From the journal RSC Chemical Biology Peer review history

20-Oct-2020

Dear Dr Brik:

Manuscript ID: CB-ART-10-2020-000179
TITLE: In vivo modulation of ubiquitin chains by N-methylated non-proteinogenic cyclic peptides

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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Claudia Höbartner
Associate Editor, RSC Chemical Biology
Inst of Organic Chemistry, University of Würzburg

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

Reviewer 1

The manuscript by Rogers and Nawatha et al. presents K48-linked ubiquitin chain-binding peptides made partly out of non-proteinogenic amino acids and their biological characterisation. The work follows closely the outline of a 2019 Nature Chemistry paper published by the same group of authors, where ubiquitin chain-binding macrocycles were first introduced, but significantly expands on these earlier results. The two peptides Ub4a and Ub4e presented in the current manuscript contain several non-proteinogenic amino acids which also tightly bind tetraubiquitin chains (9 vs 6 nM Kd), but show higher apoptosis induction in four tumour cell lines tested when compared to the previous hit peptide Ub4ix. Importantly, the authors demonstrate that Ub4a attenuates tumour growth in a mouse model to a similar extent as the proteasome inhibitor bortezomib. Overall, the molecules and their binding mode are carefully and comprehensively characterised through a suite of biochemical assays and protein NMR spectroscopy.

This work firmly establishes ubiquitin chains themselves as a strong candidate as an anti-cancer target and introduces molecules suitable to test this hypothesis in different biological models. I am thus fully convinced that the manuscript will find the attention of the broader chemical biology and ubiquitin communities, and the interest of the readership of the journal. The manuscript is very well written, data are presented in well composed and logically structured figures, the literature is adequately referenced and detailed methods are provided. I thus strongly support publication in RSC Chemical Biology after minor revisions addressing the following mostly technical aspects and additional controls.

- Since the very C-terminal residues in the K48-Ub-trimer (Figure 2C) also show high CSPs, I wonder whether the free C-terminus of the chain contributes to binding (which would explain why the distal ubiquitin in tetramers in Fig. 4B shows little interaction). Can the authors comment on what makes ubiquitin moieties 1-3 (as opposed to 2-4) unique the binding partners of the peptides? Experimentally, could the authors use their alpha-globin-tetraUb substrate to show that also this substrate is protected from cleavage by OtuB1 (similar to Fig. 3a) in an equimolar ratio? This would directly test whether also protein conjugates (as opposed to free chains) are bound efficiently.
- The finding that HEK cells are less susceptible to apoptosis induction is potentially very insightful, but requires strengthening through controls. Is uptake in HEK cells impaired compared to the other cells? Do both Ub4a and Ub4e show much less apoptosis in HEK cells compared to a tumour cell line?
- In Fig. S11, the authors assess the ability of the peptide to protect K11 and K63-linked diUb chains which are readily cleaved by DUBs. However, since the peptides only weakly binding K48-diUb chains, the better and stronger control would be to test at least one tetramer of a different linkage to support the linkage specificity. Since the peptides seem to pick up the relative orientation of hydrophobic patches on ubiquitin (as opposed to the linkage itself, which is typical), comparing to one other longer chain would strengthen the specificity argument considerably.

- On page 7, the authors write “only the first three Ub units” which I found confusing as I typically count from the proximal side. Rephrasing to “proximal trimer” or similar (as in the discussion) would increase clarity.
- If bortezomib and Ub4a share the same mechanism of both leading to the accumulation of polyubiquitinated proteins, one would expect only a weak additive effect on the viability of cells when they are treated with both molecules. While not necessary for the revision, I am curious whether the authors observed this?
- On page 9, Fig. 4E,F (not 5) should be referenced.
- Caption of Fig. 1D. Which concentrations were used in the SPR experiments?
- Caption of Fig. 3A+B. Can the authors add the method of detection (coomassie stain?)
- Figure 3B: Are the first two lanes -/+ proteasome? If so, this should be added to the figure.
- Figure S4: What is the difference between the first SPR panels on both A and B? Are these replicates of tetra-Ub?
- Figure S6: K48 should be removed on the side of the gel as the species shown have different linkages.

Reviewer 2

Deregulation of the ubiquitin-proteasome system has been associated with the development of various cancers. Accordingly, the components of this system represent attractive therapeutic targets, and, indeed, proteasome inhibitors have been employed in the treatment of certain types of cancer, though with considerable negative side effects. Thus, additional strategies for targeting the ubiquitin-proteasome system are clearly needed. By a combination of chemically synthesized defined ubiquitin chains and cyclic peptide libraries, the authors previously reported on the identification of cyclic peptides that bind with high affinity to K48-linked ubiquitin chains thereby interfering with ubiquitin chain function in vitro and in cell culture experiments. However, the cyclic peptides were made up by canonical proteinogenic amino acids limiting their suitability for therapeutic approaches. In this manuscript, Rogers et al. generated a non-proteinogenic cyclic peptide library by replacing certain canonical amino acids by respective D-stereomers or N-methylated versions. By a smart screening/amplification procedure, the authors identified cyclic peptides that bind with high affinity and specificity to K48-linked ubiquitin tetramers, which represent the major recognition signal for the proteasome. Binding of the peptides to the tetramers was carefully analyzed by NMR revealing for instance that the distal ubiquitin moiety does not make an important contribution to the interaction. At the functional level, binding of the peptides to ubiquitin tetramers interferes with their disassembly by deubiquitylating enzymes and with the degradation of ubiquitylated proteins in vitro and in cell culture experiments. Moreover, the peptides induce apoptosis in several cancer-derived cell lines and, even more remarkably, administering the peptides to mice interferes with tumor growth. All in all, this is an exciting study and if the following issues are constructively addressed, it will make a nice contribution to RSC Chemical Biology.

1) For future readers, it would be informative to briefly compare the affinities of the previously identified "canonical" cyclic peptides with those of the newly identified ones.

2) Fig. 4C: It is somewhat surprising that in contrast to a proteasome inhibitor, the cyclic peptides do not induce apoptosis in HEK293 cells. Have the authors checked whether peptide treatment results in the accumulation of ubiquitin conjugates? If so, what was the outcome? In any case, the data are difficult to interpret, and since they do not appear to make an important contribution to the main message of the manuscript, I would suggest to remove these.

3) "Plasma stability": To appreciate the results obtained, the stability of the "canonical" cyclic peptides should be determined in comparison to the newly identified ones.

4) Figs 4B, 4C, 4D, 4F: Since error bars are indicated, it should be stated how often the individual experiments were performed and whether these were technical replicates or indeed independent experiments (technical replicates are not very informative).

5) Abstract, 2nd sentence: Molecules capable of modulating the function of Ub chains...

This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters.

Dear Claudia, Thank you for sharing with the us the referees reports.
We are very excited to learn about the outcome. Below we address each of the reviewer’s points (in grey). Our comments are in black, and the changes to the paper are in red. We look forward to hear your final decision.
Best wishes Ashraf

REVIEWER REPORT(S):
Referee: 1
Comments to the Author
The manuscript by Rogers and Nawatha et al. presents K48-linked ubiquitin chainbinding peptides made partly out of non-proteinogenic amino acids and their biological characterisation. The work follows closely the outline of a 2019 Nature Chemistry paper published by the same group of authors, where ubiquitin chain-binding macrocycles were first introduced, but significantly expands on these earlier results. The two peptides Ub4a and Ub4e presented in the current manuscript contain several nonproteinogenic amino acids which also tightly bind tetraubiquitin chains (9 vs 6 nM Kd), but show higher apoptosis induction in four tumour cell lines tested when compared to the previous hit peptide Ub4ix. Importantly, the authors demonstrate that Ub4a attenuates tumour growth in a mouse model to a similar extent as the proteasome inhibitor bortezomib. Overall, the molecules and their binding mode are carefully and comprehensively characterised through a suite of biochemical assays and protein NMR spectroscopy.
This work firmly establishes ubiquitin chains themselves as a strong candidate as an anti-cancer target and introduces molecules suitable to test this hypothesis in different biological models. I am thus fully convinced that the manuscript will find the attention of the broader chemical biology and ubiquitin communities, and the interest of the readership of the journal. The manuscript is very well written, data are presented in well composed and logically structured figures, the literature is adequately referenced and detailed methods are provided. I thus strongly support publication in RSC Chemical Biology after minor revisions addressing the following mostly technical aspects and additional controls.
- Since the very C-terminal residues in the K48-Ub-trimer (Figure 2C) also show RSC Chemical Biology Page 2 of 61 high CSPs, I wonder whether the free C-terminus of the chain contributes to binding (which would explain why the distal ubiquitin in tetramers in Fig. 4B shows little interaction). Can the authors comment on what makes ubiquitin moieties 1-3 (as opposed to 2-4) unique the binding partners of the peptides? Experimentally, could the authors use their alpha-globin-tetraUb substrate to show that also this substrate is protected from cleavage by OtuB1 (similar to Fig. 3a) in an equimolar ratio? This would directly test whether also protein conjugates (as opposed to free chains) are bound efficiently.
Author Response: This is an interesting point. Indeed, in the NMR experiments the ‘free’ C-terminus of the Ub chain does show significant CSPs. This could be due to the cyclic peptide directly interacting with the C-terminus. This would be a reasonable assumption that the Ub chain targets used in the peptide selection also had this free C-terminus. Because if interaction with the C-terminus is required for binding, the cyclic peptide might not bind to Ub chains attached to cellular targets. However, both peptides Ub4a and Ub4e are able to protect K48Ub4-a-globin from degradation by the proteasome, suggesting that the peptides are still able to bind Ub chains, even when the C-terminus is tethered to a target. Moreover, we note that CSPs, like those at the C-terminus, do not necessarily mean direct contact with the cyclic peptide and could be instead due to conformational changes upon binding. In this regard, the C-terminal G76 of the distal Ub unit also exhibits CSPs or CSPs+signal attenuations in both the tetramer and the trimer.
- The finding that HEK cells are less susceptible to apoptosis induction is potentially very insightful, but requires strengthening through controls. Is uptake in HEK cells impaired compared to the other cells? Do both Ub4a and Ub4e show much less apoptosis in HEK cells compared to a tumour cell line?
Author Response: We agree with this comment and our observation should be follow up on with more extensive experimentation. However, we think a fuller exploration of this issue is beyond the scope of this paper. Because of huge time and effort would be required to gain such insights, it would be better included as part of a separate thorough study in the future. To reflect this, we have simply stated the result, without discussion. Changes: From: “Interestingly, we found that Ub4a cyclic peptides do not induce apoptosis in the noncancer HEK-293 cell line (embryonic kidney derived), unlike the direct proteasome inhibitor MG132 (Fig. 4C). This suggests that these cyclic peptides have some degree of specificity for inducing apoptosis in tumor cells.” To: Page 3 of 61 RSC Chemical Biology “Interestingly, under these conditions the Ub4a cyclic peptide did not induce apoptosis in the HEK-293 cell line (embryonic kidney derived), whereas treatment with the direct proteasome inhibitor MG132 did induce apoptosis (Fig. 4C).” And have changed the title of the section From: “Ub4a shows selective cancer cell killing” To: ”Ub4a can induce apoptosis in cancer cell lines”
- In Fig. S11, the authors assess the ability of the peptide to protect K11 and K63- linked diUb chains which are readily cleaved by DUBs. However, since the peptides only weakly binding K48-diUb chains, the better and stronger control would be to test at least one tetramer of a different linkage to support the linkage specificity. Since the peptides seem to pick up the relative orientation of hydrophobic patches on ubiquitin (as opposed to the linkage itself, which is typical), comparing to one other longer chain would strengthen the specificity argument considerably.
Author response: We agree completely that this would be a better and stronger control. But we hope the reviewer will appreciate that it takes a significant amount of time to make just one purelinkage tetra-Ub (K48). Repeating this for multiple other linkages was beyond our means in this work. We do not expect, but cannot rule out, tight binding/protection of longer alternativelinked chains. Even if some binding/protection of dimers might be expected if this were the case. To capture this, we have added this note to the figure legend: “We assume that, at these high (µM) peptide concentrations, some degree of binding/protection of alternatively-linked dimers would be observed, if these cyclic peptides also bound longer alternatively-linked Ub chains.”
- On page 7, the authors write “only the first three Ub units” which I found confusing as I typically count from the proximal side. Rephrasing to “proximal trimer” or similar (as in the discussion) would increase clarity.
Author Response: We have changed to “only first three Ub units (counting from the proximal end)”
- If bortezomib and Ub4a share the same mechanism of both leading to the accumulation of polyubiquitinated proteins, one would expect only a weak additive effect on the viability of cells when they are treated with both molecules. While not necessary for the revision, I am curious whether the authors observed this?
Author Response: We are curious too. This is an interesting experiment for us to try for future publications.
- On page 9, Fig. 4E,F (not 5) should be referenced.
Author response: Done
- Caption of Fig. 1D. Which concentrations were used in the SPR experiments?
Author response: Add to figure legend: “Binding traces shown for 17, 33, 66, 130 and 260 nM cyclic peptide”
- Caption of Fig. 3A+B. Can the authors add the method of detection (coomassie stain?)
Author response: The method was Western blot, anti-Ub and anti-HA respectively. These details have been added to the figure legend.
- Figure 3B: Are the first two lanes -/+ proteasome? If so, this should be added to the figure.
Author response: Changed.
- Figure S4: What is the difference between the first SPR panels on both A and B?
Author response: Added: “SPR traces for Ub4a binding K48Ub4 (two repeat titrations) and K48Ub2.”
- Figure S6: K48 should be removed on the side of the gel as the species shown have different linkages.
Author Response: Done
*****
Referee: 2
Comments to the Author
Deregulation of the ubiquitin-proteasome system has been associated with the development of various cancers. Accordingly, the components of this system represent attractive therapeutic targets, and, indeed, proteasome inhibitors have been employed in the treatment of certain types of cancer, though with considerable negative side effects. Thus, additional strategies for targeting the ubiquitin-proteasome system are clearly needed. By a combination of chemically synthesized defined ubiquitin chains and cyclic peptide libraries, the authors previously reported on the identification of cyclic peptides that bind with high affinity to K48-linked ubiquitin chains thereby interfering with ubiquitin chain function in vitro and in cell culture experiments. However, the cyclic peptides were made up by canonical proteinogenic amino acids limiting their suitability for therapeutic approaches. In this manuscript, Rogers et al. generated a nonproteinogenic cyclic peptide library by replacing certain canonical amino acids by respective D-stereomers or N-methylated versions. By a smart screening/amplification procedure, the authors identified cyclic peptides that bind with high affinity and specificity to K48-linked ubiquitin tetramers, which represent the major recognition signal for the proteasome. Binding of the peptides to the tetramers was carefully analyzed by NMR revealing for instance that the distal ubiquitin moiety does not make an important contribution to the interaction. At the functional level, binding of the peptides to ubiquitin tetramers interferes with their disassembly by deubiquitylating enzymes and with the degradation of ubiquitylated proteins in vitro and in cell culture experiments. Moreover, the peptides induce apoptosis in several cancer-derived cell lines and, even more remarkably, administering the peptides to mice interferes with tumor growth. All in all, this is an exciting study and if the following issues are constructively addressed, it will make a nice contribution to RSC Chemical Biology. 1) For future readers, it would be informative to briefly compare the affinities of the previously identified "canonical" cyclic peptides with those of the newly identified ones.
Author Response: Added p10: Indeed, the non-canonical rich cyclic peptides described here have similar binding affinities to previously discovered, largely canonical, cyclic peptides11. Yet, the former has smaller macrocycles and had appreciably lower molecular mass, suggesting that the non-canonical amino acid collection is in fact superior at producing high-affinity, selective cyclic peptides.
2) Fig. 4C: It is somewhat surprising that in contrast to a proteasome inhibitor, the cyclic peptides do not induce apoptosis in HEK293 cells. Have the authors checked whether peptide treatment results in the accumulation of ubiquitin conjugates? If so, what was the outcome? In any case, the data are difficult to interpret, and since they do not RSC Chemical Biology Page 6 of 61 appear to make an important contribution to the main message of the manuscript, I would suggest to remove these.
Author response: Agreed, this is somewhat surprising, and deserves further study to justify the outcome clearly. To reflect this, we have simply stated the result, without discussion. From: “Interestingly, we found that Ub4a cyclic peptides do not induce apoptosis in the noncancer HEK-293 cell line (embryonic kidney derived), unlike the direct proteasome inhibitor MG132 (Fig. 4C). This suggests that these cyclic peptides have some degree of specificity for inducing apoptosis in tumor cells.” To: “Interestingly, under these conditions the Ub4a cyclic peptide did not induce apoptosis in the HEK-293 cell line (embryonic kidney derived), whereas treatment with the direct proteasome inhibitor MG132 did induce apoptosis (Fig. 4C).” And have changed the title of the section: From: “Ub4a shows selective cancer cell killing” To: ”Ub4a can induce apoptosis in cancer cell lines”
3) "Plasma stability": To appreciate the results obtained, the stability of the "canonical" cyclic peptides should be determined in comparison to the newly identified ones.
Author response: This comparison is interesting to perform, but the current situation, where two scientists who made major contribution to this work have moved to other positions, has prevented us from running this experiment quickly. However, it should be stated that the measurement of plasma stability of peptides often faces difficulties of consistent outcome. It is possible to run serum stability instead but as stated above the situation made us difficult to perform experiments in short period of time. When we pursue further studies on the peptide to gain more insights, we will include such a study.
4) Figs 4B, 4C, 4D, 4F: Since error bars are indicated, it should be stated how often the individual experiments were performed and whether these were technical replicates or indeed independent experiments (technical replicates are not very informative).
Author response: Figs: 4A, 4B, 4C and 4D: 3 triplicates independent experiments.
5) Abstract, 2nd sentence: Molecules capable of modulating the function of Ub chains...
Author Response: Agreed, changed.

30-Nov-2020

Dear Dr Brik:

Manuscript ID: CB-ART-10-2020-000179.R1
TITLE: In vivo modulation of ubiquitin chains by N-methylated non-proteinogenic cyclic peptides

Thank you for submitting your revised manuscript to RSC Chemical Biology and for your patience in waiting for my response. After considering the changes you have made, I am pleased to accept your manuscript for publication. I have copied any final comments from the reviewer(s) below and ask you to take this into consideration for preparing your final files.

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With best wishes,

Claudia Höbartner
Associate Editor, RSC Chemical Biology
Institute of Organic Chemistry, University of Würzburg

Reviewer 2

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Reviewer 1

I stand by my previous statement that this manuscript will be very suitable for RSC Chemical Biology and will receive my strong support for publication after minor revisions.

The authors were asked by both reviewers to address a few minor yet important issues, some overlapping between both reviewers, with additional experiments. However, while these issues have been commented on by the authors, they have not been resolved.

- Leaving out the discussion on the HEK cell data, but still showing the data, does not solve the issue that it is unclear whether the lack of apoptosis induction is due to lack of permeability. The authors have made the necessary reagents (fluorescein-tagged peptides) and have an assay to test permeability (Fig. 3C), so I think it should be straight forward to experimentally adress the issue for HEK cells. Alternatively, the author should follow reviewer 2's advice of leaving the data out.

- On alternatively-linked tetra-Ub chains: I appreciate that the authors agree that this would be a better and stronger control. These chains are also commercially available ($150) and have been made before by the lead author's lab, so I hope the authors manage to obtain the amounts for three lanes on a gel (like Fig 3a). However, if the experiment cannot be carried out in these times, then I would understand, and the authors discuss this issue suitably in the revised manuscript.

- The authors were asked by reviewer 2 to compare their determined plasma stability to the stability of a canonical cyclic peptide. This is a reasonable request, since the main rational of the improved peptides is improved in vivo activity. While there can be many reasons why the new peptides have increased bioactivity when compared to the previous generation, it would not be standard to publish a control in a future study as proposed by the authors.