Dear Prof. Tilley,

Thank you very much for submitting your manuscript "A potent and selective reaction hijacking inhibitor of Plasmodium falciparum tyrosine tRNA synthetase exhibits single dose oral efficacy in vivo" for consideration at PLOS Pathogens. 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,

Audrey Ragan Odom John, MD, PhD

Academic Editor

PLOS Pathogens

Tracey Lamb

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: In the manuscript titled “A potent and selective reaction hijacking inhibitor of Plasmodium falciparum tyrosine tRNA synthetase exhibits single dose oral efficacy in vivo,” Xie et al explore ML901 derivatives to identify a candidate with improved selectivity for future antimalarial development. This led to the discovery of ML471, which exhibits activity against the P. falciparum asexual blood stage, sexual stage and liver stage. Activity against clinical isolates (falciparum and vivax) as well as drug resistant P. falciparum was confirmed. The possibility that ML901 exhibits cytotoxicity due to human E1 targeting was explored and ML471 was found to have decreased targeting of human UAE. ML471 was tested in an in vivo mouse malaria model where it had single dose oral efficacy against P. falciparum and rapid killing, meeting current MMV recommendations for new candidate selection criteria. Though it is acknowledged that it does not meet goals for reduced propensity of resistance. Lastly, structural studies were completed that provide insights into the mechanism of selectivity. The experiments presented are robust, and the authors nicely frame their findings in the context of previously published work and the current state of targeting tRNA synthetases. This impactful and timely manuscript will be of broad interest to the pathogen and drug development communities.

Reviewer #2: The manuscript is very well written and serves to be a very good example of the strength of collaborative work. The authors have indeed done a commendable amount of work and have very clearly justified every aspect of the compound testing that they performed. All of their claims are very well justified with the experimental results they have. The significance of this work can be observed by virtue of the establishment of a whole “pipeline” to effectively scrutinize multiple aspects of a compound in drugging the malarial parasite. This work, with its immense width-and-depth analyses positions itself to form a basis of future works utilizing the concept of reaction-hijacking prodrugs in more use-cases. The novelty of chemical classes being discovered and synthesized to have desirable bioactive profiles promises to continue to be an evergreen reiterative process to push the limits of molecular medicine.

**********

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: Figure 4A, could a control (no drug) trace be added to demonstrate no peak is observed in parasites without compound addition.

- The magnitude of a Tm shift (driven by thermodynamics) is not proportional with ligand binding affinity (pg 10, line 248). Perhaps reword this to discuss a more stable conformational change induced by the small molecule. Alternatively, ITC or MST could be used to measure the Kd. It is also recommended to remove this suggestion (that enhanced thermal stability indicated tighter binding) in line 337.

Reviewer #2: No major issues detected in the manuscript.

**********

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: - Is there a structural rationale for the 8 analogs tested?

-Can the authors speculate on the loss of efficacy observed between the sexual and asexual stage? Also, has the activity of ML471 or ML901 on parasite invasion been investigated?

- pg 7, line 107 could the notation be added to indicate it was at 72 hr for the IC50.

- On pg 9, line 210 it is stated that dosing led to “no evidence of toxicity.” Could the assessment for toxicity be clarified?

-Could Table S9 include previously published metrics for compounds that have favorable Minimum Inoculum for Resistance values for comparison?

- These compounds bind/modify the protein in the nM range but inhibit ATP consumption in the uM range. Could a rational for this be included?

- Conventionally prodrugs (or proinhibitors) require modification by cytochrome P450s, or other enzymes, to yield active molecules. Here, it seems pro-inhibitor is indicating the molecule pre modification of the target? To the best of my knowledge, this terminology has not been used for covalent kinase inhibitors.

Reviewer #2: Line 218 – For the in vitro resistance evolution experiment, what was the rationale for the authors to use two differential methods of inducing resistance?

Line 587 – Please mention the ratio of “Enzyme: Compound” for both the enzymes tested and the associated rationale of performing the assay at different ratios for the Pf and Hs enzymes. Currently only the PfTyrRS parameters are mentioned.

Line 761 – Please provide a full legend for the Table contents as to what is each column value depicting.

Line 766 – Please provide a full legend for the Table contents as to what is each column value depicting.

Line 772 – Please provide a full legend for the Table contents as to what is each column value depicting. Please also move the description of the assay parameters to the DSF section in the methods. Possible to add a table in the supplementary tables mentioning the raw melt temperatures by virtue of which the delta Tms have been calculated?

Please add all the relevant graphs in the supplementary figures for the supplementary tables S1, S2, S3, S4 and S5.

Please refer to every graph, figure or table wherever a related point is referred to in the text. In some cases, it might get difficult for a reader to connect two points which might be obvious to specialists in the field.

**********

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

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.

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, we recommend that you deposit your 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Prof. Tilley,

We are pleased to inform you that your manuscript 'A potent and selective reaction hijacking inhibitor of Plasmodium falciparum tyrosine tRNA synthetase exhibits single dose oral efficacy in vivo' has been provisionally accepted for publication in PLOS Pathogens.

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 Pathogens.

Best regards,

Audrey Ragan John, MD, PHD

Academic Editor

PLOS Pathogens

Tracey Lamb

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Reviewer Comments (if any, and for reference):