PONE-D-20-13905

Drug targeting CYP2E1 for the treatment of ASH

PLOS ONE

Dear Dr. Diesinger,

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Pavel Strnad

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Dear Dr. Diesinger and colleagues,

This abstract discusses the manuscript “Drug targeting CYP2E1 for the treatment of ASH” for publication in PLOS ONE. The authors describe the generation of small compounds and their possible use in the treatment of ASH, targeting the key cytochrome CYP2E1. Scope of the manuscript appears to the evaluation of the compounds in a cell culture and in vivo model. In general the study appears technically sound, structured, well written and of general interest to the scientific field. Nevertheless, a couple of inconsistencies have to be addressed and we would herewith encourage the authors to revise those points before publication:

Figure2: In case there is no reference standard available, it might allow easier comparison if the values are normalized to control HepG2 instead of plotting Fluorescence Intensity units, as those might differ between equipment/ lab. Please add corresponding data for I-an and UDCA in cell lines. Did you perform Pharmacokinetic studies in vivo? As reference for the reader, how does the drug concentration in cell culture correlate to serum/ in vivo concentrations?

Figure 5: ALT/ AST values appear very low compared to literature, please check if the shown measurements are correct.

Figure 6: Is the total magnification 100x? Please provide pictures with a comparable background (white) correction. Also for histological evidence of steatosis/ fibrosis we advise to perform an Oil red O/ Sirius red staining which would be quantifiable.

Supplementary Table 6: The control value provided for TNF appears beneath those stated in other literature. In addition, the standard curve for the ELISA used for TNF evaluation appears to have limit of 100pg/µl. Please show the standard curve for this experiment.

To add to the functional assays performed in this study it appears reasonable to provide some gene expression/ western blot data regarding the downstream effects of the drug on CYP2E1.

HepG2 cell line: Please provide further information regarding HepG2 E47 in a supplement, e.g. PCR/ WB showing the successful transfection.

As a general remark, we understand the experimental setup of administering the drug daily alongside the LDC diet. Nevertheless, treating an organism while inducing ASH is hardly translatable to the human situation. Did you do perform any experiment where disease progression was ongoing for some time before the treatment was started? Could you speculate on how this would influence the efficacy of the treatment e.g. stop disease progression/ reverse steatosis … ?

In case those minor points are addressed in a minor revision, we believe the manuscript provides a sound basis for the further evaluation of ASH therapeutics.

Reviewer #2: The present manuscript (PONE-D-20-13905) by Diesinger et al. entitled “Drug targeting CYP2E1 for the treatment of ASH” focused on developing new small molecule inhibitors of Cyp2e1 enzymatic activity that could be utilized for treatment of alcoholic steatohepatitis (ASH). The authors first designed novel CYP2E1 inhibitors using rational drug design. Then, they tested two promising compounds in vitro and in vivo using a rat, Lieber-DeCarli diet-based model of alcoholic liver disease (ALD). Concomitant administration of the tested drug along with alcohol diet improved liver injury and steatosis. Overall, major findings are supported by the data. Formally, the manuscript is little hard to read and follow. Improvements would be helpful especially in the results section which read way too much as a list of change percentages and does not educate the reader about the data or their meaning. Figures are not well labeled and thus not informative unless one reads the text, which makes it harder to read and understand. I have several comments that can further improve this manuscript and are listed below:

Major comments:

1. The title of the article as well as some parts of the paper that relate to ASH are imprecise. The authors present data to support the effect of their drugs in alcohol-induced liver injury and steatosis, but not steatohepatitis. None of the markers of liver inflammation were assessed. Most importantly, the model the author used is rather a model of early stages of ALD.

2. One of my major concerns is why the authors chose to test the potentials drug in a preventive treatment dosing as opposed to using the drugs for reversing an established ALD. This would be more important information that is relevant to clinical practice. In my opinion, this is a major limitation of the present study.

3. What were the bodyweight changes during the treatment study?

4. What was the final liver weight and liver-to-body weight ratio?

5. Was diet intake measured and caloric intake the same? These data should be included in the manuscript.

6. Figures are not easy to understand and follow. To make figures more intuitive, the authors can label graphs and images with more details (e.g., parameters assessed, use graph titles, treatment, legends, etc.)

Minor comments:

1. Any abbreviation, such as ASH, should be avoided in the title to improve understanding of the general audience. But as noted above, ASH should not even be mentioned as a major focus of the paper.

2. Replace “steatosis hepatitis” with appropriate terms – page 3, twice.

3. In the introduction, liver enzymes are not spelled out to introduce the abbreviation. Also, the abbreviations are not the most commonly used ones in the liver literature.

4. Actually, at multiple places of the manuscript several abbreviations are not introduced. Too many abbreviations make the manuscript less well readable, and especially if the abbreviations are quite specific to the field, e.g. chemistry vs. hepatology.

5. Many references throughout the manuscript were not inserted; see words ‘Error” in the text.

6. Listing % values in figure legend is not very informative and should be omitted.

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Reviewer #1: No

Reviewer #2: No

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Drug targeting CYP2E1 for the treatment of early-stage alcoholic steatohepatitis

PONE-D-20-13905R1

Dear Dr. Diesinger,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Pavel Strnad

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: We feel the Quality of the manuscript has greatly improved with the added Information in the figures. We would have liked to see further Experiments on the subject, but still believe the data included is sufficient for publication.

Reviewer #2: The authors have adequately addressed my comments and concerns, and made appropriate changes to the manuscript.

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7. 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