Design and synthesis of Nrf2-derived hydrocarbon stapled peptides for the disruption of protein-DNA-interactions

Bianca Wiedemann; Dominic Kamps; Laura Depta; Jörn Weisner; Jana Cvetreznik; Stefano Tomassi; Sascha Gentz; Jan-Erik Hoffmann; Matthias P. Müller; Oliver Koch; Leif Dehmelt; Daniel Rauh

doi:10.1371/journal.pone.0267651

Peer Review History

Original SubmissionJanuary 18, 2022
2 Apr 2022 Decision Letter - Alessio Lodola, Editor PONE-D-22-01699Design and Synthesis of Nrf2-Derived Hydrocarbon Stapled Peptides for the Disruption of Protein-DNA-InteractionsPLOS ONE Dear Dr. Rauh, Thank you for submitting your manuscript to PLOS ONE. After careful consideration also based on my own reading, we feel that it has scientific merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Editor's suggestions1) Could the authors give more details about the homology modeling procedure in the main text (now is reported in the SI)? It is a critical part of the work as it has inspired the design and synthesis of the peptide library.2) The authors commented on the flexibility of their approach which could lead to agents able to interfere with RNA activity. I agree with their vision, but I suggest them to also mention the potential selectivity issues emerging from the direct targeting of nucleic acids with peptides. Please submit your revised manuscript by May 17 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. 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This work was co funded by the German Federal ministry for Education and Research (NGFNPlus and e:Med) (Grant No. BMBF 01GS08104, 01ZX1303C), the Deutsche Forschungsgemeinschaft (DFG), the German federal state North Rhine Westphalia (NRW) and the European Union (European Regional Development Fund: Invest In Your Future) (EFRE-800400), NEGECA (PerMed NRW) and EMODI.” We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. 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Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors describe the design, synthesis and testing of stapled peptides meant to target a specific DNA sequence. The authors show convincingly that the peptides bind to DNA, albeit they are not specific to the targeted sequence but bind to other DNA sequences equally well. The experiments are generally described and carried out well, thus the results are well worse publishing in PLOS one. (Note: I am not an expert in peptide synthesis, therefore I can’t comment on this part of the work and my comments relate only to the non-synthesis parts of the manuscript.) However, the following minor comments should be addressed before publication: 1) The authors treat cells with a linear peptide and a stapled peptide. For the latter one, enrichment in the nucleus was observed and therefore the authors concluded that the enrichment is due to stapling. However, both peptides were used at the same concentration, even so the binding affinity for the stapled peptide for DNA is about 6-fold higher than for the linear one. The authors should therefore discuss if the observed enrichment in the nucleus could be rather driven by affinity than the stapling itself. 2) The result and discussion section ends rather sudden. In the last sentence, it says that the authors decided to embark in a second round of optimization. However, the optimization is not described in the paper. Is there something lacking in the manuscript or is the second round of optimization a future project? 3) Page 7, line 148, “DNA hybridization was performed as described previously”. Is there are reference lacking or do the authors rather mean “above” instead of “previously”? 4) Page 10, line 216, this should read “… is able to induce a mobility shift” 5) Next line: “… at a lower concentration in respect to the unstapled peptide 5 ..”. This is unclear. Should this read “… at a lower concentration than the unstapled peptide 5 …”? 6) Figure S1: Which colour hast the model and which colour the NMR structure? ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes:** Ruth Brenk [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0267651.r001
Revision 1
11 Apr 2022 Author Response Response to Decision Letter: Wiedemann et al. [PONE-D-22-01699] Editor’s suggestions 1. Could the authors give more details about the homology modeling procedure in the main text (now is reported in the SI)? It is a critical part of the work as it has inspired the design and synthesis of the peptide library. We agree that the homology model is an essential part of our presented work. We now include the homology modeling procedure in the main text (line 103-111). 2. The authors commented on the flexibility of their approach which could lead to agents able to interfere with RNA activity. I agree with their vision, but I suggest them to also mention the potential selectivity issues emerging from the direct targeting of nucleic acids with peptides. We thank the editor for this comment. We now address the selectivity issues in the conclusion. The text reads:” However, due to the overall negative charge of nucleic acids, great care must be taken to ensure that nonspecific electrostatic interactions do not interfere with the desired specific binding, requiring careful design and evaluation of the stapled peptide library.“ Reviewer #1: The authors describe the design, synthesis and testing of stapled peptides meant to target a specific DNA sequence. The authors show convincingly that the peptides bind to DNA, albeit they are not specific to the targeted sequence but bind to other DNA sequences equally well. The experiments are generally described and carried out well, thus the results are well worth publishing in PLOS one. We are very grateful and thank the reviewer for the positive assessment of our manuscript. 1. The authors treat cells with a linear peptide and a stapled peptide. For the latter one, enrichment in the nucleus was observed and therefore the authors concluded that the enrichment is due to stapling. However, both peptides were used at the same concentration, even so the binding affinity for the stapled peptide for DNA is about 6-fold higher than for the linear one. The authors should therefore discuss if the observed enrichment in the nucleus could be rather driven by affinity than the stapling itself. We thank the reviewer for the comment and agree that the wording needs to be more precise to clarify this. What we meant to say is that stapling increases the affinity of the peptide and thereby leads to stronger binding and enrichment in the nucleus. The manuscript now reads:” In contrast, only small amounts of linear peptide FITC-5 were detectable in cells and no enrichment in the nucleus could be observed for peptide FITC-5, suggesting that nuclear enrichment of the peptides is dependent on stapling and the resulting higher affinity towards DNA.” 2. The result and discussion section ends rather sudden. In the last sentence, it says that the authors decided to embark in a second round of optimization. However, the optimization is not described in the paper. Is there something lacking in the manuscript or is the second round of optimization a future project? This point is well taken. We will focus on another round of improved stapled peptides guided by our presented design and results in the future. To clarify this, we rephrased the sentence:” Based on these findings, we will focus on another round of improved stapled peptides guided by our presented design and results in the future, maintaining the stapling system featured by peptide 4 (i,i+7) and focusing on the modification of aa of the aforementioned group c) (see above). 3. Page 7, line 148, “DNA hybridization was performed as described previously”. Is there are reference lacking or do the authors rather mean “above” instead of “previously”? We corrected for this. The text now reads:” DNA hybridization was performed as described above.” 4. Page 10, line 216, this should read “… is able to induce a mobility shift” We corrected for this. 5. Next line: “… at a lower concentration in respect to the unstapled peptide 5 ..”. This is unclear. Should this read “… at a lower concentration than the unstapled peptide 5 …”? We corrected for this. The sentence now reads: “Additionally, the stapled peptide 4 is able to induce a mobility shift of DNA at a lower concentration than the unstapled peptide 5 (Fig. S6 8).” 6. Figure S1: Which colour has the model and which colour the NMR structure? We added the colours to make this more clear. Attachments Attachment Submitted filename: Response to Reviewers_Wiedemann et al._PLOS ONE.docx https://doi.org/10.1371/journal.pone.0267651.r002
13 Apr 2022 Decision Letter - Alessio Lodola, Editor Design and Synthesis of Nrf2-Derived Hydrocarbon Stapled Peptides for the Disruption of Protein-DNA-Interactions PONE-D-22-01699R1 Dear Dr. Rauh, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Alessio Lodola, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0267651.r003
Formally Accepted
4 May 2022 Acceptance Letter - Alessio Lodola, Editor PONE-D-22-01699R1 Design and Synthesis of Nrf2-Derived Hydrocarbon Stapled Peptides for the Disruption of Protein-DNA-Interactions Dear Dr. Rauh: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr Alessio Lodola Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0267651.r004

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