From the journal RSC Chemical Biology Peer review history

17-Oct-2022

Dear Prof Wright:

Manuscript ID: CB-COM-09-2022-000199
TITLE: A fluorescent photoaffinity probe for formyl peptide receptor 1 labelling in living cells

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.

I have carefully evaluated your manuscript and the reviewers’ reports, and the reports indicate that major revisions are necessary. In particular, I agree with the reviewers that additional assays including competition assays would strengthen the claims of the work. I also note the comments on the references.

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Prof Gonçalo Bernardes

Associate Editor, RSC Chemical Biology

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Reviewer 1

Field et al describe in the submitted manuscript a novel photoaffinity probe for the GPCR FPR1. The authors show that FPR1 is efficiently engaged by the probe by using flow cytometry as well as fluorescent microscopy read out. The authors confirm that covalent modification is achieved upon UV irradiation by SDS-PAGE. Furthermore, they perform experiments to determine the efficiency of photoaffinity labeling, which turns out to be 20-25%.
I think this work is of good technical quality. The ease of preparation of these probes as well as the importance of formyl peptide receptors in immunology will make them attractive tools for future studies of this class of GPCRs. I therefore recommend publication, but with below minor comments.

Minor comments:
Abstract: the authors use the term photoaffinity probe but also photocrosslinking chemical probe. Photocrosslinking is more often used for forming links between proteins for structural proteomics, so I recommend the use of photoaffinity probes to prevent any confusion.

Not all curves in the histogram of Fig S2b are well visible. For clarity, this panel may be split into several different panels (e.g. one for each concentration of fMLF, or something with a clearer difference in coloring).

P6: typo: To determine whether Probe-TAMRA exhibited similar behaviour to and…-> remove ‘to’.

Figure 4 and Fig S3-6: The authors perform a range of photoaffinity labeling experiments with their TAMRA-probe with SDS-PAGE read out. While they have a range of controls, including mock transfected cells, there was no control with a competitive ligand such as fMLF (which was included as competitor in the flow cytometry experiments). While it may not ultimately be necessary to have this control, it is usual to include it, as it is a good way to confirm specific target modification. Did the authors try this? Or would this not be successful as fMLF is non-covalent and would get outcompeted by probe binding?

The authors claim that there are only a handful of such probes available, and the authors refer to 3 papers. As I am not much into GPCRs, I was wondering if these are the only three papers reporting photoaffinity probes for GPCRs or if these were just examples. When I did a quick literature search, I found more reported probes, such as against VPAC1, various adrenergic receptors (most early work before the advent of activity and affinity-based protein profiling, by using radioactive isotope-labelled compounds with arylazides or benzophenones). Would it make more sense to refer to a review article on GPCR photoaffinity labeling (if such review exists) and perhaps also name a few recent examples?

Reviewer 2

Wright and coworkers report a photo-crosslinking fluorescent chemical probe for Formyl peptide receptor 1 (FPR1), which is a critical component of the innate immune response to bacterial infections and a promising target in inflammatory diseases. They show that the probe binds and covalently crosslinks to FPR1 at nanomolar concentrations in living cells, and thus can be a useful tool for visualisation and characterization of this receptor.
Major comments:
1) Competition assays: it would be interesting to keep the concentration of the probe-TAMRA at 10 nM and increase the concentration of the tracer-FITC.
2) What could be the reason for the same affinity of probe-TAMRA compared to tracer-FITC? According to the X-ray structures (refs. 13 and 14), is the fluorophore exposed to the solvent? This point should be discussed in the paper.
3) Page 7-8: 'There is a small amount of FPR1-independent binding, in flow cytometry data (Figure 2a) so it is possible that this band corresponds to another protein that also binds the probe.' According to these data, the probe-TAMRA does not have high specificity as stated in the abstract. What could the other protein be? Is it a non-specific binding? This point should be discussed in the manuscript
4) It would be interesting for the reader to show the crosslinking reaction in a ChemDraw scheme. Why is the irradiation time 30 s?
5) Why are there differences between Figure 4 (compound 2) and Figure 2b? It is only fluorescence at 525 nm versus 583 nm.
6) The 1H NMR, 13C NMR, and HRMS of compounds 1-7 and the probe-TAMRA and tracer-FITC should be included in the Supplementary information.
7) According to the data, the hydrated version of the aldehyde is not detected in probe-TAMRA and tracer-FITC. This is somewhat odd, especially after purification by HPLC.
8) There are no clear bands in Figure S3 b and Figure S5. What is the explanation for this?


Minor comments:
Use "s", "min" and "h" for seconds, minutes and hours, respectively.
Please check the quantities used in the supporting information. For example: 2.11 mL. I think it should be 2 mL, 1.066 g should be 1.07 g.
Figure 3: Probe2.0TAMRA should be probe-TAMRA.

We thank the reviewers for their careful consideration of our manuscript and suggestions for improvements. We have performed additional experiments to show that the probe can be outcompeted by other FPR1 ligands as assessed by in-gel fluorescence. We agree that this adds additional confidence that the labelled protein is FPR1 and we also think this could be a useful way of evaluating ligands and drug-like modulators in the future.

Jack White, who assisted in the additional included experiment, has been added as a co-author.

Below are changes and clarifications in response to the reviewer comments.

************
REVIEWER REPORT(S):
Referee: 1

Comments to the Author
Field et al describe in the submitted manuscript a novel photoaffinity probe for the GPCR FPR1. The authors show that FPR1 is efficiently engaged by the probe by using flow cytometry as well as fluorescent microscopy read out. The authors confirm that covalent modification is achieved upon UV irradiation by SDS-PAGE. Furthermore, they perform experiments to determine the efficiency of photoaffinity labeling, which turns out to be 20-25%.
I think this work is of good technical quality. The ease of preparation of these probes as well as the importance of formyl peptide receptors in immunology will make them attractive tools for future studies of this class of GPCRs. I therefore recommend publication, but with below minor comments.

Minor comments:
Abstract: the authors use the term photoaffinity probe but also photocrosslinking chemical probe. Photocrosslinking is more often used for forming links between proteins for structural proteomics, so I recommend the use of photoaffinity probes to prevent any confusion.
- Two instances changed: in the abstract and in the main text.

Not all curves in the histogram of Fig S2b are well visible. For clarity, this panel may be split into several different panels (e.g. one for each concentration of fMLF, or something with a clearer difference in coloring).
- Data has now been split into two panels – one which shows the lower concentrations of fMLF, where no change is seen, and one where the changes in response to higher concentrations of competitor are more clearly visible. Colours have been altered too for greater clarity.

P6: typo: To determine whether Probe-TAMRA exhibited similar behaviour to and…-> remove ‘to’.
- Corrected

Figure 4 and Fig S3-6: The authors perform a range of photoaffinity labeling experiments with their TAMRA-probe with SDS-PAGE read out. While they have a range of controls, including mock transfected cells, there was no control with a competitive ligand such as fMLF (which was included as competitor in the flow cytometry experiments). While it may not ultimately be necessary to have this control, it is usual to include it, as it is a good way to confirm specific target modification. Did the authors try this? Or would this not be successful as fMLF is non-covalent and would get outcompeted by probe binding?
- We agree that this would be an interesting experiment and so have carried this our, using fMLF, BocMLF and fMLFF (another potent reported ligand) and analysing by in-gel fluorescence. Concentrations of fMLF and BocMLF comparable with those used in our flow cytometry experiments do indeed give a drop in labelling intensity, and fMLFF abrogates labelling. We thank the reviewer for the suggestion.

The authors claim that there are only a handful of such probes available, and the authors refer to 3 papers. As I am not much into GPCRs, I was wondering if these are the only three papers reporting photoaffinity probes for GPCRs or if these were just examples. When I did a quick literature search, I found more reported probes, such as against VPAC1, various adrenergic receptors (most early work before the advent of activity and affinity-based protein profiling, by using radioactive isotope-labelled compounds with arylazides or benzophenones). Would it make more sense to refer to a review article on GPCR photoaffinity labeling (if such review exists) and perhaps also name a few recent examples?
- We apologise for the poor phrasing. There are only a few fluorescent probes reported that have been shown to crosslink to GPCRs on living cells. We have rephrased this part of the text (conclusion) to hopefully make clearer where the value in our approach lies. There is indeed a long history of crosslinking probes for GPCRs and we have mentioned this.

Referee: 2

Comments to the Author
Wright and coworkers report a photo-crosslinking fluorescent chemical probe for Formyl peptide receptor 1 (FPR1), which is a critical component of the innate immune response to bacterial infections and a promising target in inflammatory diseases. They show that the probe binds and covalently crosslinks to FPR1 at nanomolar concentrations in living cells, and thus can be a useful tool for visualisation and characterization of this receptor.

Major comments:
1) Competition assays: it would be interesting to keep the concentration of the probe-TAMRA at 10 nM and increase the concentration of the tracer-FITC.
- The 1:1 competition experiment by flow cytometry is shown in figure 2d. We agree that it would be nice to have the full concentration range for the inverse competition to that in Fig2e and FigS2, but decided that with limited resources available at present for performing additional experiments, analysing competition assays by gel (new figure 4c) would add greater value to the manuscript. We believe that we have provided a high level of evidence that Probe-TAMRA binds to FPR1 in the manuscript.

2) What could be the reason for the same affinity of probe-TAMRA compared to tracer-FITC? According to the X-ray structures (refs. 13 and 14), is the fluorophore exposed to the solvent? This point should be discussed in the paper.
- This is a good question and we have added some discussion to the manuscript. There are no published structures of fluorescent peptides bound to FPR1, although based on the structures of longer peptides one would predict the fluorophore to be positioned near the entrance to the binding pocket and not necessarily contributing many specific interactions (i.e. exposed to solvent as the referee suggests). Consistent with this, a TAMRA variant of the Tracer-FITC has been reported to have a low nM binding affinity (Science 1979), suggesting that these fluorophores at least are interchangeable. The fluorophore could be making some interactions with FPR1 but the nature of these is unknown.

3) Page 7-8: 'There is a small amount of FPR1-independent binding, in flow cytometry data (Figure 2a) so it is possible that this band corresponds to another protein that also binds the probe.' According to these data, the probe-TAMRA does not have high specificity as stated in the abstract. What could the other protein be? Is it a non-specific binding? This point should be discussed in the manuscript
- Unfortunately we don’t know what this protein is and it is difficult to identify based on the data we have. The additional experiment we have performed to evaluate gel-based competition suggests that it is not competed by known FPR1 ligands so it may be a probe-specific binder. We have added some discussion on this point. We have also moderated our language around the specificity of probe-TAMRA, although we do believe that it is astonishingly selective for a photoaffinity probe. For example, see Kleiner et al. Angew Chem 2017, 56, 1396, where the authors demonstrate that photoaffinity probes have a variety of background binders in the proteome; some of this may be driven by the preference for alkyl diazirines to label acidic residues – see West et al. JACS 2021, 143, 6691. It is possible that the off-target band is a protein particularly prone to diazirine photolabelling (e.g. ALDH1B1, found as a diazirine off-target by Kleiner et al., which has a molecular weight of 57 kDa), but this is pure speculation at present.

4) It would be interesting for the reader to show the crosslinking reaction in a ChemDraw scheme. Why is the irradiation time 30 s?
- We have added a scheme to figure 1 to illustrate this. We chose 30 s because this is what had been optimised previously for this device in experiments crosslinking proteins with diazirines (we have included a statement to this effect and the relevant reference).

5) Why are there differences between Figure 4 (compound 2) and Figure 2b? It is only fluorescence at 525 nm versus 583 nm.
- Figure 2b is flow cytometry data looking at TAMRA fluorescence (emission at 585 nm) whereas Figure 4b is looking at FITC fluorescence (emission at 525 nm). We have edited the legend of Figure 4 to hopefully make this clearer. We speculate that there is a small amount of fluorescence bleed-through of TAMRA into the FITC emission channel, which gives rise to the shift of peak that the reviewer has noted in Figure 4 for sample 2 (cells incubated with TAMRA only). This is not ideal but the x-axis is a log scale so this is minor and we judged that it was not sufficient to cause a problem in analysing flow cytometry experiments. In the future, it would be interesting to explore alternative fluorophores that might be better suited to imaging applications and/or (picking up on point 2) fluorophores that show changes in fluorescence upon probe binding.

6) The 1H NMR, 13C NMR, and HRMS of compounds 1-7 and the probe-TAMRA and tracer-FITC should be included in the Supplementary information.
- Compounds 1-7 are known in the literature (references are given) so we did not strictly need to include them in the manuscript at all, but we appreciate that inclusion of such data is useful for future researchers and so have included the 1H NMR spectra as an appendix.
For the peptides, we have now included the HRMS spectra and apologise for this omission. We have attempted to get a 1H NMR for probe-TAMRA but had little material remaining and the spectrum was too weak. If the reviewer feels strongly that this is needed then we can resynthesise the probe and obtain this. However, NMR data would not normally be obtained as standard for peptidic probes, as this is typically hard to interpret due to overlapping signals. Usually HRMS and analytical HPLC are sufficient to confirm identity and purity.

7) According to the data, the hydrated version of the aldehyde is not detected in probe-TAMRA and tracer-FITC. This is somewhat odd, especially after purification by HPLC.
- We did not observe evidence for the hydrate of the formyl group in our data for these probes. However, the hydrate of amides is known to be highly disfavoured so we would not expect to see this under these analytical conditions.

8) There are no clear bands in Figure S3 b and Figure S5. What is the explanation for this?
- GPCRs such as FPR1 are typically glycosylated and the heterogeneity in the N-glycans cause the protein to run as a diffuse band by SDS-PAGE. This is confirmed by our experiment shown in FigS4a, where the glycosidase PNGaseF causes the diffuse band to resolve into a much tighter one. The tighter band can also be observed in Figure 4c. The variations in how diffuse the band appears in different experiments may be due to the fact that we are using transient transfection of cells, which can give small variations in receptor expression/trafficking on a day-to-day basis, or due to minor variations in running SDS-PAGE gels.

Minor comments:
Use "s", "min" and "h" for seconds, minutes and hours, respectively.
- Main text and SI checked and corrected.

Please check the quantities used in the supporting information. For example: 2.11 mL. I think it should be 2 mL, 1.066 g should be 1.07 g.
- Changed in a few places.

Figure 3: Probe2.0TAMRA should be probe-TAMRA.
- Thank you – changed.

10-Jan-2023

Dear Prof Wright:

Manuscript ID: CB-COM-09-2022-000199.R1
TITLE: A fluorescent photoaffinity probe for formyl peptide receptor 1 labelling in living cells

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.

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Reviewer 1

With the changes made to the manuscript, the authors have addressed all my concerns.

Reviewer 2

The authors properly replied to all concerns. I recommend the publication of this manuscript.