From the journal RSC Chemical Biology Peer review history

Berlin, 10 August 2022

Dear Dr Poulsen:

Manuscript ID: CB-ART-06-2022-000161
TITLE: The covalent reactivity of functionalized 5-hydroxy-butyrolactams is the basis for targeting of fatty acid binding protein 5 (FABP5) by the neurotrophic agent MT-21

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Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

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Reviewer 1

This manuscript evaluates a novel reactive group, 5-hydroxybutyrolactams, as a potential covalent warhead. In detailed studies, the authors study the solution reactivity of these warheads with thiols and amines, demonstrating that these groups can in fact form covalent adducts with thiols. The stability of each of the enantiomers is also investigated, and shown to be stable under the experimental conditions used. The reactivity of these warheads within a proteome is then investigated and confirms that covalent protein modification can occur. Surprisingly, these probes are promiscuous in cell lysates, but selective for a ~15kD protein in live cells. This protein target is identified to be FABP5, and labeling of FABP5 by the probe is competed by the known FABP5 ligand SBFI-26. The authors attempt to monitor FABP5 inhibition in cells without success.

Together, this manuscript describes a new reactive group that shows interesting reactivity in cells. This manuscript also fully characterizes the binding site of FABP5 with this warhead. Given the lack of covalent modifiers for FABP5, this study sets the stage for developing more selective FABP5 binders based on this initial lead compound. I recommend publication of this manuscript upon addressing the minor comment below.

--Can the authors confirm that all of the protein targets observed for MT-21 alk in cell lysates (Figure 4a) are cysteine targets? This could be achieved by a simple experiment where the cell lysates are pre-treated with iodoacetamide prior to labeling with MT-21. Alternatively, a MS experiment enriching for MT-21 alk labeled peptides with analysis of site of labeling would also confirm this fact.

Reviewer 2

In the submitted manuscript, Svenningsen et al describe covalent chemical probes for 5-hydroxy-3-acyl-butyrolactams with a bioactive analog of a natural product. The authors demonstrate that this scaffold is cysteine reactive. Through a series of chemical proteomics experiments, they elucidate that the chemical probe based on this scaffold binds a specific cysteine residue in fatty acid-binding protein 5. Interestingly, this only takes place in live cells, cells, specifically C120, and the authors provide a computational model of the binding mode. The authors also find that this probe affects the lipid uptake in cells, but through a mechanisms independent of FABP5.
Overall, I found this an interesting, in-depth and well-designed chemical biology study that reveals the reactivity of a scaffold that occurs in various natural products. I therefore think that publication in RSC Chemical Biology is appropriate and I recommend publication. I have some comments that the authors should take into consideration when preparing the final manuscript.
1. P11/fig 3a: S-Conj3 and S-Conj2 seems to miss a CH2 group between the sulfur and the CO2
2. Page 13, figure 4a-b: it is interesting that the target labeling in live cells is quite selective, but I was puzzled why the target is not labeled at all in lysates (or at least very low in comparison with the other proteins that are labeled in the lysate). There is no clear explanation given in the results & discussion or in the conclusion (except for a hint on membrane association of the MT-21 probe. When I first saw the results in fig 4ab, I thought it could be a metabolite that is causing the labeling instead. However, I think this is not likely, given the fact that the authors ID-ed the modification site by MS/MS and the MW matches the proposed mechanism. The target itself may give some hint, though. As mentioned in the text, C120 and C127 form a (reversible) disulfide bond. Is it possible that the free C120 only occurs in the cell (because of the reducing environment) and that it’s rapidly oxidized when making a lysate? I saw in the experimental section that the lysates were prepared without the addition of DTT to protect from oxidation. I wondered if: (1) would the use of DTT during cell lysis restore the reactivity of FABP5 against MT-21 alk? (2) Naturally, the C127A mutant cannot form a disulfide. Would the FABP5 C127A mutant display reactivity against MT-21 alk in a cell lysate? This may elucidate why the probe reactivity against FABP5 is selective to whole cells, and the authors may consider doing such experiment.

Typos and other small errors:
P3, abstract: the identification of new classes of reactive groups are -> is
P5, intro: “a lasting, valuable resource for continued…” lasting…continued ..doesn’t read well. Perhaps delete the first
P5: despite of their enzymatic… -> “Despite” or “In spite of”
P5: in principal -> in principle
P17: 2x refs in brackets with ref in front of them. Please just use superscript as for all other references.
P17: found to loose -> found to lose
P19: need to be form -> need to be formed



SI, page 13: scale up of butyl amine conjugate: “See fig s2 for analytical data” -> it’s not in Fig S2 (that’s the enantiomer data), but in the section with all spectra.

Dear Prof. Süssmuth,

Thanks for your e-mail and the reviewer reports. Our response to the comments from the reviewer are below as well as descriptions of the action we have taken.

Best regards,
Thomas

--
Reviewer 1:
--Can the authors confirm that all of the protein targets observed for MT-21 alk in cell lysates (Figure 4a) are cysteine targets? This could be achieved by a simple experiment where the cell lysates are pre-treated with iodoacetamide prior to labeling with MT-21. Alternatively, a MS experiment enriching for MT-21 alk labeled peptides with analysis of site of labeling would also confirm this fact.

We thank the reviewer for the nice feedback. We performed the suggested experiment and observed that most proteins were not blocked by iodoacetamide, indicating that these are not cysteine targets. The amino acid residues could be cysteines not efficiently labelled by iodoacetamide. We have included these results in the supporting information (new Fig. S9) and described them in the main text.

Reviewer 2:
1. P11/fig 3a: S-Conj3 and S-Conj2 seems to miss a CH2 group between the sulfur and the CO2

The figure has been corrected. The same mistake was found in the supporting information and was corrected.

2. Page 13, figure 4a-b: it is interesting that the target labeling in live cells is quite selective, but I was puzzled why the target is not labeled at all in lysates (or at least very low in comparison with the other proteins that are labeled in the lysate). There is no clear explanation given in the results & discussion or in the conclusion (except for a hint on membrane association of the MT-21 probe. When I first saw the results in fig 4ab, I thought it could be a metabolite that is causing the labeling instead. However, I think this is not likely, given the fact that the authors ID-ed the modification site by MS/MS and the MW matches the proposed mechanism. The target itself may give some hint, though. As mentioned in the text, C120 and C127 form a (reversible) disulfide bond. Is it possible that the free C120 only occurs in the cell (because of the reducing environment) and that it’s rapidly oxidized when making a lysate? I saw in the experimental section that the lysates were prepared without the addition of DTT to protect from oxidation. I wondered if: (1) would the use of DTT during cell lysis restore the reactivity of FABP5 against MT-21 alk? (2) Naturally, the C127A mutant cannot form a disulfide. Would the FABP5 C127A mutant display reactivity against MT-21 alk in a cell lysate? This may elucidate why the probe reactivity against FABP5 is selective to whole cells, and the authors may consider doing such experiment.

We thank the reviewer for the detailed feedback and considerations. We performed the suggested experiments and included them in the supporting information (new Fig. S9). We have also added a section to the text describing the observations and interpretations. Briefly, addition of DTT to lysis buffer resulted in strongly reduced labelling with MT-21 alk, probably due to the reaction between the thiols of DTT and MT-21 alk – the same was observed with TCEP. We tried to remove DTT by first precipitating the proteins followed by re-dissolution in 2% SDS and then labelling with MT-21 alk, although this risks re-oxidation of the disulphide. Indeed, we observed no appearance of a band corresponding to FABP5. In a more direct experiment, we expressed the FLAG-FABP5 C127A mutant (which cannot form a disulphide) and labelled lysate from these cells, but still we did not see a band around 15 kDa appear. Possible explanations could be that fatty acids released during cell lysis might compete bindng to FABP5, or that membrane associations, which we also explained in the original text, is required for the interaction between MT-21 (alk) and FABP5.

3. Typos and other small errors:
P3, abstract: the identification of new classes of reactive groups are -> is
P5, intro: “a lasting, valuable resource for continued…” lasting…continued ..doesn’t read well. Perhaps delete the first
P5: despite of their enzymatic… -> “Despite” or “In spite of”
P5: in principal -> in principle
P17: 2x refs in brackets with ref in front of them. Please just use superscript as for all other references.
P17: found to loose -> found to lose
P19: need to be form -> need to be formed

SI, page 13: scale up of butyl amine conjugate: “See fig s2 for analytical data” -> it’s not in Fig S2 (that’s the enantiomer data), but in the section with all spectra.

We thank the reviewer for correcting these mistakes. They have been fixed.

Berlin, 2 September 2022

Dear Dr Poulsen:

Manuscript ID: CB-ART-06-2022-000161.R1
TITLE: The covalent reactivity of functionalized 5-hydroxy-butyrolactams is the basis for targeting of fatty acid binding protein 5 (FABP5) by the neurotrophic agent MT-21

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Prof. Dr. Roderich Süssmuth
Technische Universität Berlin
Faculty II - Mathematics and Natural Sciences
RSC Chemical Biology Associate Editor

Reviewer 1

The authors have addressed my prior comments; I recommend publication of this manuscript in its current form.

Reviewer 2

The authors have performed additional experiments that now show that the lack of protein labeling is likely not because of the participation of C120 in a disulfide bond. This further supports the idea put forward by the authors that the membrane environment may play a role in the labeling by probe MT-21-alk. As I have no further suggestions or concerns, I recommend acceptance of this manuscript.