From the journal RSC Chemical Biology Peer review history

11-May-2020

Dear Dr Sieber:

Manuscript ID: CB-COM-04-2020-000048
TITLE: Tranylcypromine specificity for monoamine oxidase is limited by promiscuous protein labelling and lysosomal trapping

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

Sieber and colleagues reported their efforts in profiling off-targets of the MAO inhibitor drug, tranylcypromine, by chemical proteomics. They synthesized two version of probes, one with the photocrosslinking group and the other one not. Quantitative chemical proteomics identified not only the targets that are covalently modified by the probe, but also those are non-covalently bound the tranylcrypromine group. This led to the discovery that the drug can be trapped in lysosomes that can be released by a combination use of the lysosome-targeting drug. As the chemical proteomic pipeline is up to the standard, the novel finding lies in the lysosome localization of tranylcypromine which will provide new insights to future optimization of the drug to reduce its side effect. The reviewer supports its publication with the following issues addressed.
1） As submitted in a communication format, the writing is quite concise but the figures are unproportionally huge. This might be just a formatting issue, but the reviewer feels that reducing the figure sizes should give more room for the authors to elaborate their work in a bit more details.
2） What is the justification for choosing the 100uM probe concentrations for their profiling experiments? If the authors have done any optimization on the dosing concentration, data should be reported.
3） The competition experiments reported in figure S2 are quite important for any type of target deconvolution experiment, regardless whether the probe is photo-labile or not. However, the data were not analyzed with enough attention in the current format. As the manuscript is mainly focusing on tranylcyproamine, the reviewer suggests that the main text and figures should be devoted to FBP2 and FBPP2 probes. A possible rearrangement is to move some of the figure S3 to the main text and move Figure 2B to SI. This will put the probe enrichment and competition data for FBP2 side-by-side, or the authors can report targets that are both enriched by the probe and competed by the original drug.
4） The same competition analysis should be done for FBPP2. And since it is a photo-labile probe and is designed to capture non-covalent interactions, the authors are advised to do more analysis to compare the target list between FBP2 and FBPP2. Are those lysosome-localizing targets only showing up in the FBPP2 probe pull down? Would the acid environment affect the reactivity of the probe to covalently react with targets in lysosomes?
5） Gel images in Figure S3 are suggestive evidences to show covalent labeling of these targets by FBP2, however, proper controls such as untreated lanes, competed lanes or even simply concentration dependent lanes should be provided.

Reviewer 2

The manuscript by Dreschel et al reports on proteome-wide (off)target profiling and visualization of subcellular compartmentalization of the marketed anti-depressant, Tranylcypromine, using chemoproteomic and cellular imaging strategies. The work represents the first gel-free LC-MS/MS study for off-target profiling of tranylcypromine.

Specifically, the authors i) combine alkyne probe-labeling and LC/MS-MS to inventory covalent off-targets of the title compound; ii) exploit photoaffinity labeling to profile non-covalent interactors, and iii) utilize in-cell Click chemistry to image apparent lysosomal trapping of the said drug. They reason that tranylcypromine exhibits several cellular off-targets as well as reduced activity due to this observed lysosomal trapping.

As such, the findings from a study like this could potentially lead to annotation of the MAOI's side-effects to the identified off-targets, Further, the imaging experiments suggest that co-administration of lysosomal drugs might offer a potential strategy to "decrease tranylcypromine dosage and thereby lower undesired side effects".

The study, however, has some obvious gaps that warrant addressing before the manuscript can be accepted for publication.Major revisions are therefore suggested.

1. For example, the authors do not mention if the design of probes FBP2 and FBPP2 is supported by established structure-activity relationships. Likewise, no biological characterization of either probe is provided; e.g. binding constants or biochemical/cellular IC50's vis-a-vis MAOA/B, to suggest that the probes may serve as efficient and potent surrogates for the parent compound. It is, therefore, unclear as to whether the reduced enrichment, per se, of MAOA by FBP2 (and complete absence of enrichment for MAOB; Fig 2C) is due to tranylcypromine's own promiscuity and reduced affinity for these targets or due to those changes affected by alkyne-chain installation on the rather small structure. While competitive chemoproteomics experiments (Fig S2) address this point partially, the authors note that they were unable to correlate off-target binding to activity, at least for the couple of targets that they further investigated (ALDH2 and HMOX2, Fig S3).

2. No evidence has been provided as to whether the lysosomal reversible off-targets are specific or non-specific; i.e. the photaffinity labeling study performed with probe FBPP2 (Fig 3A) require being done in a competitive mode to ascertain specificity.

3. While chloroquine treatment is shown to reduce enrichment of lysosomal targets by FBPP2 (Fig 3B) and affect the release from lysosomal compartments of probe FBP2 (Figure 3C and Figure S4), a more direct experimental evidence of lysosomal trapping of tranylcypromine may be obtained by pre-treatment with the drug itself. It is worth pointing out that even small structural changes have the potential to significantly perturb cellular penetration and distribution of a small molecule clickable imaging probe (Ghosh and Jones, MedChemComm, 2014)

Lysosomal trapping is indeed a common phenomenon associated with drugs bearing basic amine groups. A seminal study on this aspect has been published by Zuhl et al (Nat Comm, 2016, pp 1-16), wherein the authors combined photoaffinity labeling and chemoproteomics to reveal the lysosomal target Cathepsin D as the principal driver of ocular toxicity of BACE-1 inhibitors. The current authors are advised to include this work as a reference.

Dear Cai-Guang,

Thank you very much for your feedback on our manuscript. We appreciate the positive evaluation by the reviewers and their constructive suggestions to further improve our manuscript for possible publication in RSC Chemical Biology.
Reviewer 1 states that “the novel finding lies in the lysosome localization of tranylcypromine which will provide new insights to future optimization of the drug to reduce its side effect”. The reviewer provides specific points to be addressed prior to publication. We are also grateful for comments from reviewer 2 who sees “a study like this could potentially lead to annotation of the MAOI's side-effects to the identified off-targets”. Specific changes were recommended. The comments were addressed by a series of additional experiments. Please find below a detailed point-to-point response to the referees’ remarks. We incorporated all changes directly in the revised manuscript and highlighted them in green color.

Reviewer 1:
We thank the reviewer for his positive feedback and the valuable suggestions.

1) As submitted in a communication format, the writing is quite concise but the figures are un-proportionally huge. This might be just a formatting issue, but the reviewer feels that reducing the figure sizes should give more room for the authors to elaborate their work in a bit more details.

We thank the reviewer for this comment. We resized the main figures to provide a more coherent overall appearance. Additionally, we edited major parts of our manuscript, in order to elaborate our work in more detail. For example, we elaborated the labeling workflow in more detail.

2) What is the justification for choosing the 100uM probe concentrations for their profiling exper-iments? If the authors have done any optimization on the dosing concentration, data should be reported.

We thank the reviewer for pointing this out. We therefore prepared a new figure S2, in which we provide concentration depended gel-based labeling results. 100 uM provides sufficient labling intensity and less background compared to 200 uM. Experimental details were added to the method section.

3) The competition experiments reported in figure S2 are quite important for any type of target deconvolution experiment, regardless whether the probe is photo-labile or not. However, the data were not analyzed with enough attention in the current format. As the manuscript is mainly fo-cusing on tranylcyproamine, the reviewer suggests that the main text and figures should be de-voted to FBP2 and FBPP2 probes. A possible rearrangement is to move some of the figure S3 to the main text and move Figure 2B to SI. This will put the probe enrichment and competition data for FBP2 side-by-side, or the authors can report targets that are both enriched by the probe and competed by the original drug.

We thank the reviewer for this great suggestion and for recognizing the importance of these experiments. We largely restructured main figure 2 to now show enrichment and competition data of FBP2 and tranylcypromine side-by-side. Supplementary figures S3 and S4 were modified accordingly.

4) The same competition analysis should be done for FBPP2. And since it is a photo-labile probe and is designed to capture non-covalent interactions, the authors are advised to do more analysis to compare the target list between FBP2 and FBPP2. Are those lysosome-localizing targets only showing up in the FBPP2 probe pull down? Would the acid environment affect the reactivity of the probe to covalently react with targets in lysosomes?

We thank the reviewer for this comment. We included competition data with tranylcypromine and also carefully re-evaluated results between FBP2 and FBPP2; this data is provided as new supplementary figure 6 and discussed in the text. As we came to the conclusion, that protein hits in the experiments using the photo-labile probe are not specific targets of tranylcypromine but rather a result of binding-events after irradiation due to spatial proximity (trapped in lysosome) we also rewrote corresponding parts of the manuscript to stretch this point further.

5) Gel images in Figure S3 are suggestive evidences to show covalent labeling of these targets by FBP2, however, proper controls such as untreated lanes, competed lanes or even simply concentration dependent lanes should be provided.

We apologize for this mistake and added concentration dependent labeling results with untreated lanes as control (new figure S5)

Response to the comments of reviewer 2:

1) For example, the authors do not mention if the design of probes FBP2 and FBPP2 is support-ed by established structure-activity relationships. Likewise, no biological characterization of either probe is provided; e.g. binding constants or biochemical/cellular IC50's vis-a-vis MAOA/B, to suggest that the probes may serve as efficient and potent surrogates for the parent compound. It is, therefore, unclear as to whether the reduced enrichment, per se, of MAOA by FBP2 (and complete absence of enrichment for MAOB; Fig 2C) is due to tranylcypromine's own promiscui-ty and reduced affinity for these targets or due to those changes affected by alkyne-chain instal-lation on the rather small structure. While competitive chemoproteomics experiments (Fig S2) address this point partially, the authors note that they were unable to correlate off-target binding to activity, at least for the couple of targets that they further investigated (ALDH2 and HMOX2, Fig S3).

We thank the reviewer for this suggestion. The design of the probes is based on previous SAR data (now cited in ref 17) suggesting flexibility for modifications at the aromatic ring system. We included this citation in the manuscript. Furthermore, we acquired recombinant MAOA and MAOB and determined apparent IC50 values for probe and parent drug. While MaoA is inhibited with nearly the same potency as with tranylcypromine (300-500 nM), the inhibition of MAOB drops from 100 nM to 2.3 uM suggesting a more restricted accessibility for the affinity tag in the latter case. Thus, MAOA is a high affinity target of the probe and the proteome results should be representative for drug binding. Findings were included in the main text of the manuscript and depicted in figure S1. Experimental details were included in the methods section.

2) No evidence has been provided as to whether the lysosomal reversible off-targets are specific or non-specific; i.e. the photaffinity labeling study performed with probe FBPP2 (Fig 3A) require being done in a competitive mode to ascertain specificity.

See reviewer 1 comment 4)

3) While chloroquine treatment is shown to reduce enrichment of lysosomal targets by FBPP2 (Fig 3B) and affect the release from lysosomal compartments of probe FBP2 (Figure 3C and Figure S4), a more direct experimental evidence of lysosomal trapping of tranylcypromine may be obtained by pre-treatment with the drug itself. It is worth pointing out that even small structural changes have the potential to significantly perturb cellular penetration and distribution of a small molecule clickable imaging probe (Ghosh and Jones, MedChemComm, 2014)

We thank the reviewer for this suggestion. It is certainly true, that even minor changes in the structure of drugs can lead to significantly perturbed cellular properties. We therefore included the above mentioned characterization and control experiments. With the newly performed binding assays using both isoforms, MAOA and MAOB, we proved sufficient binding to the target protein; with the added MS-based competition experiments using tranylcypromine, we directly showed, that release of the probe from the lysosome is a direct consequence of cloroquine treatment, and can not be observed when pretreating the cells with tranylcypromine.

4) Lysosomal trapping is indeed a common phenomenon associated with drugs bearing basic amine groups. A seminal study on this aspect has been published by Zuhl et al (Nat Comm, 2016, pp 1-16), wherein the authors combined photoaffinity labeling and chemoproteomics to reveal the lysosomal target Cathepsin D as the principal driver of ocular toxicity of BACE-1 in-hibitors. The current authors are advised to include this work as a reference.

The corresponding citation was added to the main manuscript.

We hope that these revisions allow for publication in RSC Chemical Biology.

Best wishes,

Stephan Sieber

24-Jul-2020

Dear Dr Sieber:

Manuscript ID: CB-COM-04-2020-000048.R1
TITLE: Tranylcypromine specificity for monoamine oxidase is limited by promiscuous protein labelling and lysosomal trapping

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,

Dr Cai-Guang Yang
Associate Editor
RSC Chemical Biology

Reviewer 1

The authors have adequately addressed my concerns and the revised manuscript has been improved a lot. I think it is ready to be accepted.