From the journal RSC Chemical Biology Peer review history

Berlin, 18. June 2021

Dear Dr Abell:

Manuscript ID: CB-ART-05-2021-000113
TITLE: A cell permeable bimane-constrained PCNA-interacting peptide

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,
Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

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

Reviewer 1

The manuscript describes the development of PCNA-binding peptidomimetics derived from the PIP binding domain of p21. Macrocyclisation was achieved through dithiol bis-alkylation using a series of five different linkers. The synthesis of these molecules is presented in scheme 1. The purity of peptidomimetics 3,4 and 5 are above 90%, however the purity of peptides 1 (81%), 2 (84%), 6 (88%) and 7 (81%) is below the expected level for a journal such as RSC Chemical Biology. This is a particular concern regarding the most active compound 7.
The peptidomimetics were assessed for binding to PCNA using SPR, with peptidomimetic 7 demonstrating good binding affinity (571 nM), yet interestingly not as good as the linear control peptide (102 nM). This suggests an induced fit binding mechanism and that peptide flexibility is a requirement for binding. The mechanism of binding was then investigated using X-ray crystallography and computational docking. The conclusions from this study are well rationalised using the data obtained. NMR was then used for conformational analysis of peptidomimetic 7. The conclusion that the peptide is flexible, yet adopts the bioactive 310-helix in solution is not valid because the compound is not pure, linear deletion impurities would corrupt this data. However, this could again suggests peptide flexibility is a key structural characteristic for binding. The most impactful aspect of the work is the cell imaging. Peptidomimetic 7 is cell permeable, although gets stuck in endosomes and so does not actually get to the nucleus where the target is located! The native linear control does not enter the cells. This is extremely significant for a number of reasons. This data suggests that constrained, yet flexible peptides can be cell permeable. Peptidomimetic 7 is constrained by a bimane based linker that is fluorescent. The molecule therefore does not need to be modified for cell imaging experiments. To my knowledge, this is the first time this has been achieved and is an exceptional new design strategy for conformationally constrained peptides. The impact of this work, if the compound purity is addressed, would likely resonate through the peptide community and beyond and thus would absolutely be appropriate for publication in RSC Chemical Biology. As such, this needs to be highlighted in the abstract, in the main body of the manuscript and in the conclusions.

Other corrections:
• Scheme 1 – compounds 5,6 and 7 synthesis, not clear why e and f required for compounds 3 and 4 but not 5, 6 and 7.
• Table 1, present standard error as ± rather than a separate column, for example peptide 1 – 102.3±5.3
• ‘observation is line with our previous study’, should read ‘observation is in line with our previous study’
• explain i,i+4 vs i,i+3 constraint is applied. This is explained in the previous paper however should be rationalised for 310-helix here.
• Why was the longer p21 peptide and not peptide 1 crystalised and used as the model for docking?
• discuss why a crystal structure of their highest affinity compound (7) was not obtained.
• a good addition to the conformational analysis would be circular dichroism particularly when the secondary shifts observed using NMR were noted to be small.
• Page 2, paragraph 1, line 2: The three letter code of glutamic acid (Glu) should be included, in keeping with Lys and Dab. Similarly, this should be considered for cysteine later in the paragraph.
• Page 2, paragraph 1, line 11: Surface Plasmon Resonance does not require capitalisation.
• Figure 1 legend, A: (3) suggests a compound indicator number. The colour coding shows three PCNA monomers, so either replace with (three) or remove altogether to avoid confusion.
• Figure 1 legend, part B: Inter-Domain Connecting Loop does not require capitalisation.
• Page 2, paragraph 2: Amino acid three letter codes are not being used. This varies throughout the manuscript and needs to be consistent.
• Scheme 1 legend, 3-7b: Provide reagent equivalents for acetylation conditions, in keeping with other reactions detailed in this legend.
• Page 3, paragraph 2, line 3: Remove “however,” to simplify sentence.
• Page 4, paragraph 1, line 2: Remove comma after “macrocyclic peptide 3”.
• Page 4, paragraph 1, line 4: Adjust “The more rigid trans-butenyl-based linker in peptide 5, resulted in a KD value for PCNA of 2.82 M and the binding affinity for PCNA of aromatic m-xylene linked peptide 6, with a 9 atom linker, was lower again at 3.86 M” to “The more rigid trans-butenyl-based linker in peptide 5 resulted in a KD value for PCNA of 2.82 M, and the binding affinity for PCNA of aromatic m-xylene linked peptide 6 (9 atom linker) was lower again at 3.86 M” to improve the flow of the sentence.
• Page 4, paragraph 1, line 6: Restructure to “Considered together, these observations suggest…”.
• Page 4, paragraph 1, line 9: Remove comma after “9 atoms”.
• Page 4, paragraph 2, line 5: Include a note on why crystal structures were not obtained for macrocycles 4 and 7, particularly as peptide 7 is the macrocycle with highest binding.
• Page 5, paragraph 1, line 9: Remove the comma after “(4.4 Å)”.
• Page 5, paragraph 2, line 4: Change to “(Figure S4):” and then list the H-bond interactions, separated by ;.
• Page 5, paragraph 3, line 6: The sentence beginning with “Additionally, this conformation does not appear well stabilised” needs to be rephrased, for clarity.
• Page 6, paragraph 1, line 2: Place the comma before “except” instead of after.
• Page 6, paragraph 2, line 10: Should read “a 2.7 Å hydrogen bond”.
• Page 9, paragraph 1, line 9: Remove “however,” and include “indicates”.
• Page 9, paragraph 1, line 10: Remove “importantly”.
• Abstract image: Linker structures cover PCNA image below.
• Supplementary page 4, fluorescein attachment section: N-terminal requires italicisation.
• Supplementary page 5, paragraph 1, line 1: E. coli requires italicisation.

Expt
• Table 1 peptides 1 and 2 sequences (page 4) don’t match experimental.
• L-amino acids, L needs to be font size 10 vs 12 (throughout)
• Provide yields of peptides in method.
• Provide characterisation data with method for peptide 8
• Provide RP-HPLC spectra to demonstrate peptide purity <95%

Reviewer 2

The manuscript by Abell and coworkers describes the structure-based design of macrocyclic peptides targeting the PCNA protein which is critically involved in DNA replication and repair. Based on previously described macrocyclic peptides (Chem. Eur. J. 2018 – by the same team) a broader set of crosslinks was now tested and very toughly investigated using X-ray crystallography, SPR, NMR and modelling. The findings shed light on the structural prerequisites for high affinity binding – it should be noted that highest affinity derivative 7, exhibits still lower affinity than the linear wt sequence. The study is concluded with cell-permeability studies, which would indeed be an informative addition – however, the experimental setup is not appropriate (details below). Overall, the study is well performed and reported and given the points below have been addressed, I recommend publication in ACS Chem Biol.
Major:
1) Investigating the cellular uptake of labelled peptides after cell fixation tends to generate false positive results as membrane associated peptides show a high tendency to spread after fixation. That is why, it is not used for this purpose anymore. Alternatively, live-cell confocal or MS-based read-outs would be more suitable. If a fluorescent label is used for readout, the same fluorophore should be used. In addition, it is crucial to include a positive control, e.g. a cell-penetrating peptide.
Minor:
2) It would be informative to show one of the crosslinked structures reported earlier (Chem. Eur. J. 2018) as they served as direct precursors and involved the same modification sites.
3) The corresponding affinity SE values in Table 1 should have the same decimal digits: e.g. “769+-78“ instead of “769.1+-78“

Manuscript ID: CB-ART-05-2021-000113
TITLE: A cell permeable bimane-constrained PCNA-interacting peptide

Dear Prof. Dr. Roderich Süssmuth,

We have carefully read all of the reviewer’s comments and thank them for their thorough, insightful and complementary feedback. We have considered all of their suggestions and acted on all comments, with a point-by-point response to all comments included below. The Reviewer’s comments are in bold, our response is directly below in regular text. Any changes made to the manuscript are indicated in blue font, and changes to the manuscript text are italicised. Additionally, we have included an amended version of the manuscript and supplementary information, in their final edited form, and a separate copy with all changes highlighted by “Track Changes”. In addition to the Reviewer’s feedback we have amended a couple of small typographical errors in the text, and updated the details for recently released publications in the Reference list.

We believe the revised manuscript demonstrates a significant improvement, and hope that you find it acceptable for acceptance in RSC Chemical Biology. Thank you for your consideration and we look forward to your response.

Kind regards,
Aimee Horsfall, on behalf of all authors





Referee: 1
Comments to the Author
The manuscript describes the development of PCNA-binding peptidomimetics derived from the PIP binding domain of p21. Macrocyclisation was achieved through dithiol bis-alkylation using a series of five different linkers. The synthesis of these molecules is presented in scheme 1. The purity of peptidomimetics 3,4 and 5 are above 90%, however the purity of peptides 1 (81%), 2 (84%), 6 (88%) and 7 (81%) is below the expected level for a journal such as RSC Chemical Biology. This is a particular concern regarding the most active compound 7.
In light of the reviewer’s comments regarding the peptide purity, reviewed the data and have concluded that the HPLC data included correlated to an earlier scouting experiment, and not our final experimental data. We apologise for this oversight, and have corrected this mistake. We have included the correct HPLC purity data in the experimental of each peptide, and the HPLC traces, starting on Page 7 of the ESI. We would also like to point out that the purity and quality of peptide 7, the most active compound, is evident in the NMR provided – in particular the 2D spectra provided which show no evidence of deletion products.

The peptidomimetics were assessed for binding to PCNA using SPR, with peptidomimetic 7 demonstrating good binding affinity (571 nM), yet interestingly not as good as the linear control peptide (102 nM). This suggests an induced fit binding mechanism and that peptide flexibility is a requirement for binding. The mechanism of binding was then investigated using X-ray crystallography and computational docking. The conclusions from this study are well rationalised using the data obtained. NMR was then used for conformational analysis of peptidomimetic 7. The conclusion that the peptide is flexible, yet adopts the bioactive 310-helix in solution is not valid because the compound is not pure, linear deletion impurities would corrupt this data.
We have included CD spectra for all macrocyclic peptides (ESI Page 21), as is suggested below, that supports the idea of flexible macrocyclic that adopt a number of conformers.

However, this could again suggests peptide flexibility is a key structural characteristic for binding. The most impactful aspect of the work is the cell imaging. Peptidomimetic 7 is cell permeable, although gets stuck in endosomes and so does not actually get to the nucleus where the target is located! The native linear control does not enter the cells. This is extremely significant for a number of reasons. This data suggests that constrained, yet flexible peptides can be cell permeable. Peptidomimetic 7 is constrained by a bimane based linker that is fluorescent. The molecule therefore does not need to be modified for cell imaging experiments. To my knowledge, this is the first time this has been achieved and is an exceptional new design strategy for conformationally constrained peptides. The impact of this work, if the compound purity is addressed, would likely resonate through the peptide community and beyond and thus would absolutely be appropriate for publication in RSC Chemical Biology. As such, this needs to be highlighted in the abstract, in the main body of the manuscript and in the conclusions.
We thank the reviewer for their encouraging comments, and we have included an additional sentence in both the abstract and main text to better highlight the significance of this design.
Abstract, page 1: “The inherent fluorescence of the bimane moiety present in peptide 7 allowed it to be directly imaged in the cell uptake assay, without attachment of an auxiliary fluorescent tag. This highlights a significant benefit of using a bimane constraint to access conformationally constrained macrocyclic peptides.”
Conclusions, page 12 “Furthermore, these results emphasise the utility of the bimane moiety as a peptide linker as it can influence peptide structure, and the resulting peptides can be directly imaged without further derivatisation to investigate cell uptake of a bimane peptidomimetic. This allows the behaviour of the molecule of therapeutic interest to be assessed, instead of a related analogue that includes a fluorescent tag; and eliminates the need for an additional synthetic step. Attachment of an auxiliary fluorophore such as fluorescein to a peptide, can impair the target binding affinity (as seen for peptide 9) and may alter cellular permeability or intracellular distribution relative to the untagged analogue.”

Other corrections:
Scheme 1 – compounds 5,6 and 7 synthesis, not clear why e and f required for compounds 3 and 4 but not 5, 6 and 7.
The saturated alkyl bromides display low reactivity with thiols on-resin, as described by Zhang et al. (Angewandte 2018, Ref 21). We personally verified that addition of 1,3-dibromopropane or 1,4-dibromobutane with DIPEA in DMF to the peptidyl-resin did not yield any desired macrocyclic product. We have included a sentence to further clarify the difference in macrocyclization conditions.
Page 3: “Reaction of the resin-bound peptides with 1,3-dibromopropane or 1,4-dibromobutane required addition of NaI to generate the more reactive alkyliodides in situ,21 whereas reaction with trans-1,4-dibromobut-2-ene, m-dibromoxylene or dibromobimane proceeds under mild basic conditions (in DMF with DIPEA).23.”

Table 1, present standard error as ± rather than a separate column, for example peptide 1 – 102.3±5.3
This has been corrected, please see Table 1, Page 4.

‘observation is line with our previous study’, should read ‘observation is in line with our previous study’
This has been corrected
Page 12: “This observation is in line with our previous study19”

explain i,i+4 vs i,i+3 constraint is applied. This is explained in the previous paper however should be rationalised for 310-helix here.
Our structure guided design in the Design & Synthesis section describes this decision, page 2: “Positions 145 and 149 were chosen for modification as these side-chains are close in space when bound to PCNA (Figure 1C), and were successfully linked in a p21 lactam bridge peptide in our earlier study to stabilise the 310-helical binding conformation.19”.
To further clarify our design we added a second sentence:
Page 2: “This structure-informed design gave rise to an i-i+4 constraint, although i-i+3 constraints are more commonly reported for 310-helices. ”

Why was the longer p21 peptide and not peptide 1 crystalised and used as the model for docking?
The longer peptide, p21(141-155) was crystallised for a previous publication (Horsfall, J Biol Chem, 2021) and we have indicated that this peptide was from a different publication with appropriate references throughout the Results & Discussion. We were unfortunately not able to obtain a crystal structure of the shorter p21 variant, p21(143-154) aka Peptide 1, and so the crystal structure of p21(141-155) served as an appropriately similar starting point for our modelling experiment of peptide 1. However, the modelling experiments for macrocyclic peptides 4 and 7, which we were also unfortunately unable to obtained diffracting crystals of, were conducted using the crystal structure of peptide 3 as the starting structure as stated at the start of the Structural Analysis section (Page 5).
In order to clarify this, we have inserted the following sentences:
Page 5: “Attempts to obtain a co-crystal structure of peptides 1, 4 and 7 bound to PCNA were unsuccessful at this time. Peptide 1 was however modelled onto the PCNA surface, to confirm the short peptide interacted with the protein surface in the same manner (see Figure S1), and was constructed from the previously published structure of p21141-155 bound to PCNA (PDB: 7KQ1).13”

discuss why a crystal structure of their highest affinity compound (7) was not obtained.
We did endeavour to obtain a co-crystal structure of peptide 7 bound to PCNA, and despite much optimisation, though we did obtain crystals, these were not of sufficient quality to collect a diffraction pattern. We have mentioned our attempts to gain these crystals at the start of the Structural Analysis section as highlighted in our response above, also see the additional sentence added to Page 5 of the manuscript.
Page 5: “Attempts to obtain a co-crystal structure of peptides 1, 4 and 7 bound to PCNA were unsuccessful at this time.”

a good addition to the conformational analysis would be circular dichroism particularly when the secondary shifts observed using NMR were noted to be small.
We have collected CD data which is in support of flexible structures that adopt a number of conformers and have included these spectra in the ESI (Page 21), and included the following call-out to these data in the main-text.
Page 9: “Circular dichroism of peptide 7 displays a deep minimum near 200 nm (Figure S8) which is also consistent with a flexible structure. The computationally modelled structure of 7 bound to PCNA indicates the peptide backbone adopts the classic 310-helix defining hydrogen bonds expected for a PIP-box peptide when bound to PCNA. This importantly demonstrates that the bimane linker does not restrict the peptide from adopting this key conformation, consistent with previous reports.25”

Page 2, paragraph 1, line 2: The three letter code of glutamic acid (Glu) should be included, in keeping with Lys and Dab. Similarly, this should be considered for cysteine later in the paragraph.
In light of this Reviewer’s comment further down, regarding the inconsistency of the use of three-letter codes or expanded amino-acid names, we have instead chosen to remove reference to (Dab) and (Lys) on Page 2. Please see the comment below for further explanation, regarding the review commented on Page 2 Paragraph 2.

Page 2, paragraph 1, line 11: Surface Plasmon Resonance does not require capitalisation.
This has been changed as suggested:
Page 2: “…their affinity for PCNA determined by surface plasmon resonance (SPR).”

Figure 1 legend, A: (3) suggests a compound indicator number. The colour coding shows three PCNA monomers, so either replace with (three) or remove altogether to avoid confusion.
This has been corrected
Page 2, Figure 1 caption: “A Ring-shaped PCNA with three peptides (cyan) bound to the PIP-box binding site. PCNA monomers (three) shown..”

Figure 1 legend, part B: Inter-Domain Connecting Loop does not require capitalisation.
This has been changed as suggested:
Page 2, Figure 1 caption: ” B Single PCNA subunit with two domains shown in shades of grey and inter-domain connecting loop (IDCL)…”

Page 2, paragraph 2: Amino acid three letter codes are not being used. This varies throughout the manuscript and needs to be consistent.
We have reviewed our use three-letter codes compared to expanded amino-acid names. We have removed the mention to Lys and Dab in the Introduction (Page 2) and now believe we have been consistent throughout our manuscript. We have used three-letter amino-acid codes only when it refers to a specific peptide/protein residue and is followed immediately by the position number (e.g Phe150, His44). Expanded amino-acid names are used throughout the discussion as we feels this provides better readability and clarity.

Scheme 1 legend, 3-7b: Provide reagent equivalents for acetylation conditions, in keeping with other reactions detailed in this legend.
This change has been made as suggested:
Page 3, Scheme 1 caption: “…b. Acetylation: Ac2O (50 equiv), DIPEA (50 equiv), DMF, 15 min. c. Cleavage: 92.5:2.5:2.5:2.5 TFA/TIPS/DODT/H2O, 2 h….”

Page 3, paragraph 2, line 3: Remove “however,” to simplify sentence.
This has been changed as suggested:
Page 3: “The cysteine-modified peptide 2 displayed significantly reduced PCNA affinity, a result that is consistent with earlier reports….”

Page 4, paragraph 1, line 2: Remove comma after “macrocyclic peptide 3”.
We have instead included a comma following ‘propyl linker’ to improve the grammar of this sentence.
Page 4: “Macrocyclic peptide 3, with its propyl linker, gave the second highest affinity for PCNA at 769 nM, whereas the larger….”

Page 4, paragraph 1, line 4: Adjust “The more rigid trans-butenyl-based linker in peptide 5, resulted in a KD value for PCNA of 2.82 uM and the binding affinity for PCNA of aromatic m-xylene linked peptide 6, with a 9 atom linker, was lower again at 3.86 uM” to “The more rigid trans-butenyl-based linker in peptide 5 resulted in a KD value for PCNA of 2.82 uM, and the binding affinity for PCNA of aromatic m-xylene linked peptide 6 (9 atom linker) was lower again at 3.86 uM” to improve the flow of the sentence.
We have modified the sentence as suggested:
Page 4: “The more rigid trans-butenyl-based linker in peptide 5 resulted in a KD value for PCNA of 2.82 uM, and the binding affinity for PCNA of aromatic m-xylene linked peptide 6 (9 atom linker) was lower again at 3.86 uM.”

Page 4, paragraph 1, line 6: Restructure to “Considered together, these observations suggest…”.
We have modified the sentence as suggested:
Page 4: “Considered together, these observations suggest that the longer and more rigid linkers result in a lower binding affinity for PCNA.”

Page 4, paragraph 1, line 9: Remove comma after “9 atoms”.
We have modified the sentence as suggested:
Page 5: “The bimane linker contains 9 atoms but the bimane moiety, in contrast to the xylene ring, is not rigid and is able to flex along the plane of symmetry…”

Page 4, paragraph 2, line 5: Include a note on why crystal structures were not obtained for macrocycles 4 and 7, particularly as peptide 7 is the macrocycle with highest binding.
We have included a comment in paragraph 1 of the Structural Analysis section as noted above in the above comment, regarding obtaining crystal structures.
Page 5: “We did endeavour to obtain co-crystals of peptides 1, 4 and 7, however were unsuccessful at this time. Peptide 1 was also modelled onto the PCNA surface, to confirm the short peptide interacted with the protein surface in the same manner (Figure S1), and was constructed from the previously published structure of p21141-155 bound to PCNA (PDB: 7KQ1)13.”

Page 5, paragraph 1, line 9: Remove the comma after “(4.4 Å)”.
We have modified the sentence as suggested:
Page 5: “The propyl linker sits above Pro253 of PCNA (4.4 Å) and Phe150 of the peptide…”

Page 5, paragraph 2, line 4: Change to “(Figure S4):” and then list the H-bond interactions, separated by ;.
We have modified the sentence in question to read:
Page 5: “Three intermolecular hydrogen bonds are observed between peptide 5 and PCNA to anchor the macrocycle onto the surface (Figure S4). These are between: the Met147 amide NH and the mainchain carbonyl of His44 (2.3 Å); the Gln144 side-chain to the Ala252 carbonyl (2.8 Å) and the Pro253 carbonyl and Cys145 amide NH (3.4 Å).”

Page 5, paragraph 3, line 6: The sentence beginning with “Additionally, this conformation does not appear well stabilised” needs to be rephrased, for clarity.
We agree and have modified this sentence into two sentences which we believe are less ambiguous.
Page 6: “Additionally, this conformation does not appear well stabilised as there are only three other intramolecular interactions present. The first between the Ser146 side-chain and carbonyl; second between the Thr148 side-chain and carbonyl; and third between the Thr148 side-chain and the Tyr151 carbonyl (Figure 3E, yellow).”

Page 6, paragraph 1, line 2: Place the comma before “except” instead of after.
We have modified the sentence as suggested
Page 6: “….PCNA shows the conserved residues are positioned similarly to p21141-155, except the…”

Page 6, paragraph 2, line 10: Should read “a 2.7 Å hydrogen bond”.
We have modified the sentence as suggested
Page 6: “…made between peptide 7 and PCNA, except for a 2.7 Å hydrogen bond of Arg143 to Asp257 and…”

Page 9, paragraph 1, line 9: Remove “however,” and include “indicates”.
We have modified the sentence as suggested
Page 9: “…the computationally modelled structure of 7 bound to PCNA indicates the peptide backbone…”

Page 9, paragraph 1, line 10: Remove “importantly”.
We believe that this is point need to be emphasised and would prefer to retain the word ‘importantly’ in this sentence.

Abstract image: Linker structures cover PCNA image below.
We have modified the Table of Contents image to rectify this. Please see attached.

Supplementary page 4, fluorescein attachment section: N-terminal requires italicisation.
This has been corrected.
ESI Page 4 (synthesis of Peptide 8): “…preswelled resin (0.1 mmol) with resin-bound peptide with N-terminal free amine…”

Supplementary page 5, paragraph 1, line 1: E. coli requires italicisation.
This has been corrected
ESI Page 5: “A glycerol stock of E. coli BL21 (λDE3) cells carrying a hPCNA-pMCSG19 plasmid …”

Expt
Table 1 peptides 1 and 2 sequences (page 4) don’t match experimental.
We apologise for this mistake and have included the correct structures for the Experimentals of peptides 1, 2 and 8 in the ESI Pages 1, 2 and 4 respectively.

L-amino acids, L needs to be font size 10 vs 12 (throughout)
We have replaced two instances of this in the Experimental provided in the ESI document.

Provide yields of peptides in method.
In all cases the entire batch of peptide was not purified, only sufficient to carry out the required experiments was isolated from the crude, and so we are not able to calculate an accurate final yield. However, we do not believe this detracts from the chemistry reported.

Provide characterisation data with method for peptide 8
We apologise for this omission, the data has now been included:
Page 4 of ESI : “HRMS (ESI+) Expected [M+4H]4+ for C88H114N22O26S2: 490.7007, observed: [M+3H]3+ 490.6728.”

Provide RP-HPLC spectra to demonstrate peptide purity <95%
We have included the correct HPLC purity data in the experimental of each peptide, and the HPLC traces, starting on Page 7 of the ESI. We have reviewed the data and have concluded that the HPLC data included correlated to an earlier scouting experiment, and not our final experimental data. We apologise for this oversight, and have corrected this mistake.

Referee: 2
Comments to the Author
The manuscript by Abell and coworkers describes the structure-based design of macrocyclic peptides targeting the PCNA protein which is critically involved in DNA replication and repair. Based on previously described macrocyclic peptides (Chem. Eur. J. 2018 – by the same team) a broader set of crosslinks was now tested and very toughly investigated using X-ray crystallography, SPR, NMR and modelling. The findings shed light on the structural prerequisites for high affinity binding – it should be noted that highest affinity derivative 7, exhibits still lower affinity than the linear wt sequence. The study is concluded with cell-permeability studies, which would indeed be an informative addition – however, the experimental setup is not appropriate (details below). Overall, the study is well performed and reported and given the points below have been addressed, I recommend publication in ACS Chem Biol.

Major:
1) Investigating the cellular uptake of labelled peptides after cell fixation tends to generate false positive results as membrane associated peptides show a high tendency to spread after fixation. That is why, it is not used for this purpose anymore. Alternatively, live-cell confocal or MS-based read-outs would be more suitable. We have performed a time course experiment, where the T47D breast cancer cell line was treated with either 1 μM or 5 μM of p21F (cF-GRKRRQTSMTDFYHSKRRLIFS-NH2, where cF=carboxyfluorescein), a longer variant of the p21-derived peptides used in this manuscript, for 4, 24 and 48 hours. The cells were harvested and fixed (note: all samples were fixed for exact same amount of time of 10 minutes) as described in the methods. The results (attached, please see end of document) show that at 5 μM treatment, peptide accumulates on the membrane of the cells at 4 hour treatment. At 24 hour treatment there is still some accumulation on the membrane, but the peptide is mostly inside the cells. With 48 hours of treatment we observe most of the peptide being inside the cytoplasm. If the fixation of the cells was going to generate false positive results due to spread of the membrane associated peptides, we would not see this time course effect and all time points would have looked identical. Thus, we conclude that effects of fixation on membrane peptide spread is limited and our method is appropriate to investigate intracellular uptake of our similar p21 peptides presented here. We moved away from using T47D cells for this manuscript, and the blue nuclear DAPI stain would obscure the blue fluorescence of the bimane fluorophore, and instead used the MDA-MB-468 cells that express the nuclear-localised red fluorophore, mKate. We believe the behaviour of the T47D cells discussed here is comparable to the MDA-MB-468 cells used in the manuscript.

If a fluorescent label is used for readout, the same fluorophore should be used. In addition, it is crucial to include a positive control, e.g. a cell-penetrating peptide.
Additionally, we have included peptide 9 that includes both the bimane macrocycle, and a fluorescein tag, to serve as a control for this experiment as demonstrate that our imaging results are not influenced by the fluorophore readout. All corresponding experimental data for peptide 9 is included in the ESI. Peptides 8 and 9 have been added to Table 1. We have included discussion around this peptide in the Cellular Imaging section:
Page 9/10: “Two controls peptides were prepared: a linear p21143-154 peptide with an N-terminal fluorescein appended (peptide 8, Table 1); along with a derivative of peptide 7 that is macrocyclised with the bimane linker and also includes an N-terminal fluorescein (peptide 9, Table 1). Peptides 8 and 9 were subjected to the same cell uptake experiment to compare to cell uptake of the linear and cyclised analogues, and the impact of a fluorescein tag.”
Page 10: “Imaging peptide 9 on both the GREEN and BLUE channels, revealed blue punctate fluorescence throughout the cytoplasm, as for peptide 7. Green fluorescence was also observed throughout the cytoplasm for the peptide 9 treated cells, which is colocalised with the blue fluorescence. Together this indicates that peptide 9 is also cell permeable, with a similar distribution to peptide 8. The green fluorescence was somewhat diffuse, suggesting that some free fluorescein may be present.
Page 10: “Here we demonstrate that an N-terminal fluorescein tag appended to peptide 7, as in peptide 9, dramatically decreases the binding affinity for the protein target (PCNA, Table 1), though in this case the cellular uptake and distribution is not significantly impacted.”

Minor:
2) It would be informative to show one of the crosslinked structures reported earlier (Chem. Eur. J. 2018) as they served as direct precursors and involved the same modification sites.
We agree that this would be a useful comparison however believe the best comparison of these peptides are within their family of dithiol bis-alkylation linkers, which behave quite differently to the lactam linker, and as such is not necessary to include in this study. We have included the lactam linker, for direct comparison, for the next generation of mimetics in a separate future publication.

3) The corresponding affinity SE values in Table 1 should have the same decimal digits: e.g. “769+-78“ instead of “769.1+-78“
We have made this correction, please see Table 1.

Berlin, 17. July 2021

Dear Dr Abell:

Manuscript ID: CB-ART-05-2021-000113.R1
TITLE: A cell permeable bimane-constrained PCNA-interacting peptide

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Technische Universität Berlin
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************

Reviewer 1

The corrections suggested have all been implimented. I congratulate the authors on this fantastic science.

Reviewer 2

The authors have addressed all reviewer comments appropriately, and I recommend publication in RSC Chem Biol. I have one more minor suggestion: It would be more appropriate to show Kd values in Table 1 in µM including 3 significant digits.

19 July 2021

Dear Prof. Dr. Roderich Süssmuth,

We are delighted to have our manuscript accepted for publication in RSC Chemical Biology and have incorporated the final suggested change to our manuscript.
We have resubmitted an amended copy of the manuscript showing this change as "Track Changes", as well as a finalised copy.

We look forward to seeing the final version published online.

Kind regards,
Aimee Horsfall, on behalf of all authors




REVIEWER REPORT(S):
Referee: 1
The corrections suggested have all been implemented. I congratulate the authors on this fantastic science.

Referee: 2
The authors have addressed all reviewer comments appropriately, and I recommend publication in RSC Chem Biol. I have one more minor suggestion: It would be more appropriate to show Kd values in Table 1 in µM including 3 significant digits.
We have made this adjustment as suggested, please see Table 1.

Berlin, 19. July 2021

Dear Dr Abell:

Manuscript ID: CB-ART-05-2021-000113.R2
TITLE: A cell permeable bimane-constrained PCNA-interacting peptide

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Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor