From the journal RSC Chemical Biology Peer review history

Berlin, 17. June 2021

Dear Dr Wu:

Manuscript ID: CB-REV-05-2021-000115
TITLE: Insights into autopalmitoylation: targets, druggability, and inhibitors

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.

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Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

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

Reviewer 1

The review article “Insights into autopalmitoylation: targets, druggability, and inhibitors” by Hu et al provides a readable and comprehensible introduction to the topic of interest. I am not very familiar with this topic, but I found this article very well-structured and illustrated with highly informative figures. Length and depth were appropriate and I found the article highly interesting. I suggest publication of this manuscript after consideration of some minor points:
1) Page 4 (pdf) lines 6-7: “Nonetheless, it is generally accepted that PATs, at least most of them, are autopalmitoylated.” This is an unfortunate statement if it comes without a reference to support it.
2) Page 9 (pdf): Within one paragraph reference 74 was assigned to Mitchell and coworkers and another time a work by Deschenes et al. was mentioned. It took me a while to understand that the authors mean the same paper. This is quite confusing for the reader. The authors should choose one of the two options (Mitchell and coworkers or Deschenes et al) in order to minimize confusion.
3) Figure 4 contains a markup (red lines), which should be deleted.
4) The assay illustrated in Figure 6 is pretty smart. The formation of CO2 is making the coupled step irreversible, which might be worth mentioning.

Reviewer 2

Major revisions in structure and content of this review are recommended, and the spelling and grammar needs significant revision.

TERMINOLOGY
1. Palmitoylation can refer to N- S- or O-palmitoylation, if the type of bond is not specified it is ambiguous to the reader to which one the authors refer to.
2. Autopalmitoylation is imprecise terminology, especially as RFX3 uses stearic acid as a substrate so is therefore autostearoylated, not autopalmitoylated. The zDHHCs are auto-S-acylated, palmitate is just one of several lipids attached. Auto-S-acylation feels more appropriate.
3. zDHHC and DHHC, the readers should decide which PAT acronym to use and not mix acronyms at random; DHHC is for the mouse gene, zDHHC for the human.

CONTENT:

Introduction
1. S-palmitoylation should be described as S-acylation as it encompasses multiple fatty acid chain lengths.
2. All descriptors of atoms e.g. S-palmitoylated, the S of sulphur must be italicised.
3. The authors state that “5,000-10,000 putative S-palmitoylated”. Within the source paper referenced those authors state “The joint dataset indicates that 11% (2,339 proteins) of the human proteome may undergo S-palmitoylation” (emphasise ‘may’ – the databases/datasets likely include a great deal of noise from false postitives). The authors state this is likely an underestimation – firstly, on what basis and with what evidence is this claimed? And second, where do the authors get their figures from? 5,000 proteins are mentioned in the referenced manuscript but this figure is from multiple species.

Autopalmitoylation of PATS and other proteins

1. This section should be reordered to list the proteins which have been confidently assigned as autoacylated before those where there is less evidence, i.e. TEAD, RFX and Bet3 first followed by the others. It makes more sense to have the proteins which are more confidently assigned first, and will be less confusing to the reader.
2. This section would benefit from some description of the known function of autoacylation of the proteins. For TEAD/RFX this is later in the manuscript under “Functions of non-enzymatic protein autopalmitoylation in diseases” but it makes more sense for it to be included here when describing each protein rather than the effect of inhibiting the protein on different diseases.
3. A list or Table of zDHHC’s where autoacylation has actually been detected and the paper which that comes from would be useful information for readers. The authors state, “Nonetheless, it is generally accepted that PATs, at least most of them, are autopalmitoylated”. Which ones are generally accepted? What is the evidence? It is very poor practise to make statements such as these without substantiation.


Autopalmitoylation in diseases

The function of PAT in diseases:
1. This section would benefit from some kind of distinction from when changes in the autoacylation of the PAT affect its function (e.g. zDHHC3 phosphorylation), and when diseases are impacted from the function/loss of function of the enzyme when autoacylation levels of the enzyme are not directly impacted.
2. In this section the authors are just stating the connections between different PATs and the disease but the presentation lacks insight into the substrate function (when the substrates are known) and the implications of S-acylation in their function. The text is again a little disconnected.
3. It should be clearly stated throughout the review that 2-BP is known to have multiple off-target effects beyond covalent targeting of PATs – indeed, there is clear evidence in the literature that 2BP (and other reactive lipid ‘probes’) target 100s of proteins before touching PATs. The apparent impact of 2BP is very likely through lipid metabolism and little if at all on PATs, and is highly variable between conditions. It is not a selective inhibitor of PATs and as such, the authors should not associate it to PAT inhibition as this misunderstanding has been and continues to be highly detrimental to the field.


Functions of non-enzymatic protein autopalmitoylation in diseases:
1. In both of the examples given, autopalmitoylation is not the driving force behind the disease and is just a peripheral consequence of upstream activation of the Hippo and Hedgehog pathways. Is there any evidence that autopalmitoylation of these proteins is altered in different disease states or are they constitutively lipidated?
2. This section seems out of place as it briefly describes the function of S-acylation and then the phenotypes of different inhibitors on the function of the protein. If the small molecules blocking TEAD S-palmitoylation, are mentioned in this section they should be referenced to the known structures (Fig 5).


Targeting autopalmitoylation for drug discovery:
1. There are other examples of lipid binding pockets being exploited to target a protein which would add weight to the argument of them being “druggable”, e.g. UNC119 (Garivet, Cell Chem Bio, 2019, 842-851), NOTUM (Brommage, Bone Res, 2019, 2., Mahy, J med chem, 2020, 12942). There is also evidence for targeting the protein substrate binding site rather than the lipid pocket, e.g NMT inhibitors (Mousnier, Nat chem, 2018, 599) which can avoid issues of non-selectivity due to conserved acyl-CoA binding motifs.

Strategies for targeting autopalmitoylation:
Inhibitors of DHHC family PATs:
1. The authors state “Under cellular conditions, 2-BP might be metabolically converted to 2-BP-CoA.” There is convincing evidence that it is (Davda, ACS chem biol, 2013, 1912-1917).
2. This section would benefit from a comment on the level of validation that these compounds have seen e.g. target engagement, selectivity profiles, secondary assays etc as currently there is no assessment on which compounds are actually fit for purpose or not.
3. Within the figure there are numerous stereogenic centres which are not defined. Please update this to reflect that they are either racemic or the correct stereoisomers.
4. The structure of tunicamycin is incorrect, it is missing a hydroxyl group between the tetrahydrofuran of the nucleoside and the sugar. Also there is an undefined R group within the sugar structure.
5. Compound 1 shown in the Fig 5. from Tang et al. is described in the original literature as Compound 2, not 1.
6. Within the text when describing the results from Tang et al. the authors state “Instead of blocking TEADs-YAP interactions, 1 surprisingly surrogates palmitic acid to stabilize cellular level of TEADs. However, endogenous pan-TEAD did not show significant change after treatment of 1.” It is not clear what the authors mean here as the experiment with both overexpressed TEAD2 and endogenous TEADs are not described therefore there is no context for this observation.

Inhibitors of non-enzymatic palmitoylation of proteins:
1. Grouping the inhibitors within Fig 5 by known mechanisms of action would be useful to readers.

Missing content that would improve the manuscript
1. A figure showing state of the art methods to identify what is a truly autopalmitoylated protein and what are just artefacts and a table of what are believed to be autopalmitoylated proteins and the paper/methods used to determine this (including zDHHC autoacylation).
2. The manuscript would benefit from some descriptions of covalent inhibitors and their screening methods. It might be expected that if these proteins are able to undergo autoacylation then there would be some inherent increased reactivity for the cysteine sites of modification. This would fit well with the latter part of the manuscript where covalent TEAD inhibitors have already been developed.

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

July 28th, 2021

Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor
chembio@rsc.org

Dear Prof. Süssmuth,

Thank you very much for processing our review article to RSC Chemical Biology “Insights into autopalmitoylation: targets, druggability, and inhibitors ” by Hu, et al.

We sincerely thank you and the reviewers for the constructive comments. We have now revised the article according to the reviewers’ suggestions, which greatly helped us to improve the article. The detailed “Response to the reviewers” is also attached for your references. In general, we have included new Table 1 to summarize known protein auto-S-fatty acylation, and have re-organized section of “auto-S-fatty acylation in diseases” according to Reviewer 2’s comments. We believe the revised version has addressed all of the questions from the reviewers, and should be ready to publish.

We think this is a comprehensive overview of this field, which should be of interest to the broad chemical biology and drug discovery communities.



Thanks again for your time and efforts.

Sincerely Yours,

Xu Wu, Ph.D

Reply to the reviewers’ comments

Referee: 1

Comments to the Author:
The review article “Insights into autopalmitoylation: targets, druggability, and inhibitors” by Hu et al provides a readable and comprehensible introduction to the topic of interest. I am not very familiar with this topic, but I found this article very well-structured and illustrated with highly informative figures. Length and depth were appropriate and I found the article highly interesting. I suggest publication of this manuscript after consideration of some minor points:
We thank the reviewer for the positive view of our manuscript. The minor revisions are addressed point by point below:

1)Page 4 (pdf) lines 6-7: “Nonetheless, it is generally accepted that PATs, at least most of them, are autopalmitoylated.” This is an unfortunate statement if it comes without a reference to support it.
Response: We thank the reviewer for the suggestion. We have added references here and adopted another statement: “Taken together, it has been confirmed biochemically that ZDHHC2, ZDHHC3, ZDHHC15, and ZDHHC20 could undergo auto-S-fatty acylation as part of their catalytic processes. It is speculated that other ZDHHC proteins could also be auto-S-fatty acylated; however, more evidence is still needed to confirm that auto-fatty acylation is a conserved mechanism involved in PATs-mediated catalysis.”

2)Page 9 (pdf): Within one paragraph reference 74 was assigned to Mitchell and coworkers and another time a work by Deschenes et al. was mentioned. It took me a while to understand that the authors mean the same paper. This is quite confusing for the reader. The authors should choose one of the two options (Mitchell and coworkers or Deschenes et al) in order to minimize confusion.
Response: We thank the reviewer for their recommendation. The assay here was developed by Mitchell and coworkers, while Deschenes et al. used this assay to screen inhibitors. To clear the confusion, we added a reference to quote the work by Deschenes et al.

3)Figure 4 contains a markup (red lines), which should be deleted.
Response: Thank you, we deleted the markup.

4)The assay illustrated in Figure 6 is pretty smart. The formation of CO2 is making the coupled step irreversible, which might be worth mentioning.
Response: We thank the reviewer for this insightful feedback. We described the formation of CO2 to highlight the assay.



Referee: 2

Comments to the Author:
Major revisions in structure and content of this review are recommended, and the spelling and grammar needs significant revision.
We thank the reviewer for their suggestions and elaborative reading of our manuscript. The major and minor revisions are addressed point-by-point below.

TERMINOLOGY
1.Palmitoylation can refer to N- S- or O-palmitoylation, if the type of bond is not specified it is ambiguous to the reader to which one the authors refer to.
Response: We thank the reviewer for bringing up this important point. We used auto-S-fatty acylation throughout this manuscript to clear the confusion.

2.Autopalmitoylation is imprecise terminology, especially as RFX3 uses stearic acid as a substrate so is therefore autostearoylated, not autopalmitoylated. The zDHHCs are auto-S-acylated, palmitate is just one of several lipids attached. Auto-S-acylation feels more appropriate.
Response: We thank the reviewer for the feedback. We used auto-S-fatty acylation throughout this manuscript.


3.zDHHC and DHHC, the readers should decide which PAT acronym to use and not mix acronyms at random; DHHC is for the mouse gene, zDHHC for the human.

Response: We thank the reviewer for bringing our attention to this issue. We used zdhhc for mouse and rat gene (for example, https://www.ncbi.nlm.nih.gov/gene/102193), and ZDHHC for the human gene (for example https://www.ncbi.nlm.nih.gov/gene/131540). We used ZDHHC for both human and mouse protein names (for example: https://www.uniprot.org/uniprot/Q810M5 , and https://www.uniprot.org/uniprot/Q8WVZ1), according to the guidelines for formatting gene and protein names from NCBI and the publisher.

CONTENT:

Introduction
1.S-palmitoylation should be described as S-acylation as it encompasses multiple fatty acid chain lengths.
Response: We thank the reviewer for this feedback. We used S-fatty acylation when generally talking about S-fatty acylation and used S-palmitoylation for proteins reported to be palmitoylated. Under the Introduction, we also mentioned “Among the fatty acyl groups utilized in the modifications, C16 palmitoyl group is the most common one. Therefore, S-fatty acylation is often referred as S-palmitoylation. However, C14 myristoyl, C18 stearoyl, and other unsatuarated acyl groups (C18:1 oleoyl) are also found in protein S-fatty acylation. Therefore, S-fatty acylation is a more accurate terminology to describe such modification, and will be used in this review”.

2.All descriptors of atoms e.g. S-palmitoylated, the S of sulphur must be italicised.
Response: Thank you, we corrected this typo.

3.The authors state that “5,000-10,000 putative S-palmitoylated”. Within the source paper referenced those authors state “The joint dataset indicates that 11% (2,339 proteins) of the human proteome may undergo S-palmitoylation” (emphasise ‘may’ – the databases/datasets likely include a great deal of noise from false postitives). The authors state this is likely an underestimation – firstly, on what basis and with what evidence is this claimed? And second, where do the authors get their figures from? 5,000 proteins are mentioned in the referenced manuscript but this figure is from multiple species.
Response: We thank the reviewer for bringing our attention to this issue. As you suggested, we adopted proper statement here: “The public database SwissPalm predicts roughly 10% of the human proteome are putative S-fatty acylated proteins.”


Autopalmitoylation of PATS and other proteins

1.This section should be reordered to list the proteins which have been confidently assigned as autoacylated before those where there is less evidence, i.e. TEAD, RFX and Bet3 first followed by the others. It makes more sense to have the proteins which are more confidently assigned first, and will be less confusing to the reader.
Response: We thank the reviewer for this insightful suggestion. As you suggested, we rearranged and streamlined this section. In the revised version, we first discussed confirmed auto-S-fatty acylated proteins, including Bet3, TEAD, RFX3, followed by additional non-enzymatic proteins with weak evidence. At last, we described auto-S-fatty acylated ZDHHC proteins with reported evidence. We have added Table 1 to summarize the reports.


2.This section would benefit from some description of the known function of autoacylation of the proteins. For TEAD/RFX this is later in the manuscript under “Functions of non-enzymatic protein autopalmitoylation in diseases” but it makes more sense for it to be included here when describing each protein rather than the effect of inhibiting the protein on different diseases.
Response: We thank the reviewer for the suggestion. Following the comments, we streamlined “Functions of non-enzymatic protein auto-S-fatty acylation in diseases”, where we focus more on functions of protein fatty acylation in diseases, rather than inhibiting the proteins. However, since we have more detailed discussion of TEAD as a target, it might be easier for readers to understand their functions with the disease link.


3.A list or Table of zDHHC’s where autoacylation has actually been detected and the paper which that comes from would be useful information for readers. The authors state, “Nonetheless, it is generally accepted that PATs, at least most of them, are autopalmitoylated”. Which ones are generally accepted? What is the evidence? It is very poor practise to make statements such as these without substantiation.
Response: We thank the reviewer for bring this issue to our attention. In Table 1, we showed which ZDHHC is detected to be auto-S-fatty acylated and what methods are to determine it. We also revised the statement: “Taken together, it has been confirmed biochemically that ZDHHC2, ZDHHC3, ZDHHC15, and ZDHHC20 could undergo auto-S-fatty acylation as part of their catalytic processes. It is speculated that other ZDHHC proteins could also be auto-S-fatty acylated10; however, more evidence is still needed to confirm that auto-fatty acylation is a conserved mechanism involved in PATs-mediated catalysis”



Autopalmitoylation in diseases

The function of PAT in diseases:
1.This section would benefit from some kind of distinction from when changes in the autoacylation of the PAT affect its function (e.g. zDHHC3 phosphorylation), and when diseases are impacted from the function/loss of function of the enzyme when autoacylation levels of the enzyme are not directly impacted.
Response: We thank the reviewer for bringing this issue to our attention. In this section, we focused on ZDHHC proteins which have altered auto-S-fatty acylation levels in diseases. Instead, we would not thoroughly discuss PAT enzymes if changes of their auto-S-fatty acylation levels are unknown in the context of diseases. Since the functions of PATs (mostly through their substrates) have been extensively reviewed previously, we have shortened this section.

2.In this section the authors are just stating the connections between different PATs and the disease but the presentation lacks insight into the substrate function (when the substrates are known) and the implications of S-acylation in their function. The text is again a little disconnected.
Response: We thank the reviewer for the feedback. We added descriptions on how the function of substrates is regulated by S-acylation when discussing auto-S-fatty acylation of PATs. As described above, we have only focused on PATs which auto-S-fatty acylation are affected in diseases.

3.It should be clearly stated throughout the review that 2-BP is known to have multiple off-target effects beyond covalent targeting of PATs – indeed, there is clear evidence in the literature that 2BP (and other reactive lipid ‘probes’) target 100s of proteins before touching PATs. The apparent impact of 2BP is very likely through lipid metabolism and little if at all on PATs, and is highly variable between conditions. It is not a selective inhibitor of PATs and as such, the authors should not associate it to PAT inhibition as this misunderstanding has been and continues to be highly detrimental to the field.

Response: We thank the reviewer for this feedback. Although 2-BP is currently widely used to block PAT activity, it indeed has multiple off-targets. As you suggested, we should be very careful about 2-BP. We removed descriptions about 2-BP in this section and mentioned “Global profiling of cellular targets showed 2-BP does not only engage PATs, but also targets other group of proteins, such as transporters channels, enzymes, and chaperones,77 suggesting 2-BP is promiscuous. Therefore, it is unsuitable to use 2-BP as an uncontroversial tool to target PATs”. We have added a reference here.
Functions of non-enzymatic protein autopalmitoylation in diseases:

1.In both of the examples given, autopalmitoylation is not the driving force behind the disease and is just a peripheral consequence of upstream activation of the Hippo and Hedgehog pathways. Is there any evidence that autopalmitoylation of these proteins is altered in different disease states or are they constitutively lipidated?
Response: We thank the reviewer for this insightful note. To our best knowledge, there is no direct report showing that TEAD and RFX3 autopalmitoylation levels are altered in different disease states. However, our unpublish results suggest that TEAD palmitoylation levels are upregulated in YAP-dependent cancer cell lines, such as uveal melanoma, and the detailed mechanisms are unclear. A recent paper (Kim NG, and Gumbiner BM. Proc Natl Acad Sci U S A. 2019 ,116, 9877-9882.) showed that TEADs palmitoylation levels decreased in cells under high density (Hippo pathway on) through fatty acid biosynthesis regulation and de-palmitoylation, suggesting that TEADs palmitoylation level are regulated in response to cell signaling cues.

2.This section seems out of place as it briefly describes the function of S-acylation and then the phenotypes of different inhibitors on the function of the protein. If the small molecules blocking TEAD S-palmitoylation, are mentioned in this section they should be referenced to the known structures (Fig 5).
Response: We thank the reviewer for this feedback. We reorganized and streamlined this section. We removed inhibitors related descriptions and focused on how auto-S-fatty acylation is linked to functions of the proteins and implicated in diseases here.



Targeting autopalmitoylation for drug discovery:
1.There are other examples of lipid binding pockets being exploited to target a protein which would add weight to the argument of them being “druggable”, e.g. UNC119 (Garivet, Cell Chem Bio, 2019, 842-851), NOTUM (Brommage, Bone Res, 2019, 2., Mahy, J med chem, 2020, 12942). There is also evidence for targeting the protein substrate binding site rather than the lipid pocket, e.g NMT inhibitors (Mousnier, Nat chem, 2018, 599) which can avoid issues of non-selectivity due to conserved acyl-CoA binding motifs.
Response: We thank the reviewer for this insightful suggestion. We included these examples in this section.

Strategies for targeting autopalmitoylation:
Inhibitors of DHHC family PATs:
1.The authors state “Under cellular conditions, 2-BP might be metabolically converted to 2-BP-CoA.” There is convincing evidence that it is (Davda, ACS chem biol, 2013, 1912-1917).
Response: Thank you, we added this reference here.

2.This section would benefit from a comment on the level of validation that these compounds have seen e.g. target engagement, selectivity profiles, secondary assays etc as currently there is no assessment on which compounds are actually fit for purpose or not.
Response: We thank the reviewer for this insightful feedback. As you suggested, we discussed more detailed profiles of these PATs inhibitors in this section.

3.Within the figure there are numerous stereogenic centres which are not defined. Please update this to reflect that they are either racemic or the correct stereoisomers.
Response: Thank you, we defined stereo centers in Fig 5.

4.The structure of tunicamycin is incorrect, it is missing a hydroxyl group between the tetrahydrofuran of the nucleoside and the sugar. Also there is an undefined R group within the sugar structure.
Response: Thank you, we corrected the structure of tunicamycin.

5.Compound 1 shown in the Fig 5. from Tang et al. is described in the original literature as Compound 2, not 1.
Response: Thank you, we corrected this error in Fig 5.

6.Within the text when describing the results from Tang et al. the authors state “Instead of blocking TEADs-YAP interactions, 1 surprisingly surrogates palmitic acid to stabilize cellular level of TEADs. However, endogenous pan-TEAD did not show significant change after treatment of 1.” It is not clear what the authors mean here as the experiment with both overexpressed TEAD2 and endogenous TEADs are not described therefore there is no context for this observation.
Response: We thank the reviewer for the suggestion. We revised this description: “Instead of blocking TEADs-YAP interactions, 2 surprisingly surrogates palmitic acid to stabilize cellular level of exogenous TEADs in HEK293T cells. However, endogenous pan-TEAD levels in several cancer cells did not show significant change after treatment of 2.”

Inhibitors of non-enzymatic palmitoylation of proteins:
1.Grouping the inhibitors within Fig 5 by known mechanisms of action would be useful to readers.
Response: We thank the reviewer for this suggestion. We grouped compounds in Fig 5 into covalent and reversible inhibitors.

Missing content that would improve the manuscript
1.A figure showing state of the art methods to identify what is a truly autopalmitoylated protein and what are just artefacts and a table of what are believed to be autopalmitoylated proteins and the paper/methods used to determine this (including zDHHC autoacylation).
Response: We thank the reviewer for this suggestion. We showed methods to identify auto-S-fatty acylated proteins in Table 1. We also included corresponding references and our comments in the table.

2.The manuscript would benefit from some descriptions of covalent inhibitors and their screening methods. It might be expected that if these proteins are able to undergo autoacylation then there would be some inherent increased reactivity for the cysteine sites of modification. This would fit well with the latter part of the manuscript where covalent TEAD inhibitors have already been developed.
Response: We thank the reviewer for this insightful feedback. We described the discovery and screening assay of covalent TEAD inhibitors under “Inhibitors of non-enzymatic palmitoylation of proteins”. We also emphasized “the intrinsic active cysteine in auto-S-acylation pocket makes it possible to design covalent modulators” under “Strategies for targeting auto-S-fatty acylation” as well as Abstract.

Berlin, 16. August 2021

Dear Dr Wu:

Manuscript ID: CB-REV-05-2021-000115.R1
TITLE: Insights into autopalmitoylation: targets, druggability, and inhibitors

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 for publication after revisions.

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.

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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 from CASRAI, https://casrai.org/credit/) 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 http://www.rsc.org/journals-books-databases/journal-authors-reviewers/author-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,
Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

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

Reviewer 2

The authors alterations to the manuscript have improved it significantly. There remain a number of grammatical errors, with suggested updates and other minor points listed below:

The ambiguous term palmitoylation is used throughout the manuscript, with several exceptions. As highlighted in previous comments, this should be corrected to the more accurate term S-palmitoylation (with 'S' in italics).

“With the advances of protein purification techniques, more PATs have been shown as auto-S-fatty acylated in biochemical assays. The Linder group detected significant signals of S-palmitoylation of ZDHHC2 and ZDHHC3…….” (missed 'The')

“In mice, this nonsense mutation results in a premature stop codon (L203X) and a truncated ZDHHC13 without DHQC domain… “(inserted a space)

There is a change in format on page 14 where the columns are broken by the image of Fig 1. rather than continuous. Is this intentional?

“Interestingly, the carboxyl group of palmitic acid does not form a covalent bond with, but points directly toward the conserved cysteine residue in TEAD2 structures” (several edits')

“However, specific inhibitors targeting TEADs auto-S-fatty acylation suggest that selectivity could still be achieved.”
Please can the authors provide a reference for this statement?

“For example, TEAD inhibitor (MGH-CP1) does not inhibit the ZDHHC-family of proteins…” (missing 'the').

All "et al." are missing the "." after "al".

Remove the trademark sign from BODIPY.

“Similarly, the Luo group also succeeded by elaborately taking advantage of the active cysteine adjacent to the lipid binding pocket….”

“Likewise, starting from FA, rational installation of a warhead by the Jänne and Gray groups leads to MYF-01-37….” (missing 'the')

NSCLC is not defined anywhere in the document

Compound 2 is referred to as "Compound 2", "2", and "compound 2". Please standardise this.

There are two undefined stereocentres in Fig. 5 (2-BP and MYF-01-037), these are presumably racemic, please define them unambiguously as racemic.

“In addition, a lack of robust and sensitive HTS assays, such as the assays developed for kinase....” (missing 'a')

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

Dear Prof. Süssmuth,

Thank you very much for processing our review article to RSC Chemical Biology “Insights into autopalmitoylation: targets, druggability, and inhibitors ” by Hu, et al. (CB-REV-05-2021-000115.R1).

We thank Reviewer 2 for the positive comments and detailed suggestions of the minor grammar and formatting issues. We have now revised the article according to the reviewers’ suggestions. The detailed “Response to the reviewers” is also attached for your references. We believe the revised version has addressed all of the questions from the reviewers, and should be ready to publish.
We think this is a comprehensive overview of this field, which should be of interest to the broad chemical biology and drug discovery communities.

Thanks again for your time and efforts.

Sincerely Yours,

Xu Wu, Ph.D

Reply to the reviewers’ comments
Referee: 2

Comments to the Author
The authors alterations to the manuscript have improved it significantly. There remain a number of grammatical errors, with suggested updates and other minor points listed below:
We thank the reviewer for the positive view of our manuscript. The minor revisions are addressed point by point below:
1. The ambiguous term palmitoylation is used throughout the manuscript, with several exceptions. As highlighted in previous comments, this should be corrected to the more accurate term S-palmitoylation (with 'S' in italics).
Response: We thank the reviewer for bringing this issue to our attentions. We replaced all “Palmitoylation” with “S-Palmitoylation”.
2. “With the advances of protein purification techniques, more PATs have been shown as auto-S-fatty acylated in biochemical assays. The Linder group detected significant signals of S-palmitoylation of ZDHHC2 and ZDHHC3…….” (missed 'The')
Response: Thanks. We added “The” here.
3. “In mice, this nonsense mutation results in a premature stop codon (L203X) and a truncated ZDHHC13 without DHQC domain… “(inserted a space)
Response: Thanks. We inserted a space here.
4. There is a change in format on page 14 where the columns are broken by the image of Fig 1. rather than continuous. Is this intentional?
Response: We thank the reviewer for noticing the formatting issues. We reorganized this part to avoid breaking the columns.
5. “Interestingly, the carboxyl group of palmitic acid does not form a covalent bond with, but points directly toward the conserved cysteine residue in TEAD2 structures” (several edits')
Response: We revised this sentence as suggested.
6. “However, specific inhibitors targeting TEADs auto-S-fatty acylation suggest that selectivity could still be achieved.” Please can the authors provide a reference for this statement?
Response: Thanks for this suggestion. We added a reference here.
7. “For example, TEAD inhibitor (MGH-CP1) does not inhibit the ZDHHC-family of proteins…” (missing Page 3 of 18 RSC Chemical Biology 'the').
Response: Thanks. We added “the” here.
8. All "et al." are missing the "." after "al".
Response: Thanks for bringing this issue to our attention. We added "." after "al".
9. Remove the trademark sign from BODIPY.
Response: Thanks for this suggestion. The trademark was deleted.
10. “Similarly, the Luo group also succeeded by elaborately taking advantage of the active cysteine adjacent to the lipid binding pocket….” “Likewise, starting from FA, rational installation of a warhead by the Jänne and Gray groups leads to MYF-01-37….” (missing 'the')
Response: Thanks. We added “the” here.
11. NSCLC is not defined anywhere in the document
Response: Thanks for this suggestion. We included the full name of non-small-cell lung cancer at where “NSCLC” first appears.
12. Compound 2 is referred to as "Compound 2", "2", and "compound 2". Please standardise this.
Response: Thanks for this suggestion. We uniformly used “compound 2”.
13. There are two undefined stereocentres in Fig. 5 (2-BP and MYF-01-037), these are presumably racemic, please define them unambiguously as racemic.
Response: Thanks for this suggestion. We annotated “racemic” for 2-BP and MYF-01-037 in Fig.5.
14. “In addition, a lack of robust and sensitive HTS assays, such as the assays developed for kinase....” (missing 'a')
Response: Thanks. We added “a” here.

Berlin, 22. August 2021

Dear Dr Wu:

Manuscript ID: CB-REV-05-2021-000115.R2
TITLE: Insights into autopalmitoylation: targets, druggability, and inhibitors

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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 publication of your manuscript.

You can highlight your article and the work of your group on the back cover of RSC Chemical Biology, if you are interested in this opportunity please contact me for more information.

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Thank you for publishing with RSC Chemical Biology, a journal published by the Royal Society of Chemistry – connecting the world of science to advance chemical knowledge for a better future.

With best wishes,

Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor