From the journal RSC Chemical Biology Peer review history

01-Oct-2020

Dear Dr Beekman:

Manuscript ID: CB-ART-08-2020-000148
TITLE: In silico peptide-directed ligand design complements experimental peptide-directed binding for protein-protein interaction modulator discovery

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.

I have carefully evaluated your manuscript and the reviewers’ reports, and both reviewers found that the paper addresses an interesting and important research topic. However, one of the reviewers indicated that major revisions are necessary and identified several issues and questions that need to be addressed. In addition to describing in details the in silico screening process, it will be very useful for the readers if you present docking poses and describe the interactions with Mcl-1.

Please submit a revised manuscript which addresses all of the reviewers’ comments. 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.

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Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

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

Reviewer 1

This report answers an important question raised by the authors' prior work, which was a very innovative approach to identification of small molecules which inhibit PPIs. The authors have previously worked up a computational version of that same assay, and now they are comparing those results directly. The work shows that the two methods are both successful but yield different sets of hits. I have only one question - how much computation is saved by doing steps 1 and 2 as compared to just running step 3 from the start? One correction so minor not worth changing the tick box for: the 1H spectrum from compound 3 seems to be duplicated, instead of having the 13C spectrum (p23 on the pdf I received to review)

Reviewer 2

In this manuscript Howell and Beekman revisit the Mcl-1/Noxa protein-protein interaction (PPI) for which they had applied in 2017 for the first time their clever “peptide directed binding” strategy to identify PPI-inhibitors (Ref. 24). Later, they developed this further to an in silico approach, which they have tested for the p53/hDMX or hDM2 PPI (Ref. 28). But as the two approaches were validated on two different PPIs, it has been so far impossible to judge which approach is more efficient. With the current manuscript, in which the authors applied their in silico approach to the Mcl-1/Noxa PPI, such comparison can now be made.

The authors used molecular docking to identify 10 azide and 10 alkyne fragments, resulting in 100 virtual triazoles which were docked to Mcl-1 in a second computational step. The top 10 triazoles (additionally optimized for ADMET properties) were synthesized and tested in a fluorescence polarization assay. 8 out of the 10 compounds showed and IC50 <10 µM, which is a quite impressive achievement (Table 1 and Figure 2). A thermal shift assay and a mitochondrial depolarization assay confirmed biophysical binding and cellular activity, respectively.

The manuscript is well written and reports important and interesting findings, which deserve to be published in RSC Chem. Biol. The development of PPI inhibitors is still a very a challenging topic and the progress reported in this manuscript will be highly appreciated.

However, before publication the following issues have to be addressed:

1) On page 2 in “Results and Discussion” the authors briefly mention the in silico docking process. Even after consulting the SI (which is also not very informative about this topic), I have many open questions.

The authors should give more information about the in silico screening process – as it lies at the heart of their manuscript. How many azide and alkyne fragments have been docked? Was there any bias through information from their 2017 Angew. Chem. paper (Ref. 24), which might have served as an input in their in silico procedure?

2) I am wondering why in their current manuscript the L-Fmoc-propargyglyince-OMe fragment delivered the best results (Table 1), which is in contrast to their Table 2 in their 2017 Angew. Chem. paper (Ref. 24), where exclusively the D-Fmoc-propargyglyince-OMe fragment was used for the triazole-based inhibitors? Does the absolute configuration of the amino acid building block has no important influence on binding?

3) Have the authors tried to perform an in silico docking of a virtual library of triazoles, without having identified before suitable fragments through their peptide directed binding approach? Would this not be more straightforward? Are there any disadvantages, such as more CPU time, etc. which would speak against it?

In conclusion, I can recommend the publication of this manuscript in RSC Chem. Biol. once the issues raised in this review report will have been addressed.

Minor comments:
p.3. right column on several occasions second paragraph: “Compounds 5-8” instead of “Compound 5-8”

Dear Professor Nikolovska-Coleska

Thank you for your email regarding our submitted manuscript: CB-ART-08-2020-000148. We greatly appreciate the editors and reviewers time in evaluating our manuscript for publication in RSC Chemical Biology. We were pleased to read that the reviewers also saw the importance of our work, and we thank them for suggesting changes to improve the quality of the work. We would like to particularly thank you, the editor, for your clear guidance on additions and changes to be made. This gesture offers greater clarity in the requests made by the journal, and is one we do not often see from editors. It is a gesture that we greatly appreciate and hope you and RSC Chemical Biology continue this input from the editors.

We have made changes as requested. The details of those changes are listed below.

Editor
-"I have carefully evaluated your manuscript and the reviewers’ reports, and both reviewers found that the paper addresses an interesting and important research topic. However, one of the reviewers indicated that major revisions are necessary and identified several issues and questions that need to be addressed. In addition to describing in details the in silico screening process, it will be very useful for the readers if you present docking poses and describe the interactions with Mcl-1."

We have added representative poses to the manuscript (Figure 3), and added predicted 2D ligand interaction diagrams to the SI.
The main text now has the added paragraph
“Docking poses predicted by induced fit docking suggest compounds are able to bind in the key P2 and P3 pockets. Poses for compound 1 and 2 (Figure 3), are representative of binding poses generated for compound 1-8. The aromatic groups create contacts with Phe270 in P2 and Phe228 in P3, and interaction with Arg263 either through a water bridge interaction or hydrogen bonding. In line with the design of peptide-small molecule hybrids, the azide fragment which interacts with Arg263 in the hybrids, also interacts with the azide fragment in the predicted pose for compound 1. This motif is repeated in the majority of the binding poses, but is not necessarily the predicted lowest energy conformer. Voisin-Chiret and co-workers recently highlighted the importance of the P2/P3 binding pockets and Arg263 for binding of non-peptidic ligands.42 Representative 2D ligand interaction diagrams are presented in the SI.”

As well as figure 3 and corresponding caption.

Reviewer 1
-"This report answers an important question raised by the authors' prior work, which was a very innovative approach to identification of small molecules which inhibit PPIs. The authors have previously worked up a computational version of that same assay, and now they are comparing those results directly. The work shows that the two methods are both successful but yield different sets of hits. I have only one question - how much computation is saved by doing steps 1 and 2 as compared to just running step 3 from the start?"

This question is one that was also asked by the second reviewer, and is likely one that many other readers will have. We have added the following statement to the discussion.

“This application of peptide-directed ligand design to identify candidate triazoles is computationally 158 times more efficient than generating and screening a library of possible triazoles. The approach described here screened 1,210 compounds in an induced fit manner – 214 azides, 896 alkynes and 100 triazoles – whereas screening the same fragment library as triazoles would have required the evaluation of 191,744 compounds.”

-"One correction so minor not worth changing the tick box for: the 1H spectrum from compound 3 seems to be duplicated, instead of having the 13C spectrum (p23 on the pdf I received to review)"

This error has been corrected.

Reviewer 2
-"In this manuscript Howell and Beekman revisit the Mcl-1/Noxa protein-protein interaction (PPI) for which they had applied in 2017 for the first time their clever “peptide directed binding” strategy to identify PPI-inhibitors (Ref. 24). Later, they developed this further to an in silico approach, which they have tested for the p53/hDMX or hDM2 PPI (Ref. 28). But as the two approaches were validated on two different PPIs, it has been so far impossible to judge which approach is more efficient. With the current manuscript, in which the authors applied their in silico approach to the Mcl-1/Noxa PPI, such comparison can now be made.

The authors used molecular docking to identify 10 azide and 10 alkyne fragments, resulting in 100 virtual triazoles which were docked to Mcl-1 in a second computational step. The top 10 triazoles (additionally optimized for ADMET properties) were synthesized and tested in a fluorescence polarization assay. 8 out of the 10 compounds showed and IC50 <10 µM, which is a quite impressive achievement (Table 1 and Figure 2). A thermal shift assay and a mitochondrial depolarization assay confirmed biophysical binding and cellular activity, respectively.

The manuscript is well written and reports important and interesting findings, which deserve to be published in RSC Chem. Biol. The development of PPI inhibitors is still a very a challenging topic and the progress reported in this manuscript will be highly appreciated.

However, before publication the following issues have to be addressed:

1) On page 2 in “Results and Discussion” the authors briefly mention the in silico docking process. Even after consulting the SI (which is also not very informative about this topic), I have many open questions.
The authors should give more information about the in silico screening process – as it lies at the heart of their manuscript. How many azide and alkyne fragments have been docked? Was there any bias through information from their 2017 Angew. Chem. paper (Ref. 24), which might have served as an input in their in silico procedure?"

We have expanded the results and discussion section to include the requested information as written below

“The crystal structure of Mcl-1/NoxaB (PDB ID 2NLA29) was modified to generate the virtual reactive semi peptide AAQLRRIGD-propargylglycine and covalent docking30,31 was used to model copper catalysed azide alkyne cycloaddition in silico allowing us to identify the top ten30 synthetically viable small molecule azides fragments, from a commercially available library of 214 azides, which fit into the site vacated by the other section of peptide. Similarly, the crystal structure was modified to generate α-azidoamide-KVNLRQKLLN, and covalent docking was performed to identify small molecule alkyne fragments from a commercially available library of 869 alkynes. To avoid bias the library was taken directly from the Merck catalogue of available reagents. The top 10 azide and top 10 alkyne fragments were used to create a virtual library of 100 triazoles which was docked to Mcl-1 in an induced fit manner to score and rank the library.”

We would happily take more guidance on other details which are unclear from the text, but after trying to re-evaluate our own writing other details did not emerge to us, likely because we are too close to the work.

-"2) I am wondering why in their current manuscript the L-Fmoc-propargyglyince-OMe fragment delivered the best results (Table 1), which is in contrast to their Table 2 in their 2017 Angew. Chem. paper (Ref. 24), where exclusively the D-Fmoc-propargyglyince-OMe fragment was used for the triazole-based inhibitors? Does the absolute configuration of the amino acid building block has no important influence on binding?"

This was a mistake in the production of images and has been corrected, and clarified in the text and SI. The propargyl glycine methyl ester used for small molecule synthesis was D.

-"3) Have the authors tried to perform an in silico docking of a virtual library of triazoles, without having identified before suitable fragments through their peptide directed binding approach? Would this not be more straightforward? Are there any disadvantages, such as more CPU time, etc. which would speak against it?"

This question has been addressed in response to the same question posed by reviewer 1.

In conclusion, I can recommend the publication of this manuscript in RSC Chem. Biol. once the issues raised in this review report will have been addressed.

-"Minor comments:
p.3. right column on several occasions second paragraph: “Compounds 5-8” instead of “Compound 5-8”"

These errors have been corrected throughout the text.

Again, we want to thank the reviewers and editor for their time in evaluating the manuscript, and their comments which have improved the quality of the work. We hope that the manuscript in its current form is of a quality high enough to meet the standards required for publication in RSC Chemical Biology.

Kind regards,
Andrew Beekman

07-Nov-2020

Dear Dr Beekman:

Manuscript ID: CB-ART-08-2020-000148.R1
TITLE: In silico peptide-directed ligand design complements experimental peptide-directed binding for protein-protein interaction modulator discovery

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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Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

Reviewer 2

in this revised version of the manuscript Beekman et al. have addressed all issues raised by the reviewers of the original manuscript. In my point of view the manuscript has become even stronger and is now better readable, and it should be published in RSC Chemical Biology