From the journal RSC Chemical Biology Peer review history

19-Jun-2021

Dear Dr Li:

Manuscript ID: CB-ART-04-2021-000087
TITLE: Entropy of Stapled Peptide Inhibitors in Free State is the Major Contributor to the Improvement of Binding Affinity

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.

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I look forward to receiving your revised manuscript.

Yours sincerely,
Prof Seung Bum Park

Associate Editor, RSC Chemical Biology
Professor, Chemistry Department, Seoul National University, Korea

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Reviewer 1

Stapled peptides can improve their binding affinity to the target protein by locking their bioactive conformation, and therefore is a nice family of promising inhibitors for targeting protein-protein interactions. It has long been believed that the enhanced binding affinity can benefit from the entropy gain. However, there is a lack of in-depth study on the origin of the entropy effect, which could be due to the stabilization of stapled peptide in solution, or the increased flexibility of the complex after the binding of stapled peptide. In this manuscript, the authors quantitively demonstrated for the first time how entropy plays the dominant role in stapled peptide binding, and confirmed that the entropy gain is mainly due to the constrained-helix structure of stapled peptide in solution (free state). Additionally, using PSD-95 guanylate kinase (GK) domain as a target, the authors successfully designed a highly active stapled peptide, of which the binding affinity is increased by an order of magnitude compared to its corresponding linear counterpart. In my opinion, this work is very suitable for publication in RSC Chem Biology.
Before accepting, the following issues should to be revised.
(1) It is recommended to give a general synthetic route for the involved stapled peptides.
(2) The quality of Picture 6 is low. The authors should redraw the picture to make it more readable.
(3) The membrane permeability of peptides is related to many factors, including the concentration of the tested peptide, the composition of the used buffer, incubation time, incubation temperature, and the state of the cells before imaging (live cells or fixed cells). It will be more interesting to provide live cell imaging.
(4) It is recommended to point out the specific site of FITC modification in the sequence of the stapled peptide.
(5) In page 14, 50µg/mL ,10µM and 20µM should be replaced by 50 µM, 10 µM, and 20 µM.

Reviewer 2

This manuscript makes a valuable contribution to our understanding of the role of conformational entropy in the binding affinities of stapled peptides for proteins. Such questions are of great importance given the myriad roles that protein-protein interactions (PPIs) play in biology, how difficult PPIs are to target with small molecules, and the promise of stapled peptides for filing this gap. The authors use a combination of ITC, crystallography, and free energy calculations to design/evaluate (stapled) peptide inhibitors of the guanylate kinase (GK). The main conclusion is that reducing the entropy of the unbound peptide significantly improves its binding affinity, while the entropic benefit of increasing the entropy of the bound state is negated by entropy-enthalpy trade offs.

My sense is that most people already agree with this conclusion, but maybe don’t have such clean data to support the model. The authors explain this well in the introduction, but I still think it’s the only real weakness in the manuscript. I don’t have a good suggestion on how to improve, but thought I’d mention it in case my comment inspires the authors.

My only other (extremely minor comment) is that I didn’t quite follow the logic for why the results of introducing the phosphomimetic (glutamic acid) led the authors to consider stapled peptides. Can the authors explain that jump in a sentence?

Point-by-point response to the comments made by the reviewers

We greatly appreciate the reviewers’ constructive comments and suggestions.

Reviewer 1:
(1) It is recommended to give a general synthetic route for the involved stapled peptides.
Response: A general synthetic route for the involved stapled peptides is added on page 25 (Figure 2). The detailed synthetic pathway of the involved stapled peptides is in Figure S2A & S2B.

(2) The quality of Picture 6 is low. The authors should redraw the picture to make it more readable.
Response: We thank the reviewer for the suggestion and have updated Figure 6 (currently Figure 7) to improve its resolution.

(3) The membrane permeability of peptides is related to many factors, including the concentration of the tested peptide, the composition of the used buffer, incubation time, incubation temperature, and the state of the cells before imaging (live cells or fixed cells). It will be more interesting to provide live cell imaging.
Response: Thanks for the comment and suggestion. A live cell imaging data would complement the presented imaging results obtained using fixed cells. However, in the current experimental paradigm, live cell imaging to detect penetrated peptide signals in the cells will not be feasible. In our experiments, each FITC-labelled peptide was added to the cell culture media, and thus the media contains excess amount of the peptide (i.e. non-penetrated peptides). Without washing these non-penetrated peptide away before imaging, one cannot differentiate the fluorescent signals originated from the penetrated peptide vs the non-penetrated peptide in the media

(4) It is recommended to point out the specific site of FITC modification in the sequence of the stapled peptide.
Response: The specific site of FITC modification in the sequence of the stapled peptide is added on page 32 (Figure9).

(5) In page 14, 50µg/mL ,10µM and 20µM should be replaced by 50 µM, 10 µM, and 20 µM.
Response: The above mentioned are revised accordingly.

Reviewer 2:
Explain why the results of introducing the phosphomimetic (glutamic acid) led the authors to consider stapled peptides.
Response: Based on our ITC experiment, replacement of the phosphorylated serine to glutamic acid diminished the binding affinity (Figure S1B). Thus, a new strategy to improve the binding affinity of the phosphomimetic peptide was required. According to the previously published crystal structure (Cell Rep, 21:13, (2017)), the phosphor-peptide forms α-helical structure when bound to the GK domain. This observation inspired us to consider stapled peptides, which can restrain the peptide conformation in α-helical structure to improve the binding affinity of the peptide inhibitors. To clarify this point, we have modified the following sentence.
“To improve the binding affinity, we propose the introduction of staple to the linear peptide, which can restrain the peptide in α-helical structure mimicking the p-SAPAP1 ligand observed in the crystal structure of GK domain bound with p-SAPAP1 (22).”

24-Jun-2021

Dear Dr Li:

Manuscript ID: CB-ART-04-2021-000087.R1
TITLE: Entropy of Stapled Peptide Inhibitors in Free State is the Major Contributor to the Improvement of Binding Affinity

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,

Prof Seung Bum Park

Associate Editor, RSC Chemical Biology
Professor, Chemistry Department, Seoul National University, Korea

Reviewer 1

The revised manuscript can now be accepted for publication.