Tanshinone functions as a coenzyme that confers gain of function of NQO1 to suppress ferroptosis

NQO1 catalyzes the reduction of tanshinones to detoxify lipid peroxyl radicals and inhibit ferroptosis both in vitro and in vivo, providing a molecular mechanism for the therapeutic effects of tanshinones, such as cardiac protection.

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Thank you for this interesting contribution to Life Science Alliance. We are looking forward to receiving your revised manuscript. --High-resolution figure, supplementary figure and video files uploaded as individual files: See our detailed guidelines for preparing your production-ready images, https://www.life-science-alliance.org/authors --Summary blurb (enter in submission system): A short text summarizing in a single sentence the study (max. 200 characters including spaces). This text is used in conjunction with the titles of papers, hence should be informative and complementary to the title and running title. It should describe the context and significance of the findings for a general readership; it should be written in the present tense and refer to the work in the third person. Author names should not be mentioned.
--By submitting a revision, you attest that you are aware of our payment policies found here: https://www.life-sciencealliance.org/copyright-license-fee B. MANUSCRIPT ORGANIZATION AND FORMATTING: Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** Reviewer #1 (Comments to the Authors (Required)): This manuscript by Wang et al describes the isolation of tanshinones as natural products that can inhibit ferroptosis, a form of non-apoptotic cell death, in combination with the enzyme NQO1. Results from both cell-based and animal models are presented and together make a compelling argument in favor the molecular model, which in turn may explain the purported therapeutic activity of tanshinones. This is a straightforward paper with interesting results and conclusions. My comments are mostly minor: -In the Discussion the authors claim that the tanishones are the most potent ferroptosis inhibitors known to date. This seems like a bold assertion in the absence of direct comparative studies, with many other molecules being reported to have low nanomolar potency against ferroptosis (e.g., see the work of Derek Pratt, e.g., PMID: 35921655). It may be safer to simply note that these are among the most potent. -Some experiments (e.g., Figure 3C) appear to have been performed only a single time, and in other cases it is not clear whether the data are derived from experiments performed on different days or merely technical replicate wells from one experiment.
-Molecular weight markers should be added to all western blots in Figure 5.
-Line 46 and elsewhere. No need for lipid ROS to be hyphenated.
-Line 83. RTA is radical trapping antioxidant, not radical trapping agent.
-Line 199. Not clear why the Yang et al., 2014 paper is cited in connection with I/R injury. That paper appears to deal exclusively with cancer.
-The text needs some editing for language throughout. Line 64, for example, should probably read "Taking advantage..." not "Take advantage...". This is just one of many examples.

Reviewer #2 (Comments to the Authors (Required)):
In the present manuscript, Wang et al provide a very careful characterization of the mode of action of Tanshinone and its derivatives in preventing ferroptosis. The authors demonstrate that Tanshinone are extremely potent ferroptosis inhibitors both in vivo and in vitro. They proceed to show using a series of assays that the quinone oxidoreductase (NQO1) is required to reduce Tanshinone to its radical trapping-active metabolite. The model proposed are validated using a combination of biochemical assays and validated using NQO1 knockout cell lines and mice. The conclusion are sounds and of general interest. The work is of high quality and the data well presented. I only apologize the authors and the editors for the delay in reviewing this piece mostly because I have nothing to add to this very nice study.

José Pedro Friedmann Angeli
Reviewer Comments: Reviewer #1 (Comments to the Authors (Required)): This manuscript by Wang et al describes the isolation of tanshinones as natural products that can inhibit ferroptosis, a form of non-apoptotic cell death, in combination with the enzyme NQO1. Results from both cell-based and animal models are presented and together make a compelling argument in favor the molecular model, which in turn may explain the purported therapeutic activity of tanshinones. This is a straightforward paper with interesting results and conclusions. My comments are mostly minor:

Response:
We appreciate the reviewer's positive view and constructive comments to improve the quality of this manuscript.

Major comments:
(1) -In the Discussion the authors claim that the tanshinones are the most potent ferroptosis inhibitors known to date. This seems like a bold assertion in the absence of direct comparative studies, with many other molecules being reported to have low nanomolar potency against ferroptosis (e.g., see the work of Derek Pratt, e.g., PMID: 35921655). It may be safer to simply note that these are among the most potent.

Response:
We have revised related statement to "The low nanomolar EC50 of tanshinone in suppressing ferroptosis makes it among the most potent ferroptosis inhibitors known to date". (3) -Some experiments (e.g., Figure 3C) appear to have been performed only a single time, and in other cases it is not clear whether the data are derived from experiments performed on different days or merely technical replicate wells from one experiment.

Response:
As for the concentration-dependent compounds treatment experiments like Figure 3C and other EC50 values measurement experiments, data are presented as mean ± S.D., n = 3 independent repeats. EC50 values were calculated by nonlinear regression [Log(agonist) vs. response-Variable slope (four parameters)]. We have stated this clearly in the figure legends.
(4) -Molecular weight markers should be added to all western blots in Figure 5.

Response:
The molecular weight markers for all western blots have been added in Figure 5.
(5) -Line 46 and elsewhere. No need for lipid ROS to be hyphenated.

Response:
The hyphen between lipid and ROS are deleted.

Response:
We have revised this inaccurate description in the manuscript. We thank the Reviewer for pointing out our mistake and corrected this in our revised manuscript.
(7) -Line 199. Not clear why the Yang et al., 2014 paper is cited in connection with I/R injury. That paper appears to deal exclusively with cancer.
Response: This was a mistake. We have replaced the citation of Yang et al., 2014with Friedmann Angeli et al., 2014and Gao et al., 2015 (8) -The text needs some editing for language throughout. Line 64, for example, should probably read "Taking advantage..." not "Take advantage...". This is just one of many examples. Response: We apologize for the language problem. We have carefully proofread the manuscript to minimize typographical, grammatical, and bibliographical errors and have sent the manuscript to AJE company to polish the language.

Response:
Reviewer #2 (Comments to the Authors (Required)): In the present manuscript, Wang et al provide a very careful characterization of the mode of action of Tanshinone and its derivatives in preventing ferroptosis. The authors demonstrate that Tanshinone are extremely potent ferroptosis inhibitors both in vivo and in vitro. They proceed to show using a series of assays that the quinone oxidoreductase (NQO1) is required to reduce Tanshinone to its radical trapping-active metabolite. The model proposed are validated using a combination of biochemical assays and validated using NQO1 knockout cell lines and mice. The conclusion are sounds and of general interest. The work is of high quality and the data well presented. I only apologize the authors and the editors for the delay in reviewing this piece mostly because I have nothing to add to this very nice study.

Response:
We appreciate the Reviewer for the general enthusiasm and positive evaluation of our manuscript. Thank you for submitting your revised manuscript entitled "Tanshinone functions as a coenzyme that confers gain-of-function of NQO1 to suppress ferroptosis". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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