From the journal RSC Chemical Biology Peer review history

26-Jul-2021

Dear Dr Wilson:

Manuscript ID: CB-COM-06-2021-000137
TITLE: Towards Optimizing Peptide-Based Inhibitors of Protein-Protein Interactions: Predictive Saturation Variation Scanning (PreSaVS)

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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I look forward to receiving your revised manuscript.

Yours sincerely,

Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

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Reviewer 1

The manuscript submitted for publication as a communication in RSC Chem. Biol entitled “Towards Optimizing Peptide-Based Inhibitors of Protein-Protein Interactions: Predictive Saturation Variation Scanning (PreSaVS),” describes the computational workflow for sequence mutation of peptide ligands to probe binding to relevant protein targets. Previous work by the authors has delivered a computational alanine scanning mutagenesis tool (BUDE Ala Scan). The key development here is the provision of a tool that can estimate improvements in binding and not just the loss of binding. The methods were exemplified computationally and experimentally in alpha helical and beta sheet peptide based protein-protein interaction inhibitors that both exhibited equivalent binding despite bearing computationally predicted modifications to key residues. Interestingly, the modifications also affected key physical properties such as secondary structure and proteolytic stability and could be rationalised.

In my view, this is a useful and timely new tool for those working in the field of peptide drug design and in understanding protein-protein interactions. Compared with related software, this approach is straightforward and accessible to many researchers. As such, the new findings here are very likely to be adopted by the community and will be a significant asset. This will be of broad interest to a wide range of readers of your journal.

The manuscript is well written, clearly and concisely described and has been carefully proof-read as there are no typographical errors that I could see. Figures and data in ESI are displayed clearly for the reader to interpret. The methods are thoroughly described in the ESI and are clear and appropriate for the work. The conclusions and interpretations of results are consistent with the data obtained.

One question that I have, is whether the authors expect that these approaches (demonstrated in alpha helix and beta sheet) would work equally well with a less structured peptide ligand. Maybe a brief comment on this would be beneficial for readers.

Overall, my recommendation is to accept the manuscript for publication without further amendments.

Reviewer 2

The authors previously developed BUDE for energy calculations (Chem. Sci., 2017) and reported an alanine scanning method based on BUDE (ACS Chem. Biol., 2019). In this study, they further extended the usage of BUDE. A computational workflow called PreSaVS was reported. PreSaVS computationally substitutes each residue in a peptide sequence of interest to 16 of the proteinogenic amino acids and calculates the difference in binding free energy relative to the native sequence. The study tested the workflow on the NOXA-B/MCL-1 complex (2JM6) and the SIM/SUMO complex (2LAS). The paper is interesting and is written in a clear and concise manner. And I think the workflow may be useful for study PPIs. However, to fully validate that the workflow could be widely used in studying various PPIs, it is better to test the workflow on more new systems. The NOXA-B/MCL-1 complex (2JM6) and the SIM/SUMO complex (2LAS) have already been used in the study of BUDE Alanine Scanning.

Dear Professor Yang,
Please find enclosed a revised manuscript entitled “Towards Optimizing Peptide-Based Inhibitors of Protein-Protein Interactions: Predictive Saturation Variation Scanning (PreSaVS)” that we (Hetherington et al.) hope is now considered suitable for publication in RSC Chemical Biology. We thank the referees for the evaluation of the manuscript and constructive comments. Changes to the text, which include several of our own modifications to improve readability have been made with the significant changes in response to the referees highlighted yellow; below we summarize changes made in response to the comments.
Reviewer 1
One question that I have, is whether the authors expect that these approaches (demonstrated in alpha helix and beta sheet) would work equally well with a less structured peptide ligand. Maybe a brief comment on this would be beneficial for readers.
We have added the following sentence to the conclusion: ’We expect the approach to be useful for other topologies e.g. loops, and, whilst NOXA and SIM are intrinsically disordered their PPIs are well defined; thus utility of the approach for “fuzzy” interactions remains to be explored.’ We feel this more accurately reflects some of the challenges that remain to be addressed as both our model PPIs involve peptide ligands derived from intrinsically disordered regions and so are ‘less structured’, but adopt interactions with well defined interactions whereas disordered complexes are likely to prove more challenging.
Reviewer 2
I think the workflow may be useful for study PPIs. However, to fully validate that the workflow could be widely used in studying various PPIs, it is better to test the workflow on more new systems. The NOXA-B/MCL-1 complex (2JM6) and the SIM/SUMO complex (2LAS) have already been used in the study of BUDE Alanine Scanning.
We consider NOXA75-93/MCL-1 and SIM2705-2717/SUMO to be ideal exemplar model systems in this work precisely because they were demonstrably suitable in our prior study on Alanine Scanning (ACS Chem. Biol., 2019, 14, 2252). It is entirely necessary to have confidence in the behaviour of theses experimental exemplars to ensure that the predictive saturation variation scanning described here, which differs from the earlier work in exploring sequence space beyond Alanine, can be used to genuinely identify new binding information, which we show to be the case. In the context of the number of systems reported in this work, it is necessary to have a sufficient number to demonstrate the approach works; the consensus of the reviewers seems to be that the data provide this evidence. More systems could always provide further evidence, but we are of the opinion that inclusion in this submission is beyond the scope of demonstrating the validity of the approach and the manuscript warrants publication with no additional new data, so that the wider community can adopt the approach where applicable. That said, the method has already been successfully applied to alternative systems by members of our team, specifically peptide based modulators of the main protease (Mpro) of SARS-CoV-2 (https://www.biorxiv.org/content/biorxiv/early/2021/06/19/2021.06.18.446355.full.pdf). This new paper which is currently under review represents a separate study with a clear focus on drug discovery and we feel it is entirely appropriate to describe the methodology in a self-contained manuscript as we have done for the manuscript under consideration here. We have added a citation to this BioRxiv paper and modified the final sentence accordingly: ‘Ongoing studies are focussed on exploring the scope to identify affinity enhancing and/or selectivity modifying variations across a broader array of PPI targets.43’
In summary, we thank the reviewers for suggesting these changes and additions, which we feel has improved our manuscript and we hope will now be able to be accepted for publication in RSC Chemical Biology. If you should require any further information then please contact me. In the meantime, we look forward to receiving your response.
Kind Regards

Andy Wilson
Prof Andrew J Wilson PhD. BSc. (Hons) FRSC, FHEA
Astbury Centre for Structural Molecular Biology

30-Jul-2021

Dear Dr Wilson:

Manuscript ID: CB-COM-06-2021-000137.R1
TITLE: Towards Optimizing Peptide-Based Inhibitors of Protein-Protein Interactions: Predictive Saturation Variation Scanning (PreSaVS)

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,

Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

Reviewer 2

The authors have successfully addressed my concerns.