From the journal RSC Chemical Biology Peer review history

22-Nov-2023

Dear Dr Yudin:

Manuscript ID: CB-ART-10-2023-000201
TITLE: Cell penetration of oxadiazole-containing macrocycles

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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Yours sincerely,
Claudia Höbartner
Associate Editor, RSC Chemical Biology

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Reviewer 1

The authors describe the synthesis of a series of macrocyclic peptides containing an oxadiazole graft based on previously reported macrocycles but now exchanging one of the amino acids for a chloroalkane modified lysine (Lys(ct)). The chloroalkane is required for the CAPA assay that can be used to assess the cell permeability of a molecule. Next, the 8 prepared molecules were tested in the CAPA assay. Peptide 1, where a glycine in the original molecule was replaced by the Lys(ct) proved to be highly permeable especially in comparison to compound 8 which lacked the oxadiazole graft. When the position of the Lys(ct) was changed, strong effects were observed in CAPA cell uptake. Adding polar residues to the peptide also decreased the cellular uptake. Next, the PAMPA assay as a model for passive membrane permeability was used which showed that all compounds had low passive membrane permeability. To gain insight into the conformation of the peptides NMR experiments were performed. These included variable temperature experiments to determine solvent shielding of backbone amide NH groups, as well as ROESY measurements to determine solution structures based on distance constraints. These results demonstrated that the beta-turn was intact in all structures but compounds 4a and 6a had a very different conformation in the other parts. I find the paper interesting and to provide very useful insights into the difference between passive and active uptake of macrocycles. I fully agree with the conclusion that a focus on passive permeability might result in overlooking compounds that are cell permeable via other mechanisms. However, in some places in the manuscript the author do seem to contradict themselves regarding these two different mechanisms.

- One of the contradicting statements is: “This drastic change in cell penetration upon removal of the lipophilic residues suggests that these sites are essential for efficient cell penetration, consistent with a role in masking solvent-exposed amides.” This effect would only be relevant to passive permeability but relates to the CAPA assay results. I think the text here should be changed or removed to avoid confusion of the reader.
- Another part of the text where clarification is needed is the discussion relating to the HPLC retention time and the CAPA results. Concluding that more hydrophobic peptides are more permeable seems fine, but whether this has to do with the polar surface areas or conformation is not relevant anymore as long as the uptake mechanism is not passive permeability and otherwise unknown. Please alter the text here to make it more clear.
- The authors state that it is important to measure degradation of chloroalkane-tagged compounds for the CAPA assay. Although this is relevant for the chloroalkane itself so it can react with the HaloTag protein, it is not relevant for the permeability since the peptides are only exposed to the proteases once they have entered the cell. As it is now unclear whether degradation of the chloroalkane was meant the text should be changed to clarify this.
- For the NMR studies, Boc or Dde protected equivalents were used. However, no explanation for doing so is given. This needs to be added.

Reviewer 2

This is an interesting study that investigates the cell permeability of a new class of oxadiazole-containing (Odz) cyclic peptides and SAR related to this property. The authors utilized the CAPA assay previously developed by the Kritzer group along with PAMPA assays and NMR structural studies to correlate cell penetration ability with passive permeability and peptide conformation. The research is rigorous and very well described. The results, which ultimately show the impact of subtle structural/conformational changes in the peptides on the cell penetration properties of the peptide, are interesting, novel and relevant to the community working on peptide-based probes and therapeutics. Because of the high quality of the report, publication is recommended ‘as is’.

Minor corrections:
1) Abstract: “To elucidate the cell-penetrating ability of oxadiazole-containing (Odz),” is missing ‘peptides’

December 12, 2023



To: Dr. Claudia Höbartner

Dear Dr. Höbartner:

Thank you very much for your assessment of our paper.

My co-workers and I have carefully addressed all of the referee’s comments. Below you will find our responses.

Reviewer 1:

One of the contradicting statements is: “This drastic change in cell penetration upon removal of the lipophilic residues suggests that these sites are essential for efficient cell penetration, consistent with a role in masking solvent-exposed amides.” This effect would only be relevant to passive permeability but relates to the CAPA assay results. I think the text here should be changed or removed to avoid confusion of the reader.

We thank the reviewer for pointing out this confusion. To address this, we removed the “role in masking solvent-exposed amide” as this statement might confuse the readers about how this property relates to CAPA study. We have replaced this statement with “binding the membrane, promoting cellular uptake, and/or promoting endosomal escape.”

Another part of the text where clarification is needed is the discussion relating to the HPLC retention time and the CAPA results. Concluding that more hydrophobic peptides are more permeable seems fine, but whether this has to do with the polar surface areas or conformation is not relevant anymore as long as the uptake mechanism is not passive permeability and otherwise unknown. Please alter the text here to make it more clear.

We thank the reviewer for this comment. We decided to remove the statement “We surmised that this difference may be due to conformational effects” as it removes the confusion of connecting the lipophilicity data we observed from HPLC to the conformational investigation that was done.

The authors state that it is important to measure degradation of chloroalkane-tagged compounds for the CAPA assay. Although this is relevant for the chloroalkane itself so it can react with the HaloTag protein, it is not relevant for the permeability since the peptides are only exposed to the proteases once they have entered the cell. As it is now unclear whether degradation of the chloroalkane was meant the text should be changed to clarify this.

We thank the reviewer for allowing us to clarify this point. To address this concern, we added “Since it is known that exogenously added material often becomes trapped in endosomes or in the lysosome, testing for degradation by cellular proteases ensures that the peptide is not degraded in an intracellular compartment which would release a free chloroalkane and produce a false positive signal in CAPA” to clarify the justification for doing this investigation.

For the NMR studies, Boc or Dde protected equivalents were used. However, no explanation for doing so is given. This needs to be added.

We thank the reviewer for allowing us to further elaborate this point. We reworked the sentence to “Representative compounds 1a-7a, in which the Lys residues are either protected by Boc or Dde, were used due to their ease of synthesis to perform NMR investigation for their respective ct-compounds 1 to 7 (Figure 5)” which explains that the ease synthesis were the reasons why Boc and Dde protected compounds were used for NMR studies.

Reviewer 2:

Abstract: “To elucidate the cell-penetrating ability of oxadiazole-containing (Odz),” is missing ‘peptides.’

We thank the reviewer for editing the abstract. As instructed, we have added “peptides” in this sentence.


Sincerely yours,

Professor Andrei K. Yudin
FRSC (UK), FRSC (Canada)
Canada Research Chair in Medicine by Design
Associate Editor, Chemical Science

Davenport Research Laboratories
Department of Chemistry
University of Toronto
80 St. George Street
Toronto, Ontario
M5S 3H

21-Dec-2023

Dear Dr Yudin:

Manuscript ID: CB-ART-10-2023-000201.R1
TITLE: Cell penetration of oxadiazole-containing macrocycles

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your manuscript for publication in its current form.

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Claudia Höbartner
Associate Editor, RSC Chemical Biology


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