From the journal RSC Chemical Biology Peer review history

20-Jul-2020

Dear Dr Troeira Henriques:

Manuscript ID: CB-ART-06-2020-000099
TITLE: Cyclic peptide scaffold with ability to stabilize and deliver a helical cell-impermeable cargo across cancer cell membranes

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Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

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Reviewer 1

The manuscript by lawrence et al is a nice exercise of molecular grafting and targeting.
In my opinion it makes a big effort in the direction of characterization of the peptide- membrane interactions.
It is true that a 2-fold increase in selectivity is a mild targeting tool but what is really interesting is the mitochondrial accumulation that ha a big potential as a delivery tool for some mitochondrial diseases.

Reviewer 2

The work by Lawrence et al enlights important aspects of the action of CPPs but needs clarification in some aspects.

1. Consider adding "in culture" to the title to better inform readers ("...across membranes of cancer cell in culture")

2. "PF4 peptide" in the abstract is cPF4PD? A single form should be used.

3. The last two paragraphs of Introduction belong to Materials and Methods, and Results.

4. Figure 3: What equations were used to fit the data? What statistical method was used for the fittings?

5. Table 2: CC50 should be defined in the title. WT, mut and WT(rep) are not defined.

6. Table S1: Footnotes b and c should refer to Figure 3. Calculation of P/Lmax is ill described. Is P/Lmax>1 reasonable? How to explain?

To Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology


Re: CB-ART-06-2020-000099


Dear Professor Nikolovska-Coleska thank you for your email of 20th July 2020 with the editorial decision on our manuscript. We very much appreciate the referees’ analysis of the manuscript and your invitation to submit a revised version. We have addressed the suggestions from the reviewers.

A marked copy of the revised manuscript with changes highlighted in yellow and a point-by-point detailed response with description of the changes are included. This submission includes supporting information for publication.

We thank the reviewers for their helpful comments and you for editorial handling of the manuscript.

Yours sincerely,

Sónia Henriques


RESPONSE TO REVIEWERS
Thank you for your comments on the manuscript. We very much appreciate your suggestions and have addressed any concerns, as below:

Reviewer #1:
Comment 1: The manuscript by lawrence et al is a nice exercise of molecular grafting and targeting. In my opinion it makes a big effort in the direction of characterization of the peptide- membrane interactions. It is true that a 2-fold increase in selectivity is a mild targeting tool but what is really interesting is the mitochondrial accumulation that has a big potential as a delivery tool for some mitochondrial diseases.
Response: We thank the reviewer for their positive comments on this manuscript.

Reviewer #2:
Comment 1: Consider adding "in culture" to the title to better inform readers ("...across membranes of cancer cell in culture")
Response: The title has been changed to “Cyclic peptide scaffold with ability to stabilize and deliver a helical cell-impermeable cargo across membranes of cultured cancer cells”.

Comment 2: "PF4 peptide" in the abstract is cPF4PD? A single form should be used.
Response: The term “PF4 peptide/s” refers to three related peptide scaffolds; cPF4PD (disulfide cyclized dimer), PF4PD (open dimer), and ctPF4PD (thioether cyclized dimer), see page 5. To make this clearer, the text in the abstract has been changed to “We hypothesized that cPF4PD and PF4-derived peptide analogues…”.

Comment 3: The last two paragraphs of Introduction belong to Materials and Methods, and Results.
Response: The second last paragraph from the Introduction has been removed. Instead, the rationale behind the design of the PF4-pDI grafted peptides is now included in the Peptide Design section of the Results and Discussion section (page 6).
The final paragraph has been replaced with a more succinct overview that focuses on the approaches that have been included in the study to allow reporting of target-specific versus membrane-specific peptide activity (page 5).

Comment 4: Figure 3: What equations were used to fit the data? What statistical method was used for the fittings?
Response: Non-linear regressions used to fit the data (presented in Figures 3, 4 and 6) are described in the Methods and Materials section under the heading Non-linear regressions. This section has been updated to include Figure callouts for the corresponding curves. Graphpad Prism software was used to fit the curves, and the equations and statistical methods used are now provided, see pages 30-31. This information has also been included in the caption of Figure S7 in the Electronic Supplementary Information.

Comment 5: Table 2: CC50 should be defined in the title. WT, mut and WT(rep) are not defined.
Response: The title for Table 2 has been edited to “Cytotoxic concentration of PF4-derived peptides and grafted peptides required to kill 50% of cells (CC50) for cultured cell lines and isolated blood cells”. p53 status is now defined in the legend of Table 2 as “p53 status is wild-type (WT), mutant (mut), repressed (rep).”

Comment 6: Table S1: Footnotes b and c should refer to Figure 3. Calculation of P/Lmax is ill described. Is P/Lmax>1 reasonable? How to explain?
Response: Table S1 footnote reference to Figure 3 has been corrected. We have included more information about calculation and meaning of P/Lmax. This is defined as the P/L at binding saturation in the results & discussion (page 9), and calculation from fitted SPR dose-response curves (Figure 3A) is explained in the material & methods (page 31) and supplementary information (footnote b, Table S1). Thanks for noticing the inconsistency of P/Lmax>1; this binding curve does not reach saturation and therefore the P/Lmax cannot be accurately determined. This is now mentioned in supplementary information (footnote f, Table S1).

01-Oct-2020

Dear Dr Troeira Henriques:

Manuscript ID: CB-ART-06-2020-000099.R1
TITLE: Cyclic peptide scaffold with ability to stabilize and deliver a helical cell-impermeable cargo across membranes of cultured cancer cells

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,

Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

Reviewer 3

The authors have addressed all the comments satisfactorily. While not strongly potent, I agree that the development and evaluation of these peptides are of high interest as a proof-of-principle, and that the work highlights the importance of characterizing peptide–membrane interactions during the development of peptide-based drug leads. I therefore recommend acceptance of this work.