From the journal RSC Chemical Biology Peer review history

24-Feb-2022

Dear Dr Koksch:

Manuscript ID: CB-ART-01-2022-000018
TITLE: Fluorine-induced polarity increases inhibitory activity of BPTI towards chymotrypsin

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Prof Seung Bum Park

Associate Editor, RSC Chemical Biology
Professor, Chemistry Department, Seoul National University, Korea

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

Reviewer 1

This manuscript systematically investigated the site-specific incorporation of fluorinated aliphatic amino acids into BPTI. And found that enhanced inhibition towards
chymotrypsin by the two fluorinated BPTI variants compared to wild-type BPTI. This study will explore the fluorinated amino acids as bioorthogonal tools to facilitate drug development. However, there are some limitations that the authors must solve before the manuscript might be accepted for publication.

My detailed comments are as follows:
1. Fluorine is one of the halogen elements, are authors consider other halogen elements, such as I, Cl.

2. Previous studies have reported that halogen bonding has attracted increasing interest in molecular recognition in the biological system. Maybe halogen bonding also plays an important role in the increment of inhibitory activity of BPTI in this study. I suggest the authors make a supplemental introduction or discussion using the updated references in the revised manuscript.

3. As we know the peptide sequence of BPTI is positioned ideally for peptide bond hydrolysis. However, the peptide bond hydrolysis is very slower for BPTI than for peptide substrates. The main reason is that the peptide bond between P1 and P1’ would be resynthesized. So do the authors determine the influence of substituting the P1 position with fluorinated aliphatic amino acids?

Minor error:
1. Page 10, in the part of “Inhibitory activity assy”, I think “nm” should be “nM”.

Reviewer 2

Leppkes and co-workers in this manuscript report the microwave solid-phase peptide synthesis of a library of bovine pancreatic trypsin inhibitor (BPTI), a 58 amino acid protein with side-chain fluorinated aliphatic amino acids, (e.g., MfeGly, DfeGly, TfeGly, DfpGly, PfpGly) replacing Lys15. Further inhibitory activities of these BPTI variants were tested towards the serine protease alpha-chymotrypsin, from which two fluorinated BPTI were found with enhanced inhibition. In the previous study, the same group reported the BPTI variants with Abu, DfeGly, and TfeGly and their inhibitory activities towards beta-trypsin in 2015. The current studies are more or less the expansion of the previous one. To make the current manuscript more informative, some revisions are suggested:
1) Provide the overlayed CD of BPTI variants, and provide comments on the effect of fluorination on the protein conformation in the text
2) Provide the detailed synthesis and characterization of all fluorinated aliphatic amino acid building blocks if not commercially available
3) Page 4, provide a brief description of Otlewski and Smalas's result.
4) it is argued to use the polarity to explain the inhibition activity. However, how can the result of TfeGly fit in this explanation? It might be difficult, however it is suggested to come up with a more generalized model
5) Ref. 13 and 44 are the same.
6) The conclusion is suggested to include the further elaboration of the result for the general application in the field.

Reviewer 3

The manuscript by Koksch describes the effect of fluorination of residue Lys15 in BPTI in its activity to inhibit the serine protease chymotrypsin. The corresponding Lys15-mutant analogs of BPTI were synthesized chemically by SPPS, folded, characterized by X-ray crystallography. When tested for its ability to inhibit chymotrypsin, the authors found that the introduction of CXF2-functionality (where X = H- or CH3-) increased the inhibitory activity of the corresponding BPTI-mutant towards chymotrypsin.
The work presented although interesting is not that innovative, the authors seem to have extended the original work by Ye et al showing that fluorination of the P1 position in BPTI can lead to increased inhibition, which is also observed in the present manuscript. In addition, the efficient synthesis of long peptides (similar in size to that of BPTI) is now quite common in by using optimized coupling protocols. Consequently, this work would be more appropriated for a specialized journal in peptide chemistry.
Other comments:
1) The authors may want to consider providing the inhibition data on a table format and using the more common Ki rather than Ka values.

Referee: 1

Comments to the Author

This manuscript systematically investigated the site-specific incorporation of fluorinated aliphatic amino acids into BPTI. And found that enhanced inhibition towards chymotrypsin by the two fluorinated BPTI variants compared to wild-type BPTI. This study will explore the fluorinated amino acids as bioorthogonal tools to facilitate drug development. However, there are some limitations that the authors must solve before the manuscript might be accepted for publication.

My detailed comments are as follows:

1) Fluorine is one of the halogen elements, are authors consider other halogen elements, such as I, Cl.

2) Previous studies have reported that halogen bonding has attracted increasing interest in molecular recognition in the biological system. Maybe halogen bonding also plays an important role in the increment of inhibitory activity of BPTI in this study. I suggest the authors make a supplemental introduction or discussion using the updated references in the revised manuscript.

3) As we know the peptide sequence of BPTI is positioned ideally for peptide bond hydrolysis. However, the peptide bond hydrolysis is very slower for BPTI than for peptide substrates. The main reason is that the peptide bond between P1 and P1’ would be resynthesized. So do the authors determine the influence of substituting the P1 position with fluorinated aliphatic amino acids?

Minor error:
1. Page 10, in the part of “Inhibitory activity assy”, I think “nm” should be “nM”.


Response to the Referee:

The authors thank the referee for the productive input regarding the submitted manuscript. In the following section, the authors give answers to the comments and questions from the referee.

1) The authors think that the referee has raised a valid point of discussion, this is why additional text was added to the introduction of the manuscript, underlining, why fluorine was chosen as element of interest in this manuscript.

“Fluorine is quite unique compared to other halogen atoms, as the C-F bond stability is superior to those of other carbon-halogen bonds and due to its size fluorine is considered bioisosteric to hydrogen.”

2) The authors would like to thank the referee for their comment on this important point. To address this topic, a section was added to the introduction of the manuscript, with according literature sources.

“(…) as well as facilitate interactions with other functional groups through hydrogen or halogen bonding.”

3) The referee has a good point with the importance of the position of the fluorinated residue. In this study the library of all synthesized inhibitors only contains P1-substituted variants. If the referee wanted to suggest the substitution at the P1’ position, the authors did not synthesize these variants, however think this is a great idea for extending this study in the future. Nevertheless, in context of this report, it would go beyond the scope of the anticipated investigations.

Studies with varying positions of the fluorinated amino acid in substrates have been described by our group in the past years and pose as an individual topic. Here are a research article and a review by our group for further reading:

Asante, V., et al. (2014). "Impact of fluorination on proteolytic stability of peptides: a case study with α-chymotrypsin and pepsin." Amino Acids 46(12): 2733-2744.
Huhmann, S. and B. Koksch (2018). "Fine-Tuning the Proteolytic Stability of Peptides with Fluorinated Amino Acids." European Journal of Organic Chemistry 2018(27-28): 3667-3679.

Minor error:
The authors corrected the mistake from nm to nM. 
Referee: 2

Comments to the Author

Leppkes and co-workers in this manuscript report the microwave solid-phase peptide synthesis of a library of bovine pancreatic trypsin inhibitor (BPTI), a 58 amino acid protein with side-chain fluorinated aliphatic amino acids, (e.g., MfeGly, DfeGly, TfeGly, DfpGly, PfpGly) replacing Lys15. Further inhibitory activities of these BPTI variants were tested towards the serine protease alpha-chymotrypsin, from which two fluorinated BPTI were found with enhanced inhibition. In the previous study, the same group reported the BPTI variants with Abu, DfeGly, and TfeGly and their inhibitory activities towards beta-trypsin in 2015. The current studies are more or less the expansion of the previous one. To make the current manuscript more informative, some revisions are suggested:

1) Provide the overlayed CD of BPTI variants, and provide comments on the effect of fluorination on the protein conformation in the text

2) Provide the detailed synthesis and characterization of all fluorinated aliphatic amino acid building blocks if not commercially available

3) Page 4, provide a brief description of Otlewski and Smalas's result.

4) it is argued to use the polarity to explain the inhibition activity. However, how can the result of TfeGly fit in this explanation? It might be difficult; however, it is suggested to come up with a more generalized model

5) Ref. 13 and 44 are the same.

6) The conclusion is suggested to include the further elaboration of the result for the general application in the field.

Response to the Referee:

The authors thank the referee for the productive input regarding the submitted manuscript.

1) The authors think it is a good idea to show overlayed CD spectra of all BPTI variants and thus added a figure (Figure S2) to the supporting information of this manuscript, containing overlayed CD spectra of all BPTI variants. In addition to that we commented on the secondary structures of all BPTI variants in the main body of the manuscript, referring to the Supporting Information figure:

“In addition to that CD spectroscopy of all BPTI variants showed that overall global conformation is not perturbed (Figure S2).”

2) The synthesis of fluorinated aliphatic amino acids is currently uploaded to ChemRxiv as a preprint and was sent for publication. The reference containing the DOI was added to the manuscript.

3)The authors added short description of Otlwski and Smalas’s results:
“They showed, that BPTI has a relatively broad specificity, inhibiting trypsin- as well as chymotrypsin- and elastase like serine proteases. In complex with β-trypsin, the replacement of Lys15(P1) residue with apolar amino acids, like Nvl or Nle, pushes the hydrophobic side chains in the S1 specificity pocket, which hosts Asp189, leading to a six to nine orders of magnitude drop in inhibitory activity. Whereas in case of α-chymotrypsin, the affinity of wild type BPTI is quite similar to that of semisynthetic BPTI bearing Nvl or Nle instead of Lys.”

4) The authors would like to express gratitude to the referees for bringing up this point. We further elaborated this point with a passage in text, explaining more in detail the difference in the polarities of the various fluorinated amino acids.

“They calculated the electrostatic potential of various fluorinated amino acids, containing MfeGly, DfeGly, DfpGly and TfeGly and could show that MfeGly, DfeGly as well as DfpGly posses a much higher polarity in the side chain than TfeGly.”

5) The authors thank the referee for identifying this mistake.

6) The authors would like to thank the referee for his comment. Further text was added to the manuscript conclusion:

“We argue that fluorine induces polarity in otherwise hydrophobic aliphatic side chains of amino acids. Obviously, fluorine not being used by nature as building block in amino acid biosynthesis, offers the potential of creating amino acid functionalities that are compatible with natural amino acid properties and, thus, fluorinated amino acids can be accepted by natural protein binding pockets. Using these functions skillfully enables the establishment of fluorinated amino acids as bioorthogonal tools in protein engineering and drug development.” 
Referee: 3

Comments to the Author
The manuscript by Koksch describes the effect of fluorination of residue Lys15 in BPTI in its activity to inhibit the serine protease chymotrypsin. The corresponding Lys15-mutant analogs of BPTI were synthesized chemically by SPPS, folded, characterized by X-ray crystallography. When tested for its ability to inhibit chymotrypsin, the authors found that the introduction of CXF2-functionality (where X = H- or CH3-) increased the inhibitory activity of the corresponding BPTI-mutant towards chymotrypsin.

The work presented although interesting is not that innovative, the authors seem to have extended the original work by Ye et al showing that fluorination of the P1 position in BPTI can lead to increased inhibition, which is also observed in the present manuscript.

In addition, the efficient synthesis of long peptides (similar in size to that of BPTI) is now quite common in by using optimized coupling protocols.

Consequently, this work would be more appropriated for a specialized journal in peptide chemistry.

Other comments:

1) The authors may want to consider providing the inhibition data on a table format and using the more common Ki rather than Ka values.

Response to the Referee:

The authors thank the referee for the productive input regarding the submitted manuscript.

The authors would like to underline, that a novel synthesis and refolding protocol was developed compared to the study published by Ye et al. in 2015 in Chem. Sci.. Furthermore, BPTI variants containing previously undescribed fluorinated amino acids, were tested in the context of a different serine protease and varying trends to the study in 2015 are shown.
The authors agree, that with modern techniques in SPPS, the synthesis of larger proteins is easier feasible. However, the authors would like to underline that beside the synthesis of BPTI, additional analyses including inhibition assays, CD spectroscopy and X-ray analysis were performed which very well fits into the scope of this journal, as these techniques are quintessential in the world of chemical biology.



Other comment:

The authors want to thank the referee for the suggestion to change Ka to Ki, as the referee is right, Ki is used more commonly. Manuscript was changed accordingly.

06-May-2022

Dear Dr Koksch:

Manuscript ID: CB-ART-01-2022-000018.R1
TITLE: Fluorine-induced polarity increases inhibitory activity of BPTI towards chymotrypsin

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.
Actually, one of reviewers recommended the rejection of your manuscript and its transfer to other journals. Based on my own evaluation, I made a decision to overwrite his/her recommendation and accepted your manuscript. I think this manuscript can be beneficial to the general readership of RSC Chemical Biology.

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Associate Editor, RSC Chemical Biology
Professor, Chemistry Department, Seoul National University, Korea

Reviewer 3

Manuscript is better suited for a specialized journal in peptide chemistry.

Reviewer 1

The author responded well to the reviewer's questions and has revised the manuscript according to the comments. This manuscript has met the requirements to be received.