From the journal RSC Chemical Biology Peer review history

11-Nov-2021

Dear Professor Sbardella:

Manuscript ID: CB-ART-10-2021-000196
TITLE: Lysine methyltransferase inhibitors: where we are now.

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. Please also include the suggestion of reviewer 1 to add more structural insight into inhibitor binding. 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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I look forward to receiving your revised manuscript.

Yours sincerely,
Dr Andrea Rentmeister

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

Reviewer 1

This is a very nice overview and will be of value for a broad readership. The big question whether more structural insight into inhibitor binding (at least for selected cases) should go in. This is an editorial decision mostly.
minor points: regarding PRC degraders the EZH1/2 selecivitiy is not really discussed.
A potential overlap between methyl lysine reader and MTase inhibitors should be disucssed. A366 is a prime example for this and hence the selectivity of this compound has to be discussed beyond MTases. What is the stereochemistry of the alcohol in cpd )?

Reviewer 2

In this review articles, Feoli and colleagues described the efforts of a decade of developing PKMT inhibitors. The manuscript is organized according to the PKMTs with known inhibitors of high quality. For the PKMT, the inhibitors were classified according to the MOAs. The authors also briefly mentioned the biological functions of individual PKMTs to justify the importance of developing their inhibitors. The collection of these inhibitors is nearby comprehensive. Although 80% of the compounds have been reviewed by other works, the new development of PKMT inhibitors in the past several years make such a review necessary to catch up the state-of-art of the field. The reviewers recommend publishing this work after addressing the following concerns:
(1) In Figure 1, the electron arrows should attach the CH3 rather than sulfonium. In addition, the SAM cofactor has a chiral center at the sulfonium. The two issues need to be fixed.
(2) In the end of introduction, the authors only mentioned the purpose of this review. However, the similar reviews have been published by many other labs. The prior works should be mentioned. The difference and advancement of this manuscript should be highlighted.
(3) In Figure 1, ASH1L should be highlighted for the reason mentioned below.
(4) While OTS193320 and its derivative were claimed as SUV39H2 inhibitors. These compounds were not well characterized in terms of their selectivity against SUV39H2 versus other PMTs. In addition, the biology associated the set of inhibitors don’t align with the functions of SUV39H2. The authors should put caution of these compounds upon considering as SUV39H2 inhibitors.
(5) The authors failed to mention two families of SMYD2 inhibitors: JMC, 2016, 59, 11079; https://www.thesgc.org/chemical-probes/PFI-5
(6) The authors should include the newly reported ASH1L inhibitor: https://www.nature.com/articles/s41467-021-23152-6

Dear Editor,
I and the other co-authors have carefully read the comments made by the reviewers and by the Editorial Office and really appreciated this critical discussion that gave us the opportunity to revise our manuscript into a better presentation.

Therefore, according to reviewers’ comments we have thoroughly revised the manuscript.
We really hope that the manuscript is now acceptable for publication in RSC Chemical Biology.

Below, you will find a point-by-point response to the remarks of the Editorial Office and of the reviewers
Looking forward to your reply, I send you my best regards.
Sincerely Yours



Reviewer: 1

This is a very nice overview and will be of value for a broad readership. The big question whether more structural insight into inhibitor binding (at least for selected cases) should go in.

We thank the reviewer for her/his comments. We have now included in the manuscript additional structural insights into the binding mode of at least one selected inhibitor for each protein. Also, to make such insights clearer for the reader, we have added additional figures (Figures 3, 5, 8, 10, 12, 14, 17, 19, 21, 23, 25, 27, 29, 30, 33, 35, 37) depicting the key interactions as flat representations generated with LigPlot+ starting from the proper PDB file.

minor points: regarding PRC degraders the EZH1/2 selectivity is not really discussed.

We thank the reviewer for her/his comments. We have now added a brief discussion on the EZH1/2 selectivity of the degraders of PRC (p. 16).


A potential overlap between methyl lysine reader and MTase inhibitors should be disucssed. A366 is a prime example for this and hence the selectivity of this compound has to be discussed beyond MTases.

We thank the reviewer for her/his comment. Indeed, compound A-366 occupies the lysine binding site of G9a and is competitive with respect to the substrate peptide and uncompetitive with respect to the SAM co-factor. Therefore, it is not very surprising that it was also reported as a sub-micromolar inhibitor of the epigenetic methyl reader protein Spindlin1 (IC50 of 186.3 nM). In fact, the capability of the compound to bind to the same pocket of the lysine substrate of the methyltransferase makes it also able to bind to the reader protein that recognizes the methylation reaction product (Kme).
We have now added a comment at this regard in the manuscript (p. 9).

What is the stereochemistry of the alcohol in cpd )?

Unfortunately, we are not sure of what the reviewer meant with this comment. If she/he was referring to compound 9, to the best of our knowledge this compound was reported as a racemic mixture in the original paper: Scheufler et al. ACS Med. Chem. Lett. 2016, 7, 8, 730–734.

Reviewer: 2

[…] The reviewers recommend publishing this work after addressing the following concerns:.

(1) In Figure 1, the electron arrows should attach the CH3 rather than sulfonium. In addition, the SAM cofactor has a chiral center at the sulfonium. The two issues need to be fixed.

We greatly appreciated the comments and suggestions by the reviewer. The electron arrow was actually attaching the CH3 but, due to the small size of the original version of Figure 1, the reader might have had the impression of something different. We are very sorry for this and, for the sake of clarity, we have now included the Figure in a larger two-column format. We have also included the chiral center at the sulfonium of the SAM cofactor (thanks for pointing out our slip) and highlighted ASH1L to address the issue raised in comment 3.


(2) In the end of introduction, the authors only mentioned the purpose of this review. However, the similar reviews have been published by many other labs. The prior works should be mentioned. The difference and advancement of this manuscript should be highlighted.

We thank the reviewer for her/his useful suggestion. In this revised version of the manuscript, we have added the following paragraph at the end of the Introduction section (p. 1), also citing the main reviews previously published by other groups:

“In the last decade, several reviews regarding these proteins have been published, some of them focusing on structural features of the proteins, some on their involvement in different pathologies, others reporting the currently available inhibitors for some of them.15-23
In this review, we report an updated overview of the current literature regarding selected KMTs for which high quality chemical probes have been reported. For each of these proteins, we provide a summary of their biological functions, including the implications in pathological states, and describe the corresponding inhibitors on the basis of their mechanism of action. For selected inhibitors (at least one for each protein) structural insights into the binding mode are also provided.”

(3) In Figure 1, ASH1L should be highlighted for the reason mentioned below.

Done. Thanks for the suggestion (see also our replies to concerns #1 and #5).

(4) While OTS193320 and its derivative were claimed as SUV39H2 inhibitors. These compounds were not well characterized in terms of their selectivity against SUV39H2 versus other PMTs. In addition, the biology associated the set of inhibitors don’t align with the functions of SUV39H2. The authors should put caution of these compounds upon considering as SUV39H2 inhibitors.

We thank the reviewer for her/his comment and suggestion.
In this revised version of the manuscript, we have added the following paragraph at the end of the section (p. 11):

“However, it should be noted that these compounds were not well characterized by the authors in terms of their selectivity against other PMTs and, consequently, caution should be exercised when considering their use as chemical probes. Nevertheless, to the best of our knowledge they are the most potent SUV39H2 inhibitors reported to date.”

Please notice that we have also added the description of SUV39H1 inhibitor F5446 (p. 10) that we did not include in the original version.

(5) The authors failed to mention two families of SMYD2 inhibitors: JMC, 2016, 59, 11079; https://www.thesgc.org/chemical-probes/PFI-5.

We thank the reviewer for bringing this to our attention and we are sorry for the omission.
In this revised version of the manuscript, we have now included the missing SMYD2 inhibitors (now compound 70, p. 19 and Figure 18, and compound 75, p. 21 and Figure 18).

(6) The authors should include the newly reported ASH1L inhibitor: https://www.nature.com/articles/s41467-021-23152-6

Again, we thank the reviewer for her/his carefully review and for having brought to our attention this omission, helping us to improve the quality of the manuscript. In this revised version we have added the description of ASH1L inhibitors together with the biological role of the protein.

10-Dec-2021

Dear Professor Sbardella:

Manuscript ID: CB-ART-10-2021-000196.R1
TITLE: Lysine methyltransferase inhibitors: where we are now.

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.

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With best wishes,

Dr Andrea Rentmeister

Reviewer 1

The manuscript is even better now, great work