18. Line 132: inhibition (not “inhibitions”)

19. Line 134: delete “for” in “This is for the first time…”

20 Line 139: allow (not “allows”)

21. Line 143: promoters

22. Line 154: It is unnecessary to spell out name of compound/assay in Results. I suggest describing this detail only in the Materials and Methods section.

23. Figure 2A: pCMV, not “pMCV”

24. Figure 7A: “inhibior” is misspelled

25. Figure 11B: “efficay” is misspelled

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

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

[1] A letter containing a detailed list of your responses to the 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.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. 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,

Hamed Kalani

Academic Editor

PLOS Neglected Tropical Diseases

S Madison-Antenucci

Section Editor

PLOS Neglected Tropical Diseases

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Dear Dr. Zhu,

We are pleased to inform you that your manuscript 'On-target inhibition of Cryptosporidium parvum by nitazoxanide (NTZ) and paclitaxel (PTX) validated using a novel MDR1-transgenic host cell model and algorithms to quantify the effect on the parasite target' 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,

Hamed Kalani

Academic Editor

PLOS Neglected Tropical Diseases

Susan Madison-Antenucci

Academic Editor

PLOS Neglected Tropical Diseases

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