From the journal RSC Chemical Biology Peer review history

19-Jun-2021

Dear Dr Pomerantz:

Manuscript ID: CB-REV-04-2021-000085
TITLE: 19F NMR Viewed Through Two Different Lenses: Ligand-Observed and Protein-Observed 19F NMR Applications for Drug Discovery

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.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after revisions.

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I look forward to receiving your revised manuscript.

Yours sincerely,
Prof Seung Bum Park

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

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

Reviewer 1

In this review, Buchholz and Pomerantz present recent developments in ligand-observed and protein observed 19F NMR in drug discovery. After a short overview of fluorine’s role in biomedicine and a short description of the benefits and shortcomings of the fluorine atom, they cover applications of 19F NMR in FBDD. They also describe newer developments in the field including quantitative, structural and in-cell NMR supported by several case studies.
The review is very well-written and it’s clear that the authors are expert in the field. However, in some cases very long sentences can make it challenging to follow, which the authors should pay attention to in revising the manuscript. In general, the work is of high quality, hence suitable for publication in RCS Chem. Biol. after minor revisions.

In the abstract line 4, I recommend to change the sentence “…19F NMR using labeled proteins and small molecules…” to “…19F NMR using labeled small molecules and proteins…”
Page 2 line 11–13: Please insert a reference.
Page 4 line 6: Please insert a reference.
Page 5: Rephrase the sentence “To identify a suitable spy molecule generally a library… for the target is fluorinated”.
Figure 4: Rephrase figure text.
Page 6: (4.9%) should be (4.9% hit rate)
Page 6: “Current CPMG pulses……” should be “As described previously, current CMPG pulses…”
Page 7: In the example of 100-fold potency increase after fragment linking, the affinity range could be reported to give the reader an understanding of how potent the new molecule is.
Page 9 line 5: Reference for the following “…proteins as large as 180 kDa has been studied.”
Page 13: Please show the structure of V and 5.

Typos:
Page 3 line 6 and 7: “170 ppm” on the same line.
Page 3 line 12: “Even isotopes effects” should be “Even isotope effects”
Page 3: E.coli should be E. coli.
Page 3: “They concluded that that…” delete one “that”.
Page 3 column 2: “Conversely…” comma after “Conversely”.
Page 3 column 2: “… an SAR” should be “… a structure-activity relationship (SAR)…”.
Page 3 column 2: “FBDD, offers…” delete the comma.
Page 4 line 2 and 3: “300 g/mol” on the same line.
Page 4 line 9: “…as a sensitive” there are two spaces.
Page 4: “…fluorine direct binding chemical shift anisotropy and exchange for screening (FAXS)” should be “…fluorine chemical shift anisotropy and exchange for screening (FAXS)”.
Page 4 column 1 and in figure 3: n-FABS should be with italic "n"
Page 5: N-acetylserotonin: with italic "N"
Page 8 line 3–5 : “New methodologies ….. is well-suited for this this…” delete one “this”.
Page 8: “Control experiments with ... the product was be found …”delete “be”.
Page 9 last line in column 1: “However this…” comma after “However”.
Page 9 second column: “Cys 265” delete the space.
Page 10 first column: “… Six of the seven” delete a space.
Page 10 first column: “…aromatic amino acids…” delete a space.
Page 10 first column fifth and fourth last line: “40 µM” on the same line
Page 13, line 10. “20 uM binder” – I assume this is Kd, so perhaps state that.
Page 13, twice “1,4,5-substituted-imidazole” should be “1,4,5-substituted imidazole”
Page 13: >55 fold should be >55-fold.
Page 13: 9-13 fold should be 9–13-fold. And in general, EN DASH should be used for ranges throughout.
Page 13: ”Unfortunately there were…” comma after “unfortunately”.
Page 13: “D-galactose” should be ”ᴅ-galactose”.
Page 13: “N-acetyl D-galactosamine” should be with italic "N"
Page 14 line 11: “… phenylalanines In a folded protein” should be “… phenylalanines in a folded protein”.
Page 14: “…the scope of methodology” should be “the scope of this methodology”.
Page 14: “trifluoromethyl-L-phenylalanine” should be “trifluoromethyl-ʟ-phenylalanine”.
Page 14 line 14: “Alternatively 19F…” comma after “Alternatively”.
Page 14: “in vitro” in italics.
Page 15 line 2: “However only…” comma after “However”.

In general:
Et al. should be italics. On page 1, 3, 5, 6, 9, 10, 12 and 13.
“~ XX” with space on page 3 and 5. The space should be deleted.
“XX member” with space on page 6, 10 and 12. Insert a hyphen between the number and member.
Ki , KD, Koff, Kon ect. should be italics on page 5, 7, 8, 9, 12 and 13

Reviewer 2

The review on „19F NMR Viewed Through Two Different Lenses“ by Carolin Buchholz and William Pomerantz is a timely written review of the recent literature. The authors start with a well written introduction giving also the non-expert an entry port into the fundamentals of the subject. The examples given afterwards make this review a nice summer of what has happened in the recent past and an excellent starting point for others in the chemical biology / medicinal chemistry interface to start or expand their knowledge.

Only a few comments should be addressed:
- there is an overall focus on fragment-based screening. This should be covered in the title as well.
- Figure 1 should include the appropriate reference (already given in the main text)
- figure captions should be a bit more descriptive e.g. figure 2 has almost no help to understand the context of the figure to an outsider of the field. Where in this figure is NMR useful?
- page 4, T2 filter experiments are explained on a very technical level, likely for the NMR spectroscopist. Not every reader with a chemical biology can anticipate of decaying echos. It would be helpful to translate this into something observable for the non-NMR spectroscopist. E.g. once the small ligand adopts the relaxation properties of the large receptor, its peak intensities will diminish faster compared to the free non-binder …
- page 5: FAXS, please refer the reader to a limitation of this assay setup, that is the existence of an „assay wall“. Depending on the setup of the assay there is a minimum Kd that can be determined by such an approach e.g. given by the protein concentration. Even if the inhibitor would be fM at least 50% of the available protein has to be bound to generate an e.g. IC50.
- page 5: FAXS, using such an approach for fragment cocktails implies deconvolution - which is stated by the authors. However, with an lets say average hit rate of 5% of a fragment library against an average target, one would need a very low number of fragments (< 20) in one bin to make deconvolution useful and not generating more work than anticipated. This is often impractical.
- page 6, when discussing BURBOP and large chemical fragment mixtures: would the authors be concerned about fragment competition and thereby artificial reduction of the hit-rate? I wouldn’t but it would be helpful for the reader to learn more about this. In the same context, a sentence on how the chemical shift diversity (done during the selection) relates to a chemical diversity that is essential for fragment screening/druggability assessment.

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

Thank you for handling our manuscript. We have revised our review article with updated title “19F NMR Viewed Through Two Different Lenses: Ligand-Observed and Protein-Observed 19F NMR Applications for Fragment-Based Drug Discovery” and now feel it is suitable for publication in RSC Chemical Biology. We thank the reviewers for their careful consideration of our manuscript and suggestions. We address their comments below in a point-by-point fashion in our attached cover letter which have significantly improved our report.

When uploading our documents, the template appears to be adding line numbers over over text, obscuring some text. Several versions were uploaded to try to avoid this.

Dear Dr. Park,
Enclosed is our revised review article with updated title “19F NMR Viewed Through Two Different Lenses: Ligand-Observed and Protein-Observed 19F NMR Applications for FragmentBased Drug Discovery” which we feel is now suitable for publication in RSC Chemical Biology”.
We thank the reviewers for their careful consideration of our manuscript and suggestions. We address their comments below in a point-by-point fashion which have significantly improved our report.
Referee: 1
Comments to the Author
In this review, Buchholz and Pomerantz present recent developments in ligand-observed and protein observed 19F NMR in drug discovery. After a short overview of fluorine’s role in biomedicine and a short description of the benefits and shortcomings of the fluorine atom, they cover applications of 19F NMR in FBDD. They also describe newer developments in the field including quantitative, structural and in-cell NMR supported by several case studies. The review is very well-written and it’s clear that the authors are expert in the field. However, in some cases very long sentences can make it challenging to follow, which the authors should pay attention to in revising the manuscript. In general, the work is of high quality, hence suitable for publication in RCS Chem. Biol. after minor revisions.
1. In the abstract line 4, I recommend to change the sentence “…19F NMR using labeled proteins and small molecules…” to “…19F NMR using labeled small molecules and proteins…”

We have made this revision.
2. Page 2 line 11–13: Please insert a reference.
We have made this revision, A review by Ray Norton on “Applications of 19F-NMR in fragment-based drug discovery” has been added as reference 21.

3. Page 4 line 6: Please insert a reference.
We have made this revision, A perspective by Dan Erlanson “Introduction to Fragment-Based Drug Discovery” is now ref. 59
4. Page 5: Rephrase the sentence “To identify a suitable spy molecule generally a library… for the target is fluorinated”.
We turned this into two sentences for clarity. “To identify a suitable spy molecule a library of fluorinated molecules can be first screened against the target. Alternatively, a second approach can be to fluorinate a known ligand.”
5. Figure 4: Rephrase figure text.
We have updated the figure legend to improve the clarity
6. Page 6: (4.9%) should be (4.9% hit rate)
Thank you. We have updated this point.
7. Page 6: “Current CPMG pulses……” should be “As described previously, current CMPG pulses…”
Thank you. We have updated this point.
8. Page 7: In the example of 100-fold potency increase after fragment linking, the affinity range could be reported to give the reader an understanding of how potent the new molecule is.
Thank you. We have updated this sentence. As the authors are somewhat vague on the starting point. We list the final potency as ~ 74 nM.
9. Page 9 line 5: Reference for the following “…proteins as large as 180 kDa has been studied.”
We have added this reference. It is now Ref 36 from the Arthinari group.
10. Page 13: Please show the structure of V and 5.
Although we have decided not to add an additional figure for space concerns, we have updated the description to include the structural class of these molecules as 1,4,5 trisubstituted imidazoles.
11. Typos
Regarding the suggested typos, we thank the reviewer for the conscientious comments and have made all of the updates. In the three examples, where ppm was moved to a separate line, we wanted to leave those corrections for the copy editing stage as the formatting may change.
Referee: 2
Comments to the Author
The review on „19F NMR Viewed Through Two Different Lenses“ by Carolin Buchholz and William Pomerantz is a timely written review of the recent literature. The authors start with a well written introduction giving also the non-expert an entry port into the fundamentals of the subject. The examples given afterwards make this review a nice summer of what has happened in the recent past and an excellent starting point for others in the chemical biology / medicinal chemistry interface to start or expand their knowledge.
Only a few comments should be addressed:
1. there is an overall focus on fragment-based screening. This should be covered in the title as well.
We thank the reviewer for this point and have updated the titled to include Fragment-Based Drug Discovery.
2. Figure 1 should include the appropriate reference (already given in the main text)
We thank the reviewer for this point and have added the comprehensive reference string to the figure legend.
3. figure captions should be a bit more descriptive e.g. figure 2 has almost no help to understand the context of the figure to an outsider of the field. Where in this figure is NMR useful?
We thank the reviewer for this point and have updated the figure legends. Figure legend 2 is reproduced below. “Figure 1: NMR in fragment-based drug discovery. Screening of libraries of ~500–2,000 fragments by NMR can be followed up by fragment growing and/or linking, and finally optimization to FDA approved drugs as in the case of Venetoclax.”
4. - page 4, T2 filter experiments are explained on a very technical level, likely for the NMR spectroscopist. Not every reader with a chemical biology can anticipate of decaying echos. It would be helpful to translate this into something observable for the non-NMR spectroscopist. E.g. once the small ligand adopts the relaxation properties of the large receptor, its peak intensities will diminish faster compared to the free non-binder …
We thank the reviewer for this suggestion, and have amended the text with additional clarification for the non-expert, to appreciate the observables in the experiment. The text is reproduced below. “In practice, a T2-based filter experiment leads to detection of ligand binding events through the observation of a decrease in resonance intensity corresponding to the free ligand. This decrease in intensity is due to filtering out of the bound state contribution from the ligand that is in rapid chemical exchange.”
4. page 5: FAXS, please refer the reader to a limitation of this assay setup, that is the existence of an „assay wall“. Depending on the setup of the assay there is a minimum Kd that can be determined by such an approach e.g. given by the protein concentration. Even if the inhibitor would be fM at least 50% of the available protein has to be bound to generate an e.g. IC50.
We thank the reviewer for this point and have amended the sentence appropriately. This F value is a displacement value used to determine the Ki and thus an accurate IC50 needs to be determined. The edited text is reproduced below. “An attractive feature of this approach is that while spy molecules are weak affinity binders, they can be used to determine the Ki of high affinity ligands presuming sufficient protein is used to determine an accurate displacement value.73”
5. page 5: FAXS, using such an approach for fragment cocktails implies deconvolution - which is stated by the authors. However, with an lets say average hit rate of 5% of a fragment library against an average target, one would need a very low number of fragments (< 20) in one bin to make deconvolution useful and not generating more work than anticipated. This is often impractical.
We agree with the reviewer that the deconvolution steps can be time consuming, similar to protein-observed NMR experiments with fragment mixtures. We note that a deconvolution step is needed and will leave the decision of practicality of that step up to the users/readers.
6. page 6, when discussing BURBOP and large chemical fragment mixtures: would the authors be concerned about fragment competition and thereby artificial reduction of the hit-rate? I wouldn’t but it would be helpful for the reader to learn more about this.
This was indeed a concern that we believe the authors wanted to address in their supermixture of 152 compounds. We have clarified the text to note that 5 fragments hits from the original screen were included in that mixture showing that competition wasn’t a concern due to rapid exchange a weak binding fragments. Presumably this may be a concern if very high affinity interactors were present, but that is unlikely from a fragment library.
7. In the same context, a sentence on how the chemical shift diversity (done during the selection) relates to a chemical diversity that is essential for fragment screening/druggability assessment.
We note in the main text that the chemical diversity is being determined from a 2D-fingerprint analysis developed by Novartis over the years to assess chemical space and is not based solely on chemical shift. Due to the sensitivity of the fluorine chemical shift to different chemical environments and the fluorine functional group’s representation of only a small percentage of the entire molecule, we do not anticipate a strong correlation between chemical shift diversity and chemical space diversity. However, in their screen, the number of hits for a particular chemical shift range correlated with the observed hits, supporting the inclusion of a broad range of fluorinated motifs. This is now indicated in the main text. “Of these hits, the number of library members within a specific chemical shift range correlated with the number of observed hits, supporting a lack of bias towards a particular fluorinated motif.”
Sincerely,
William Pomerantz,
University of Minnesota

07-Jul-2021

Dear Dr Pomerantz:

Manuscript ID: CB-REV-04-2021-000085.R1
TITLE: 19F NMR Viewed Through Two Different Lenses: Ligand-Observed and Protein-Observed 19F NMR Applications for Fragment-Based Drug Discovery

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.

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With best wishes,

Prof Seung Bum Park

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

Reviewer 1

I think the authors have addressed all points made by the reviewers and have thus improved the manuscript. I recommend publication in RSC Chem Biol.