From the journal RSC Chemical Biology Peer review history

04-Apr-2024

Dear Dr Zhang:

Manuscript ID: CB-ART-03-2024-000062
TITLE: Natural triterpenoids aided identification of the druggable interface of HMGB1-TLR4 protein-protein interaction

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript when the comments raised by the reviewers are addressed.

Please revise your manuscript to fully address the reviewers’ comments. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

Please submit your revised manuscript as soon as possible using this link :

*** PLEASE NOTE: This is a two-step process. After clicking on the link, you will be directed to a webpage to confirm. ***

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You should submit your revised manuscript as soon as possible; please note you will receive a series of automatic reminders. If your revisions will take a significant length of time, please contact me. If I do not hear from you, I may withdraw your manuscript from consideration and you will have to resubmit. Any resubmission will receive a new submission date.

All RSC Chemical Biology articles are published under an open access model, and the appropriate article processing charge (APC) will apply. Details of the APC and discounted rates can be found at https://www.rsc.org/journals-books-databases/about-journals/rsc-chemical-biology/#CB-charges.

RSC Chemical Biology strongly encourages authors of research articles to include an ‘Author contributions’ section in their manuscript, for publication in the final article. This should appear immediately above the ‘Conflict of interest’ and ‘Acknowledgement’ sections. I strongly recommend you use CRediT (the Contributor Roles Taxonomy, https://credit.niso.org/) for standardised contribution descriptions. All authors should have agreed to their individual contributions ahead of submission and these should accurately reflect contributions to the work. Please refer to our general author guidelines https://www.rsc.org/journals-books-databases/author-and-reviewer-hub/authors-information/responsibilities/ for more information.

The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.

Please also encourage your co-authors to sign up for their own ORCID account and associate it with their account on our manuscript submission system. For further information see: https://www.rsc.org/journals-books-databases/journal-authors-reviewers/processes-policies/#attribution-id

Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Dr Sander van Kasteren
Professor Molecular Immunology
Leiden University
Associate Editor of RSC Chemical Biology

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

Reviewer 1

The paper by Zhang and co-workers utilizes a natural triterpenoid probe to characterize the interface of the HMGB1-TLR4 interaction, providing interesting immunological and structural insights into this signaling axis, with potential implications for future inhibitor design.

The study is original and potentially impactful with conclusions that seem to be supported through a variety of well-designed and complementary experimental approaches. Nonetheless, the paper would benefit from further proof-reading for improved clarity. Moreover, the present version and formatting (with the figures and captions separated from the text) does not help the reader.

There are some comments that could be addressed before acceptance of the manuscript.
Some of the computational approaches and the results regarding prediction of the HMGB1 interface as well as the associated conformational changes upon interaction could be better supported using experimental NMR studies. It would be important to perform some of these experiments to confirm the calculated data.

It would also be interesting to include future perspectives of further in vivo studies in mice that could validate these initial results.

On a separate note, I was wondering if the authors have information on the potential effect that subtle structural variations in the oleanane-type triterpene (i.e. C4 and C16-oxidation, hydroxylation) could have in PT-22 and its binding to HMGB1 and disruption of the protein-protein interaction. For instance, following Figs S1 and S3, I was curious to know if the authors evaluated the binding effect of PT-21 (C4 R1=CHO) vs PT-23 (R1=CH3) as well as PT-27 (R2=H as in PT-22) versus R2=OH as in PT-68.

Finally, this manuscript will also be improved if the authors could elaborate further the results in a better described discussion. With the suggested comments, the article will be suitable for publication in RSC Chem Bio without any major revision.

Reviewer 2

The authors report the identification of key high-mobility group box 1 (HMGB1) residues involved in the HMGB1-TLR4 binding interface using the modified triterpene PT-22 as a chemical proximity probe. The manuscript combines the methods of structural biology, immunology and chemistry and is well suited for publication in a chemical biology journal. Using photoaffinity labelling to cross-link a chemical probe (triterpene PT-22) that has high affinity for HMGB1 and disrupts the HMGB1/TLR4 interaction, the authors were able to identify K114 on the "L-shaped" B-box as the key residue involved in the protein-protein interaction. The manuscript provides insight into the HMGB1-TLR4 interaction and offers solutions for specific inhibition of the protein-protein interaction with small molecules.
The limitation of the study is the use of a 'sterile' inflammation condition in which LPS (lipopolysaccharide ) was excluded from all in vitro activation and binding experiments. HMGB1 is known to bind LPS directly, has a high affinity for LPS and is thought to act as a delivery vehicle to transport LPS from the extracellular space to the cytoplasm via RAGE-mediated endocytosis. Bacterial LPS-induced TLR4 and caspase-11 signaling mediates the release and accumulation of HMGB1 in hepatocytes, and caspase-11-mediated activation of gasdermin D leads to active HMGB1 release from hepatocytes.
The authors report that their key molecule PT-22 does not interfere with the LPS-TLR4 axis, but can inhibit the pro-inflammatory signaling induced by HMGB1 in vitro in a TLR4-dependent manner. Why didn't the authors combine LPS and HMGB1 in the in vitro studies? Simultaneous exposure to LPS and HMGB1 would mimic a more natural situation and could provide a deeper insight into the possible interplay between LPS, HMGB1, TLR4/MD-2 and a triterpene molecule PT-22. Would PT-22 still inhibit HMGB1/TLR4-mediated signaling in the presence of LPS or not?
Next, for the molecular dynamics simulation studies, the authors used the TLR4 ectodomain from PDB 3FXI (a dimeric human [TLR4/MD-2/LPS]2 complex), which is not consistent with a model of 'sterile' inflammation (in the absence of LPS). TLR4/MD-2 is a flexible protein that changes conformation upon LPS binding. The authors are strongly recommended to use other X-ray structures of the TLR4/MD-2 complex without bound ligand (e.g. PDB 5IJB or 2Z64), which are more consistent with a 'sterile' LPS-free inflammation model. Second, a co-receptor protein MD-2 is physically associated with TLR4, but it appears that MD-2 was not included in the studies...
Could the authors also indicate whether murine or human TLR4 ectodomain was used? There are pronounced species-specific differences between m- and hTLR4/MD-2 complexes, and a correlation with cellular studies performed in a mouse cell line (RAW 264.7 macrophage cell lines) should also be considered.
The authors do not specify any TLR4 regions that might be involved in HMGB1/TLR4 binding; could the authors comment on this?

Dear Editor,
We deeply appreciate these helpful reviews from you and the reviewers about our manuscript entitled “Natural triterpenoids aided identification of the druggable interface of HMGB1-TLR4 protein-protein interaction” (Manuscript ID: CB-ART-03-2024-000062). We have carefully read the comments and made the corrections point-by-point, also marked in red. The following are the replies to these questions. We hope these responses will be satisfactory and the revised manuscript will be suitable for publication.
Thank you again for your kind help and time!

Yours sincerely,

Jian Zhang, Ph.D.,
State Key Laboratory of Natural Medicines,
China Pharmaceutical University,
24# Tong Jia Xiang Street, Nan Jing, 210009, China
E-mail: wilsonzhang99@cpu.edu.cn
Tel: 86-25-86185157
Fax: 86-25-8618518


Point-to-point responses to the comments
Comments from the Editorial Office to the Author
1.RSC Chemical Biology strongly encourages authors of research articles to include an ‘Author contributions’ section in their manuscript, for publication in the final article. This should appear immediately above the ‘Conflict of interest’ and ‘Acknowledgement’ sections.
Response 1. Thanks. The Author contributions section has been added to the revised manuscript and marked in red.

Reviewer reports:
Referee: 1
Comments to the Author
The paper by Zhang and co-workers utilizes a natural triterpenoid probe to characterize the interface of the HMGB1-TLR4 interaction, providing interesting immunological and structural insights into this signaling axis, with potential implications for future inhibitor design.

1.The study is original and potentially impactful with conclusions that seem to be supported through a variety of well-designed and complementary experimental approaches. Nonetheless, the paper would benefit from further proof-reading for improved clarity. Moreover, the present version and formatting (with the figures and captions separated from the text) does not help the reader.
Response 1. We apologize for any confusion caused. The description has been polished. The layout and formatting of the text have been re-adjusted to make it easier for readers to read.

2.There are some comments that could be addressed before acceptance of the manuscript.
Some of the computational approaches and the results regarding prediction of the HMGB1 interface as well as the associated conformational changes upon interaction could be better supported using experimental NMR studies. It would be important to perform some of these experiments to confirm the calculated data.
Response 2. Thank you for your suggestion. We are designing experiments related to the expression and purification of key domains of isotopically labeled HMGB1 (A-box and B-box). In the follow-up study, the interaction between HMGB1 and probe molecules was further verified using NMR. We hope to continue to publish the research results in this journal.

3.It would also be interesting to include future perspectives of further in vivo studies in mice that could validate these initial results.
Response 3. We are grateful for your helpful advice. This article mainly focuses on the effect of direct binding of pentacyclic triterpenes to HMGB1 on HMGB1-TLR4 protein-protein interaction at the molecular and cellular levels, thus discovering the binding interface of HMGB1-TLR4. Structural optimization of the probe molecules and further in vivo activity evaluation are ongoing.

4.On a separate note, I was wondering if the authors have information on the potential effect that subtle structural variations in the oleanane-type triterpene (i.e. C4 and C16-oxidation, hydroxylation) could have in PT-22 and its binding to HMGB1 and disruption of the protein-protein interaction. For instance, following Figs S1 and S3, I was curious to know if the authors evaluated the binding effect of PT-21 (C4 R1=CHO) vs PT-23 (R1=CH3) as well as PT-27 (R2=H as in PT-22) versus R2=OH as in PT-68.
Response 4. We apologize for not addressing the query properly. Due to the weak inhibitory activity against the HMGB1-induced inflammatory response, the binding affinity of PT-21, PT-23, PT-27 and PT-68 with HMGB1 was not detected. PT-25 served as a negative control, the conformational isomer of PT-22, which has a weak binding ability to HMGB1. Accordingly, there is little inhibitory effect of PT-25 on the cytokine activity of HMGB1. The triterpene's structural variations and their impact on its binding to HMGB1 and protein-protein interaction disruption need further exploration.

5.Finally, this manuscript will also be improved if the authors could elaborate further the results in a better described discussion. With the suggested comments, the article will be suitable for publication in RSC Chem Bio without any major revision.
Response 5. Thank you, we deeply appreciate your wise suggestions and helpful comments.

Referee: 2
Comments to the Author
The authors report the identification of key high-mobility group box 1 (HMGB1) residues involved in the HMGB1-TLR4 binding interface using the modified triterpene PT-22 as a chemical proximity probe. The manuscript combines the methods of structural biology, immunology and chemistry and is well suited for publication in a chemical biology journal. Using photoaffinity labelling to cross-link a chemical probe (triterpene PT-22) that has high affinity for HMGB1 and disrupts the HMGB1/TLR4 interaction, the authors were able to identify K114 on the "L-shaped" B-box as the key residue involved in the protein-protein interaction. The manuscript provides insight into the HMGB1-TLR4 interaction and offers solutions for specific inhibition of the protein-protein interaction with small molecules.

1.The limitation of the study is the use of a 'sterile' inflammation condition in which LPS (lipopolysaccharide ) was excluded from all in vitro activation and binding experiments. HMGB1 is known to bind LPS directly, has a high affinity for LPS and is thought to act as a delivery vehicle to transport LPS from the extracellular space to the cytoplasm via RAGE-mediated endocytosis. Bacterial LPS-induced TLR4 and caspase-11 signaling mediates the release and accumulation of HMGB1 in hepatocytes, and caspase-11-mediated activation of gasdermin D leads to active HMGB1 release from hepatocytes.
Response 1. Thank you very much for your valuable advice. The study of HMGB1-LPS interaction and the discovery of corresponding inhibitors is a promising research area, and we will consider exploration in the future. Unfortunately, the current studies only focus on the discovery of specific HMGB1-TLR4 inhibitors and their binding interface, while research around HMGB1/LPS-TLR4 is ongoing.

2.The authors report that their key molecule PT-22 does not interfere with the LPS-TLR4 axis, but can inhibit the pro-inflammatory signaling induced by HMGB1 in vitro in a TLR4-dependent manner. Why didn't the authors combine LPS and HMGB1 in the in vitro studies? Simultaneous exposure to LPS and HMGB1 would mimic a more natural situation and could provide a deeper insight into the possible interplay between LPS, HMGB1, TLR4/MD-2 and a triterpene molecule PT-22. Would PT-22 still inhibit HMGB1/TLR4-mediated signaling in the presence of LPS or not?
Response 2.Thank you. The interaction of HMGB1 and LPS has been shown to play a role in various infectious diseases and traumatic injuries. In this study, we aim to find inhibitors that can specifically target HMGB1, which is representative of DAMPs in multiple aseptic inflammatory diseases. Additionally, we will conduct further research to explore the possible interplay between LPS, HMGB1, and TLR4/MD-2.

3.Next, for the molecular dynamics simulation studies, the authors used the TLR4 ectodomain from PDB 3FXI (a dimeric human [TLR4/MD-2/LPS]2 complex), which is not consistent with a model of 'sterile' inflammation (in the absence of LPS). TLR4/MD-2 is a flexible protein that changes conformation upon LPS binding. The authors are strongly recommended to use other X-ray structures of the TLR4/MD-2 complex without bound ligand (e.g. PDB 5IJB or 2Z64), which are more consistent with a 'sterile' LPS-free inflammation model. Second, a co-receptor protein MD-2 is physically associated with TLR4, but it appears that MD-2 was not included in the studies...
Response 3. We appreciate your advice. In the molecular dynamics simulation studies, we selected the TLR4 ectodomain from PDB 3FXI (MD2 and LPS were deleted in our MD studies) to predict the binding interface according to the literature [1]. The X-ray structures of the TLR4/MD-2 complex without bound ligands might be a better option. In follow-up studies, we will conduct MD simulation using PDB 5IJB or 2Z64 and compare them with the current results. In addition, MD2 will be included in future studies.
[1] Sun, S., He, M., VanPatten, S., and Al-Abed, Y. (2019). Mechanistic insights into high mobility group box-1 (HMGb1)-induced Toll-like receptor 4 (TLR4) dimer formation. J Biomol Struct Dyn 37, 3721-3730. 10.1080/07391102.2018.1526712.

4.Could the authors also indicate whether murine or human TLR4 ectodomain was used? There are pronounced species-specific differences between m- and hTLR4/MD-2 complexes, and a correlation with cellular studies performed in a mouse cell line (RAW 264.7 macrophage cell lines) should also be considered.
Response 4. Thank you. The human TLR4 ectodomain used in this study was provided by Sino Biological Inc (10146-H08B) in Beijing, China. The interaction between human TLR4 ectodomain and HMGB1 and the inhibition of small molecules on protein-protein interaction were studied by surface plasmon resonance (SPR) and competitive ELISA at the molecular level. In RAW 264.7 macrophages, we validated the feasibility of the HMGB1/LPS-TLR4 pathway using the TLR4 inhibitor TAK-242. Therefore, there is no problem with species differences in this study.

5.The authors do not specify any TLR4 regions that might be involved in HMGB1/TLR4 binding; could the authors comment on this?
Response 5. Thank you for underlining this deficiency. In this manuscript, we focus on the anti-inflammatory activity of small molecules, through directly binding to HMGB1 and then influencing the HMGB1-TLR4 interaction. In addition, in the SPR experiment, we found that the probe molecule PT-22 had no significant interaction with TLR4 (Fig. s8). Therefore, we mainly focus on the HMGB1 interface residues bound by small molecules and receptor TLR4.

28-Apr-2024

Dear Dr Zhang:

Manuscript ID: CB-ART-03-2024-000062.R1
TITLE: Natural triterpenoids aided identification of the druggable interface of HMGB1-TLR4 protein-protein interaction

Thank you for your resubmission to RSC Chemical Biology. However, having evaluated the requests from the reviewers and your response thereto, I cannot as of yet accept this paper for publication. The reviewers requested additional experiments that they felt necessary to strengthen the observations described within the paper (e.g. extra NMR experiments). I would very much like to see these points addressed with additionala data.

Please submit a revised manuscript which addresses all of the reviewers’ original concerns, where necessary with additional data. Further peer review of your revised manuscript may be needed. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

Please submit your revised manuscript as soon as possible using this link:

*** PLEASE NOTE: This is a two-step process. After clicking on the link, you will be directed to a webpage to confirm. ***

https://mc.manuscriptcentral.com/rsccb?link_removed

(This link goes straight to your account, without the need to log on to the system. For your account security you should not share this link with others.)

Alternatively, you can login to your account (https://mc.manuscriptcentral.com/rsccb) where you will need your case-sensitive USER ID and password.

You should submit your revised manuscript as soon as possible; please note you will receive a series of automatic reminders. If your revisions will take a significant length of time, please contact me. If I do not hear from you, I may withdraw your manuscript from consideration and you will have to resubmit. Any resubmission will receive a new submission date.

All RSC Chemical Biology articles are published under an open access model, and the appropriate article processing charge (APC) will apply. Details of the APC and discounted rates can be found at https://www.rsc.org/journals-books-databases/about-journals/rsc-chemical-biology/#CB-charges.

RSC Chemical Biology strongly encourages authors of research articles to include an ‘Author contributions’ section in their manuscript, for publication in the final article. This should appear immediately above the ‘Conflict of interest’ and ‘Acknowledgement’ sections. I strongly recommend you use CRediT (the Contributor Roles Taxonomy, https://credit.niso.org/) for standardised contribution descriptions. All authors should have agreed to their individual contributions ahead of submission and these should accurately reflect contributions to the work. Please refer to our general author guidelines https://www.rsc.org/journals-books-databases/author-and-reviewer-hub/authors-information/responsibilities/ for more information.

The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.

Please also encourage your co-authors to sign up for their own ORCID account and associate it with their account on our manuscript submission system. For further information see: https://www.rsc.org/journals-books-databases/journal-authors-reviewers/processes-policies/#attribution-id

Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Dr Sander van Kasteren
Professor Molecular Immunology
Leiden University
Associate Editor of RSC Chemical Biology

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

Reviewer 2

The authors provided detailed responses to the reviewers' questions in their rebuttal letter, but the appropriate revisions to the manuscript were only partially made.

A binding interface always involves two binding partners, in this case HMGB1 and TLR4. The authors report the identification of a putative binding site that may be involved in the interactions on only one binding partner, HMGB1, without specifying the binding site on the second partner, TLR4.
However, the title of the manuscript promises to show the "interface of HMGB1-TLR4 protein-protein interaction".
The abstract also states: "To map the binding interface of HMGB1 with TLR4" and "druggable interface of HMGB1-TLR4 interaction".
Authors should inform the reader already in the abstract that the potential binding site has only been identified on one binding partner, HMGB1. Authors are also encouraged to provide a more specific title that reflects the findings reported in the manuscript: e.g. high-affinity triterpenoid probe targeting HMGB1 (but not TLR4).

This information should be presented consistently in the text and figures, e.g. the side chain of K114 of HMGB1 shown to be involved in interaction with a triterpenoid molecule should be clearly identified in the text as belonging to HMGB1 (e.g. K114/HMGB1 or similar) so that it is clear to the reader (without going into detail) to which protein this residue belongs.

In view of the above, some headings in the body of the text are also misleading, such as “5. Validation of HMGB1-TLR4 interaction interface using the triterpenoid probe“ which could be changed to “Validation of HMGB1 - triterpenoid probe binding interface” or
“2. Verification of triterpenoid probe interfering with HMGB1-TLR4 interaction” which could be changed to “Verification of HMGB1-specific triterpenoid probe interfering with HMGB1-TLR4 interaction” or
“Discovery of triterpenoid probes toward HMGB1-TLR4 signaling” which could be changed to “HMGB1-specific triterpenoid probes inhibiting HMGB1-mediated TLR4 signaling”

In my previous review of the manuscript, I pointed out some limitations of the experimental settings used by the authors. Although the authors provided their responses in their letter, no corresponding revision of the manuscript was made.
Readers need to be fully informed about these limitations, such as the use of the TLR4 ectodomain alone in the studies (although TLR4 is known to exist as a complex with a co-receptor protein MD-2, which is physically associated with TLR4). First, in the absence of MD-2, the overall conformation of TLR4 may be different, which can significantly affect the protein-protein interaction; second, HMGB1 may partially occupy the space of the missing co-receptor protein MD-2 in the binding studies.

Reviewer 1

The authors have responded to some of the reviewer's remarks in their letter but have not implemented any significant revisions in the manuscript itself. As such, from a scientific point of view, the revised version of the article is essentially similar to the previous one.

This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters

Dear Editor,
We deeply appreciate these helpful reviews from you and the reviewers about our manuscript entitled “Natural triterpenoids aided identification of the druggable interface of HMGB1 occupied by TLR4” (Manuscript ID: CB-ART-03-2024-000062.R1). We have carefully read the comments and made the corrections point-by-point, also marked in red. The following are the replies to these questions. However, due to the limited experimental conditions, we could not timely add further experimental data within the paper (e.g. extra NMR experiments). We hope these responses will be satisfactory and the revised manuscript will be suitable for publication.
Thank you again for your kind help and time!

Point-to-point responses to the comments
Referee: 1
The authors have responded to some of the reviewer's remarks in their letter but
have not implemented any significant revisions in the manuscript itself. As such, from
a scientific point of view, the revised version of the article is essentially similar to the
previous one.

Response: We apologize for not addressing the query properly. We have re-adjusted
on the basis of the previous version and marked it in red.

Referee: 2

A binding interface always involves two binding partners, in this case HMGB1
and TLR4. The authors report the identification of a putative binding site that may be
involved in the interactions on only one binding partner, HMGB1, without specifying
the binding site on the second partner, TLR4.
However, the title of the manuscript promises to show the "interface of
HMGB1-TLR4 protein-protein interaction".
The abstract also states: "To map the binding interface of HMGB1 with TLR4" and
"druggable interface of HMGB1-TLR4 interaction".
Authors should inform the reader already in the abstract that the potential binding site
has only been identified on one binding partner, HMGB1. Authors are also
encouraged to provide a more specific title that reflects the findings reported in the
manuscript: e.g. high-affinity triterpenoid probe targeting HMGB1 (but not TLR4).
This information should be presented consistently in the text and figures, e.g. the
side chain of K114 of HMGB1 shown to be involved in interaction with a triterpenoid
Page 3 of 36 RSC Chemical Biology
molecule should be clearly identified in the text as belonging to HMGB1 (e.g.
K114/HMGB1 or similar) so that it is clear to the reader (without going into detail) to
which protein this residue belongs.
In view of the above, some headings in the body of the text are also misleading, such
as “5. Validation of HMGB1-TLR4 interaction interface using the triterpenoid
probe“ which could be changed to “Validation of HMGB1 - triterpenoid probe
binding interface” or
“2. Verification of triterpenoid probe interfering with HMGB1-TLR4 interaction”
which could be changed to “Verification of HMGB1-specific triterpenoid probe
interfering with HMGB1-TLR4 interaction” or “Discovery of triterpenoid probes
toward HMGB1-TLR4 signaling” which could be changed to “HMGB1-specific
triterpenoid probes inhibiting HMGB1-mediated TLR4 signaling”

Response: We apologize for any confusion caused. The description has been polished
to make it easier for readers to read.
In my previous review of the manuscript, I pointed out some limitations of the
experimental settings used by the authors. Although the authors provided their
responses in their letter, no corresponding revision of the manuscript was made.
Readers need to be fully informed about these limitations, such as the use of the TLR4
ectodomain alone in the studies (although TLR4 is known to exist as a complex with a
co-receptor protein MD-2, which is physically associated with TLR4). First, in the
absence of MD-2, the overall conformation of TLR4 may be different, which can
significantly affect the protein-protein interaction; second, HMGB1 may partially
occupy the space of the missing co-receptor protein MD-2 in the binding studies.
RSC Chemical Biology Page 4 of 36

Response: Thank you for underlining this deficiency. Unfortunately, the current
studies only focus on the discovery of specific HMGB1-TLR4 inhibitors and their
binding interface on HMGB1, while research around HMGB1-TLR4/MD2 is ongoing.
However, due to time constraints, we hope that this article can be published first.


Yours sincerely,

Jian Zhang, Ph.D.,
State Key Laboratory of Natural Medicines,
China Pharmaceutical University,
24# Tong Jia Xiang Street, Nan Jing, 210009, China
E-mail: wilsonzhang99@cpu.edu.cn
Tel: 86-25-86185157
Fax: 86-25-8618518

04-Jun-2024

Dear Dr Zhang:

Manuscript ID: CB-ART-03-2024-000062.R2
TITLE: Natural triterpenoids aided identification of the druggable interface of HMGB1 occupied by TLR4

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your manuscript for publication in its current form. In this second revision, you have sufficiently addressed my and the reviewers' concerns to merit publication.

You will shortly receive a separate email from us requesting you to submit a licence to publish for your article, so that we can proceed with the preparation and publication of your manuscript.

All RSC Chemical Biology articles are published under an open access model, and the appropriate article processing charge (APC) will apply. Details of the APC and discounted rates can be found at https://www.rsc.org/journals-books-databases/about-journals/rsc-chemical-biology/#CB-charges.

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With best wishes,

Dr Sander van Kasteren
Professor Molecular Immunology
Leiden University
Associate Editor of RSC Chemical Biology


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