From the journal RSC Chemical Biology Peer review history

31-Dec-2020

Dear Dr Barnard:

Manuscript ID: CB-REV-11-2020-000205
TITLE: Modulators of protein-protein interactions as antimicrobial agents

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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Dr Cai-Guang Yang
Associate Editor
RSC Chemical Biology

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

Reviewer 1

Kahan and co-workers have presented a review examining recent research into controlling protein-protein interactions as antimicrobial agents. The problem of antimicrobial resistance is described and the importance of new mechanism for antimicrobial action is presented with clarity. The role PPIs can play is highlighted well, and some recent insights into PPIs relevant to antibiotics is described. The conclusion highlights the challenges of getting new antibiotics approved, and the current shortcomings of the PPI field in getting to clinic.
The review is very well written, and this reviewer thinks this is a timely piece of work, with PPIs becoming a larger field, and an obvious direction for PPI researchers is towards antibiotics.
I believe this review should be published, but have a few suggestions for the manuscript.

I think it would be relevant to at least give a nod to the action of vancomycin. Although not initially described as a PPI modulator, the mode of action of vancomycin is as a PPI. Recent work has highlighted how VRE can be targeted by taking a PPI approach. (https://www.pnas.org/content/114/26/E5052). I don't think it needs to be a large addition, but this is a classic example of the effectiveness PPIs have as antibiotics, and I think a small paragraph would add interest for those who work outside of PPIs.

There has also been a lot of recent for look at the BAM protein complex. Inhibition of bamA and bamD has recently been reported as a way to affect bacterial cell was synthesis, and also acts in a PPI manner. Can the authors comment on any reasons for omission of these studies. (https://www.pnas.org/content/116/43/21748, https://www.pnas.org/content/112/7/2011)

Minor comments
Early in the work the MIC is reported in micromolar, and later is reports as micrograms per millilitre. It would make for easier reading and comparison if all the MIC values were in the same unit, preferably micromolar as it is less ambiguous.

Figures 2 and 4 would benefit from having the crystal structure labelled with the compound number (in addition to the already existing compound number in the caption). Adding the number to the picture would improve readability.

Overall this referee thinks this review is very interesting, and will be of great interest to the chemical biology community.

Reviewer 2

Kahan et al. summarized recent discoveries on modulators of bacterial protein-protein interactions potential for antimicrobial agent development in this review. The article structure is clear, and the writing is fluent, especially the conclusion part, which demonstrated the required quality to publish in RSCCB if the following issues can be resolved and improved.
General:
1. As a scientific publication, the authors need to ensure the readability and reduce the complexity of sentences, e.g. Page 3, paragraph 3, “ZipA bridges FtsZ and the bacterial membrane, therefore inhibition of this PPI interferes with the ability of the Z-ring to guide the synthesis and reshaping of the peptidoglycan wall during division with the incomplete division subsequently leading to bacterial cell death”, and Page 19, paragraph 3, “They theorised that a compound mimicking the toxin residues that interacts with the antitoxin would free the toxin to trigger growth inhibition and carried out in silico modelling of peptide profiles to direct the choice for an extensive collection of peptide libraries used to screen for hits targeting the interaction.”. They are too long and need to break to two or more sentences. Go through the whole manuscript and make necessary changes.
2. In general, the dissociation constant should be described as Kd rather than KD. Check the requirement of RSCCB to confirm it.
3. Citations should be provided in the figures for the crystal structures with PDB codes.
4. Abbreviations were provided for the words used only once, e.g. cyclohexyl-L-alanyl (Cha) and 3,4-dichlorophenyalanine (diClF). Go through the whole manuscript and delete them.
5. The authors need to unify the presentation of PPIs with “dash” or “slash”. Chapters 3.1 and 3.3 adopted dash, but 3.2 used slash.
6. The names of bacterial species should be described in full for the first time appeared in the manuscript and abbreviated in the following text. Go through the whole manuscript and make corrections.
7.
Specific:
1. Page 3, paragraph 4. What is “structure-merging approach”? It is not a common word. Consider changing it.
2. Page 3, paragraph 4. “The disagreement between PPI inhibition and activity (MIC considerably lower than the IC50) suggests that 2 and related compounds might act by additional unknown mechanisms beyond ZipA-FtsZ inhibition, requiring further mode-of-action studies before progressing the series forward.” Note that IC50 values highly depend on the concentrations used in the designed assays. The concentrations of the same inhibitor used to titrate the same enzyme at 1 nM and 1 mM would be significantly different, which are determined by the signal-to-noise ratio as well as the preference of method users. Therefore, this conclusion is rather subjective and should be deleted. Additionally, the MIC of the compound should be provided.
3. Page 4, paragraph 2. “Saccharomyces cerevisae” should be Saccharomyces cerevisiae.
4. Page 6, paragraph 3. “Insertion of a single methyl group at position 4 of the pyrrole ring”. Where is position 4? Mark it on the structure (Figure 4).
5. Page 7, paragraph 1. “Rifamycin and fidaxomicin are the only marketed antibiotics that act on bacterial transcription, but both target active sites in bacterial RNA polymerase (RNAP)”. The description was wrong, because fidaxomicin targets RNAP clamp (Clin Infect Dis. 2012, 55(Suppl 2): S127–S131; and Mol Cell. 2018, 70(1): 60–71.e15.)
6. Page 7, paragraph 2. “RNAP core enzyme (α2, β, β' and ω subunits)”. α2 should be described as 2α, because RNAP is composed by two α subunits.
7. SB series as inhibitors of the RNAP-σ PPI should be added to 3.2.i (Assay Drug Dev Technol 2:629–635.).
8. Page 8, paragraph 2. “in vitro” should be italic.
9. Page 8, paragraph 2. “β'-CH/σ70/σA2.2” was confusing. Which PPI was described?
10. Page 8, paragraph 3. “Gram-negative S. aureus USA300” was wrong.
11. Page 8, paragraph 3. The following structure optimization study of compound 15 should be added (J. Med. Chem. 2020, 63, 14, 7695–7720; and Eur J. Med. Chem. 2020, 208, 112671.).
12. Page 9, paragraph 1. “indol-3-yl-urea derivatives” was only mentioned in the text. A representative structure should be added in the figure.
13. Page 10, paragraph 2. Compounds’ function on toxin release should be added (Int. J. Mol. Sci. 2020, 21(16), 5772.).
14. Page 11, paragraph 2. The authors directly started with Type II TA system. On Page 20, the authors also mentioned type VI TA system when introducing the TplE-TplEi PPI. A brief introduction on how the TA systems were classified should be added.
15. Page 12, paragraph 1. “where the toxin alone has a 0% inhibitory value and the toxin bound to the antitoxin represents 100% inhibition. The VapC30 toxin-mimicking peptides 22 and 23 showed a greater inhibitory effect of 53% and 73% compared to the antitoxin-mimicking peptide 21 at 43%.” The authors described percentages of inhibition without concentrations of toxin, antitoxin, and peptide. The information of concentrations used in the assay should be supplemented.
16. Page 12, paragraph 1. “Peptide 28 demonstrated the best activity and was able to disrupt TA binding of VapBC26 by 80%.” As above
17. Table 2. % Inhibition of PPI at what concentration should be added. Toxin and antitoxin concentration in the assays should be provided as a footnote of the table.
18. Page 14, paragraph 1. “All four peptides inhibited the activity of VapC11 to some degree, although the inhibition was dependent on a 100-fold greater amount of peptide compared to protein.” Using different concentrations for titration of inhibitory activity is how assays are normally performed in a laboratory. A 1:1 ratio such as the acid-base reaction in chemistry does not exist in PPIs. “although…” should be deleted.
19. Page 15, paragraph 1. “with peptide 41 demonstrating 80% inhibition”. Paragraph 3. “in the presence of the EDF and MazE, MazF had activity close to 100%.” See comment 14.
20. Figure 12. “Site 1 is formed by residues D12, R25, Q79, R81, and R87 from monomer 1 and residues I83, R81 and T82 from monomer 2. Site 2 is formed by residues Q50, K55, L56, H59 and E78 from monomer 1 and residues I34, F38, S39, P40 and T41 from monomer 2.” can be deleted as they were not shown in the figure.
21. Page 17, paragraph 1. “The peptides targeting site 2 demonstrated the greatest inhibition (up to 35%)”. See comment 14.
22. Table 6. See comment 16.
23. Page 17, paragraph 2. “peptides 53 and 58 proving most successful with inhibition of 44% and 46%,” See comment 14.
24. A figure should be provided for 3.3.i.4 HipAB TA system.
25. Page 19, paragraph 3. “They experiment” should be their experiment.
26. Table 10. K100 of peptide 71 should be highlighted by color with a footnote.
27. Page 22, paragraph 2. “analogues” should be homologues.
28. Page 23, paragraph 3. “exact roles in in bacterial biology”

Response to Referee Comments:

Reviewer: 1
We thank the reviewer for their helpful suggestions and have incorporated them into the manuscript as detailed below.

1. This reviewer has suggested including a paragraph giving a nod to the action of vancomycin. Having completed a review of the relevant literature, including the papers cited by the reviewer we have decided not to include this.
Vancomycin is a critical antibiotic that has been used clinically for over 60 years.1 Although it does not function as a PPI inhibitor, the primary antibacterial mechanism of action of vancomycin is an interaction with bacterial cell wall precursors, it interacts in a similar way to a PPI.2,3 The bacterial wall precursors have a D-Ala-D-Ala dipeptide terminal, a substrate for the enzyme transpeptidase which catalyses cell wall cross-linking.4 Vancomycin binds to D-Ala-D-Ala via hydrogen bonding, inhibiting formation of the transpeptidase links.2–4 Vancomycin-resistant strains alter the peptide end to be D-Ala-D-Lac resulting in the loss of a critical hydrogen bond.5–7 Several studies have identified that an introduction of a peripheral hydrophobic group, in particular 4-chlorobiphenyl addition, overcomes the resistance.8–12 This modified vancomycin acts via a mechanism independent of D-Ala-D-Ala/D-Lac binding.8,13–15 There is likely a direct interaction between the substituted carbohydrates and proteins involved in transglycosylation which may involve the inhibition of PPIs.10,16 Whilst vancomycin is an excellent example of a successful antibiotic, since it’s mechanism of action is not directly attributed to PPI inhibition it seems tenuous to include this and would disrupt the flow of the manuscript.
1 D. P. Levine, Clin. Infect. Dis., 2006, 42, S5–S12.
2 D. H. Williams and B. Bardsley, Angew. Chem. Int. Ed., 1999, 38, 1172–1193.
3 H. R. Perkins, Pharmacol. Ther., 1982, 16, 181–197.
4 J. C. J. Barna and D. H. Williams, Annu. Rev. Microbiol., 1984, 38, 339–357.
5 C. T. Walsh, Science, 1993, 261, 308+.
6 V. L. Healy, I. A. Lessard, D. I. Roper, J. R. Knox and C. T. Walsh, Chem. Biol., 2000, 7, R109–R119.
7 C. C. McComas, B. M. Crowley and D. L. Boger, J. Am. Chem. Soc., 2003, 125, 9314–9315.
8 M. Ge, Z. Chen, H. Russell, Onishi, J. Kohler, L. L. Silver, R. Kerns, S. Fukuzawa, C. Thompson and D. Kahne, Science, 1999, 284, 507.
9 R. C. Goldman, E. R. Baizman, C. B. Longley and A. A. Branstrom, FEMS Microbiol. Lett., 2000, 183, 209–214.
10 A. Okano, N. A. Isley and D. L. Boger, Proc. Natl. Acad. Sci. U.S.A., 2017, 114, E5052.
11 A. Okano, A. Nakayama, A. W. Schammel and D. L. Boger, J. Am. Chem. Soc., 2014, 136, 13522–13525.
12 Z.-C. Wu, N. A. Isley, A. Okano, W. J. Weiss and D. L. Boger, J. Org. Chem., 2020, 85, 1365–1375.
13 L. Chen, D. Walker, B. Sun, Y. Hu, S. Walker and D. Kahne, Proc. Natl. Acad. Sci. U.S.A., 2003, 100, 5658.
14 N. E. Allen, J. N. Hobbs and T. I. Nicas, Antimicrob. Agents Chemother., 1996, 40, 2356.
15 Z.-C. Wu, M. D. Cameron and D. L. Boger, ACS Infect. Dis., 2020, 6, 2169–2180.
16 Z.-C. Wu and D. L. Boger, Acc. Chem. Res., 2020, 53, 2587–2599.

2. In response to suggestion to add the studies of targeting the BAM protein complex, an extra section has been added to the review titled: Targeting Outer Membrane Proteins.

Minor comments:
1. All the MIC values have been converted to the same unit: micromolar.
2. Crystal structures in Figures 2 and 4 have been labelled with the compound number to improve readability.

Reviewer: 2
We thank the reviewer for taking the time to give such thorough feedback on our manuscript. We have implemented all suggested corrections as detailed below.

General Comments:
1. To improve readability we have gone through the whole manuscript and changed sentences that are too long into to two or more sentences including those highlighted by the reviewer.
2. Dissociation constant now described as Kd rather than KD.
3. Citations have now been provided in the figures for the crystal structures alongside the associated PDB codes.
4. Abbreviations have been removed for the words used only once, e.g. cyclohexyl-L-alanyl (Cha) and 3,4-dichlorophenyalanine (diClF).
5. We have unified the presentation of PPIs with a “dash”.
6. The names of bacterial species have been included in full the first time they appear in the manuscript and abbreviated in the following text.
Specific comments:
1. Page 4, paragraph 1. “structure-merging approach” has been changed to “fragment-merging”
2. Page 4, paragraph 1. “The disagreement between PPI inhibition and activity (MIC considerably lower than the IC50) suggests that 2 and related compounds might act by additional unknown mechanisms beyond ZipA-FtsZ inhibition, requiring further mode-of-action studies before progressing the series forward.”

This is the justification given by the authors of this paper for not developing the series of compounds further. This conclusion has not been deleted, but the sentence has been rephrased not to give the impression that the IC50 values could have been determined at protein-limiting concentrations.
3. Page 4, paragraph 3. “Saccharomyces cerevisae” has been corrected Saccharomyces cerevisiae.
4. Page 6, paragraph 3. “Insertion of a single methyl group at position 4 of the pyrrole ring”. Position 4 has been marked on the structure (Figure 4).
5. Page 7, paragraph 1. “Rifamycin and fidaxomicin are the only marketed antibiotics that act on bacterial transcription, but both target active sites in bacterial RNA polymerase (RNAP)”. The description has been clarified.
6. Page 7, paragraph 2. “RNAP core enzyme (α2, β, β' and ω subunits)”. α2 corrected to 2α, because RNAP is composed of two α subunits.
7. The mentioned SB series as inhibitors of the RNAP-σ PPI has not been added to 3.2.i (Assay Drug Dev Technol 2:629–635.) We were aware of this study but since the paper mostly discussed assay development, with just two tested in an RNAP-sigma inhibition assay, both of which were not active against E. coli, it was decided not to include this example.
8. Page 8, paragraph 2. “in vitro” changed to italic.
9. Page 8, paragraph 3. “β'-CH/σ70/σA2.2” was clarified.
10. Page 8, paragraph 4. “Gram-negative S. aureus USA300” was double-checked, this is the name of the strain used in the paper.
11. Page 9, paragraph 1. The structure optimization study of compound 15 was added (J. Med. Chem. 2020, 63, 14, 7695–7720; and Eur J. Med. Chem. 2020, 208, 112671.).
12. Page 9, paragraph 2. A representative structure of “indol-3-yl-urea derivatives” was added to the figure. Compound 16
13. Page 11, paragraph 1. Compounds’ function on toxin release has been added (Int. J. Mol. Sci. 2020, 21(16), 5772.)
14. Page 14, paragraph 1. The authors directly state that this is a Type II TA system. Further reading identified that TplE-TplEi PPI is not a type VI system, so this has been clarified on page 24. With this in mind, it would be superfluous to discuss how TA systems were classified so this has been removed from the manuscript.
15. Page 15, paragraphs 1 and 2; page 17, paragraph 1; page 18, paragraph 3; page 19, paragraph 4 and page 20, paragraphs 2 and 4. The concentrations of the toxins, antitoxins, and peptides have been added.
16. Table 2 and Table 6. The concentration that results in the stated % Inhibition of a PPI has be added. Toxin and antitoxin concentrations in the assays have been provided as a footnote in the tables.
17. Page 16, paragraph 1. “All four peptides inhibited the activity of VapC11 to some degree, although the inhibition was dependent on a 100-fold greater amount of peptide compared to protein.” “although…” has be deleted.
18. Figure 14. “Site 1 is formed by residues D12, R25, Q79, R81, and R87 from monomer 1 and residues I83, R81 and T82 from monomer 2. Site 2 is formed by residues Q50, K55, L56, H59 and E78 from monomer 1 and residues I34, F38, S39, P40 and T41 from monomer 2.” This discussion has been deleted as the residues were not highlighted in the figure.
19. A figure has been provided for 3.3.i.4 for the HipAB TA system. Figure 15
20. Page 23, paragraph 2. “They experiment” changed to ‘their experiment’.
21. Table 10. K100 of peptide 78 has been highlighted by colour with a footnote.
22. Page 25, paragraph 2. “analogues” should be homologues.
23. Page 26, paragraph 3. “exact roles in in bacterial biology” has been corrected to “exact roles in bacterial biology”

Additional Comments:
1. We noticed a correction in section 3.4.i.4. The target for HipA was originally identified as EF-TU, however, more recent papers have identified the target to be further upstream in the translation process, the glutamyl-tRNA-synthetase (GltX).17,18 This has been corrected in the text.
17. E. Germain, D. Castro-Roa, N. Zenkin and K. Gerdes, Mol. Cell, 2013, 52, 248–254.
18. I. Kaspy, E. Rotem, N. Weiss, I. Ronin, N. Q. Balaban and G. Glaser, Nat. Commun., 2013, 4, 3001.

27-Jan-2021

Dear Dr Barnard:

Manuscript ID: CB-REV-11-2020-000205.R1
TITLE: Modulators of protein-protein interactions as antimicrobial agents

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.

You will shortly receive a separate email from us requesting you to submit a licence to publish for your article, so that we can proceed with publication of your manuscript.

You can highlight your article and the work of your group on the back cover of RSC Chemical Biology, if you are interested in this opportunity please contact me for more information.

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With best wishes,

Dr Cai-Guang Yang
Associate Editor
RSC Chemical Biology

Reviewer 2

The manuscript can be accepted for publication in RSCCB