From the journal RSC Chemical Biology Peer review history
Methyltetrazine as a small live-cell compatible bioorthogonal handle for imaging enzyme activities in situ
Round 1
Manuscript submitted on 10 Mot 2022![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/icon-letter.png)
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08-Jun-2022
Dear Prof van Kasteren:
Manuscript ID: CB-ART-05-2022-000120
TITLE: Methyltetrazine as a small live-cell compatible biorthogonal handle for imaging enzyme activities in situ
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Yours sincerely,
Prof Gonçalo Bernardes
Associate Editor, RSC Chemical Biology
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Reviewer 1
The manuscript by van de Plassche et al. describes the design of methyltetrazinylalanine as a phenylalanine analog and its use in a dipeptide-based cysteine protease inhibitor for activity-based protein profiling. The design and synthesis of this new activity-based probe were excellent and the gel-based target identification was sound. The live-cell engagement assay was not as convincing based on the imaging data presented. The work is significant as it provides an alternative chemical reporter for ABPP studies. The publication is suggested after the authors address the following concerns.
1) The authors need to explain the color schemes used in Fig. 1, especially with Fig. 1B. The raw in-gel fluorescence images are preferred.
2) If the overlaid image was used in Fig. 2, the separate images need to be presented.
3) Fig. 3: What are the differences between A and B? Co-localization image in B gave white color; are there any other images used in the overlay? Also, is there a reason not using a fluorescent lysosomal tracer in co-localization studies?
4) The live-cell target engagement using new ABPP probe was not as convincing based on the presented image data. Can the authors explain why? Are there any strategies to be employed in the future to improve the performance?
Reviewer 2
In this paper, van de Plassche and coworkers describe the use of a 4-methyltetrazine derivative of alanine in activity based probes. The authors use a pan-cathepsin probe for proof-of-principle studies and show labeling in lysates with gel experiments and in cells with microscopy imaging. Both the concept of labeling activity-based probes after incorporation into proteins by bioorthogonal reactions and the reaction of tetrazines with trans-cyclooctene are well established. Novelty of the article stems from the unprecedented structure of the tetrazine-containing amino acids, which resembles phenylalanine and that it is the first time a tetrazine is used as the bioorthogonal reaction partner linked to the ABP. The innovations and the potential usefulness of this chemistry in principle justify publication in RCS Chemical Biology. However, there are considerable shortcomings in terms of presentation and experimental design as outlined below. These first need to be addressed before the manuscript can be accepted.
- It is noteworthy that the authors use the amino acid in a liquid-phase situation. A comment would be helpful if the amino acid is compatible with solid-phase peptide synthesis conditions. If not, this would reduce the general utility of the approach and needs to be clearly communicated.
- Fig. 1a: It is not mentioned what the staining corresponds to.
- Fig. 1: The assignment of bands to cathepsin needs to be shown in the figures.
- Fig. 1b: There is no mention of what lanes contain the sCy5-TCO probe.
- It is unclear why the staining color in Fig. 1b for probe 6 is different than in Fig. 1a.
- Protein size rulers are missing in all gels.
- Quantitative evaluations of the labeling are missing for the reaction of probe 5 with cathepsins. In figure 1b, Z-FA-FMK appears to completely out-compete 5 in the direct competition experiment. This result contradicts the premise of the publication, which is that methyltetrazine-modifications are better tolerated than other bioorthogonal groups. Careful quantification is required to make that point. The authors mention a norbornene-labeled probe as an example for a bulky group that is disadvantageous for ABP labeling. A direct comparison of 5 with a norbornene-labeled probe would help validate their claims.
- The authors mention the need for cell fixation because the LAMP-1 antibody is not cell permeable. However there are several widely used lysosomes stains available that are perfectly suitable with living cells. It appears more likely that the authors chose to fix the cells because of the need to wash away the unreacted CF©500-TCO probe. Please clarify.
- Microscopy is a problematic way of comparing fluorescence intensities such as it has been done for the staining with CF©500-TCO. Flow cytometry is the preferred way of quantifying such differences.
- The colonization of the CF©500-TCO and the LAMP1-antibody is poor. The authors make the point that other reports have also found poor colocolization. If co-localization is intrinsically poor, then this would mean that the experiment was poorly designed. Maybe, the authors could use a cathepsin antibody for finding co-localization. In the current form, this experiment does not support their claims.
- In general, the quality of the microscopy experiments appears substandard.
- Page 4, line 14: The carboxamide is not oxidized in this reaction. This is a dehydration reaction.
Reviewer 3
Dear Professor Bernardes! Dear Authors!
First of all, I need to apologize for this very late review! By no means did I want to delay this process unnecessarily!
I have finally finished reading the manuscript in detail and I really like the authors' idea to substitute the phenyl ring in a dipeptide protease inhibitor with a methyltetrazine as a bioorthogonal functionality. This structural change can indeed be considered minor (at least regarding the size of the molecule) and I agree with the authors that Z-FA-FMK represents an excellent model to test the concept of phenyl/MeTz (bio)isosterism, given the limited "space" to attach an additional reactive group as it has previously been used and reported for the development of bioorthogonal probes for ABPP. The presented data is convincing but partly confusing (see below). However, given the high novelty of this work and its potential impact in the field, I recommend publication of a revised manuscript in RSC Chemical Biology.
Comments/suggestions/concerns:
- Figure 1 is confusing in that it's not clear to me which signal is shown!? How are the bands visualized in Fig. 1A? According to the main text, it's in-gel fluorescence detection and the caption says it's a competition experiment with TAMRA-DCG-04 (compound 6). Hence, I'd expect this to be the Cy3 channel. However, in Fig. 1B this fluorescent compound was used to label cysteine cathepsins (last lane) resulting in "fluorescent"/bright bands (rather than "black"). So, please indicate which color refers to which channel or method of detection.
- Please include the used concentrations and incubation/reaction times in Fig. 2.
- "inhibit cathepsins with a specificity similar to that of its parent compound" > It would be helpful to provide numbers if possible (e.g., the results from competition experiments should allow - at least some - quantification; binding IC50). In addition, is the data really showing/confirming inhibition or "just" binding!?
- Fig. 3: A competition experiment with the parent inhibitor would help to rule out any unspecific binding of the probe.
- It's certainly too much asked for, but it would have been great to see a non-substituted Tz in comparison. I agree with the authors' argument that such "Proton-tetrazines" (I'd suggest saying "H-tetrazines" or "mono-substituted tetrazines") are known to be rather unstable. However, I am not sure whether reference 30 is the correct one!? In addition, low stability has mainly been observed and reported for mono-substituted aryl-tetrazines, while in this case (replacing Ph in phenylalanine with HTz) it would be a mono-substituted alkyl-Tz, which can be quite stable compounds. Hence, I'd suggest double-checking the references and revising the discussion about MeTz vs. HTz.
- "phenylalanine in Z-FA-FMK is replaced with a methyltetrazine" > only the phenyl substituent is replaced
- please correct "biorthogonal" in the title to "bioorthogonal"
- electrophillic > electrophilic
"using a two step reaction, where it was first reacted with..." > please rephrase (e.g., using a two-step protocol, in which the starting material was first reacted with...)
- DIEA (manuscript) vs. DIPEA (SuppInfo) > I'd suggest using DIPEA.
- "After incubation, the lysates were incubated with..." > please rephrase
- please show the structure of CF(c)500-TCO in Fig. 3 (e.g., bottom-right)
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Dear Prof. Bernardes, Dear Gonçalo,
I would first like to congratulate you on your recent promotion to professor. We have also gone through the comments raised by the two reviewers of our manuscript titled “Methyltetrazine as a small live-cell compatible bioorthogonal handle for imaging enzyme activities in situ” and have performed additional experiments to address each of these comments. We have included our responses in a point-by-point manner below.
I would finally like to apologize for the delay in this response. The current speed at which we can obtain reagents proved a complicating factor in addressing these points in a more timely manner.
Yours sincerely,
Sander I. van Kasteren and Herman S. Overkleeft
REVIEWER 1
1) The authors need to explain the color schemes used in Fig. 1, especially with Fig. 1B. The raw in-gel fluorescence images are preferred.
We have changed this figure. Figure 1A/B are now grouped together and figure C has become Figure 3. We felt that separating them would clarify matters as A/B are both direct labeling experiments of remaining cathepsin activity, and figure 1C (now 3) was a two-step labeling experiment. This allowed us to clarify the figure legends to more clearly present these experiments.
The raw in-gel fluorescence data are included as supplementary figures 1 and 4 .
2) If the overlaid image was used in Fig. 2, the separate images need to be presented.
The separate images have been included as supplementary Figure S6. Please note that the splitting of figure 1 and the addition of selective cathepsin labeling experiments have led to Figure 2 being figure 4 in the new text.
3) Fig. 3: What are the differences between A and B? Co-localization image in B gave white color; are there any other images used in the overlay? Also, is there a reason not using a fluorescent lysosomal tracer in co-localization studies?
Figure 3A was the ‘classical overlay of the colours. Figure B was a pictorial representation (Costes’ mask) of the quantification of overlap between the LAMP and probe signals. We understand this looks confusing. As a result, we have included a ‘classical’ overlay picture in the main text (Figure 5) and moved the overlay images together with the quantification of the Mandel coefficient to the SI (Figure S8). We have also taken better images with improved microscope settings to enhance their quality.
The reason for not using lysotracker is on the one hand historic (we always use LAMP-1, as many of our experiments require fixing and slicing of cells which leads to lysotracker signal dissipation) and in part to allow better washing to reduce the background signal of the CF500-TCO.
4) The live-cell target engagement using new ABPP probe was not as convincing based on the presented image data. Can the authors explain why? Are there any strategies to be employed in the future to improve the performance?
We have re-run this experiment to obtain a clearer result. This has been included as supplementary figure S7. We managed to reduce the background of CF500-TCO by better washing of the cells and an increase in cell numbers used to produce the lysate. For future strategies, particularly for those involving imaging, we concur that better TCO-fluorophores are needed. In fact, we are developing such fluorophores as we speak, but this is proving non-trivial.
REVIEWER 2
- It is noteworthy that the authors use the amino acid in a liquid-phase situation. A comment would be helpful if the amino acid is compatible with solid-phase peptide synthesis conditions. If not, this would reduce the general utility of the approach and needs to be clearly communicated.
A comment is added about the utility of the amino acid in SPPS. Sadly, tetrazines are incompatible with Fmoc-based SPPS. However, their compatibility with the acid deprotection/cleave steps would suggest that they can be used in Boc-based strategies or as the last-introduced amino acid.
- Fig. 1a: It is not mentioned what the staining corresponds to.
The staining corresponds to the labeling of lysates that have been treated first with probe 5, or with the parent inhibitor. Residual cathepsin activity was then made visible with a broad-spectrum fluorophore containing cathepsin probe (called DCG-04). The legend of figure 1 has been changed to clearly reflect this.
- Fig. 1: The assignment of bands to cathepsin needs to be shown in the figures.
An additional experiment using cathepsin specific inhibitors has been performed to allow the assignment of the bands to individual cathepsins. This experiment has been included as Figure 2 and S3.
- Fig. 1b: There is no mention of what lanes contain the sCy5-TCO probe.
Figure 1b has been changed to clearly indicate all the reagents present in each lane. We hope this has clarified this figure. Please note that Fig. 1b is now Fig 3.
- It is unclear why the staining color in Fig. 1b for probe 6 is different than in Fig. 1a.
There was no reason for this. The colors have therefore been adjusted to make the coloring consistent throughout the manuscript.
- Protein size rulers are missing in all gels.
The lanes with protein size rules have been added to the figure. Additionally, raw gel images, obviously including protein size rulers, have been included throughout the SI as uncropped figures.
- Quantitative evaluations of the labeling are missing for the reaction of probe 5 with cathepsins.
The quantification of labeling of probe 5 and the parent inhibitor have been included as supplementary figure 2.
- In figure 1b, Z-FA-FMK appears to completely out-compete 5 in the direct competition experiment. This result contradicts the premise of the publication, which is that methyltetrazine-modifications are better tolerated than other bioorthogonal groups. Careful quantification is required to make that point. The authors mention a norbornene-labeled probe as an example for a bulky group that is disadvantageous for ABP labeling. A direct comparison of 5 with a norbornene-labeled probe would help validate their claims.
We are a little confused by the first part of this comment. Does the competition of Z-FA-FMK not show that our probe behaves the same as the parent molecule? We have quantified the competition with DCG04 to obtain apparent IC50-values (see point above) and have more clearly mentioned in the text that the tetrazine somewhat influences the binding, despite being the most minimal of modifications. We think that the ligation capacity offsets this reduction in apparent IC50, as the specificity of the probe is the same as the parent inhibitors. A norbornene-probe would indeed be an excellent addition to the roster. However, this will sadly have be included in a future publication as we do not have access to it at present.
- The authors mention the need for cell fixation because the LAMP-1 antibody is not cell permeable. However there are several widely used lysosomes stains available that are perfectly suitable with living cells. It appears more likely that the authors chose to fix the cells because of the need to wash away the unreacted CF©500-TCO probe. Please clarify.
Fixation indeed heled the reduction in background by washing away unreacted CF500-TCO. This has been changed in the text.
- Microscopy is a problematic way of comparing fluorescence intensities such as it has been done for the staining with CF©500-TCO. Flow cytometry is the preferred way of quantifying such differences.
We initially chose microscopy as a technique, because we wanted to investigate whether out probe could be used to localize active cathepsins in the cell. We agree with the reviewer that comparing fluorescence intensities in microscopy is suboptimal. We therefore repeated the experiments of figure 5 to further improve their quality. In addition we performed flow cytometry of the cells, which allows us to obtain compare mean fluorescence intensities under different conditions. This data is included in the main text as Figure 6.
- The colonization of the CF©500-TCO and the LAMP1-antibody is poor. The authors make the point that other reports have also found poor colocolization. If co-localization is intrinsically poor, then this would mean that the experiment was poorly designed. Maybe, the authors could use a cathepsin antibody for finding co-localization. In the current form, this experiment does not support their claims.
We have performed the suggested colocalization experiment with an anti-CatB antibody (Figure S9). The colocalization is similar to that of LAMP-1. This can be explained by the fact that probe 5 has multiple targets, and by the fact that the anti-CatB antibody is unable to distinguish between inactive zymogen precursors of CatB and its active form.
- In general, the quality of the microscopy experiments appears substandard.
We have improved the quality by repeating the experiments with improved microscope settings, cell numbers, and wash protocols (See Figure 5).
- Page 4, line 14: The carboxamide is not oxidized in this reaction. This is a dehydration reaction.
This has been adjusted in the text.
REVIEWER 3
- Figure 1 is confusing in that it's not clear to me which signal is shown!? How are the bands visualized in Fig. 1A? According to the main text, it's in-gel fluorescence detection and the caption says it's a competition experiment with TAMRA-DCG-04 (compound 6). Hence, I'd expect this to be the Cy3 channel. However, in Fig. 1B this fluorescent compound was used to label cysteine cathepsins (last lane) resulting in "fluorescent"/bright bands (rather than "black"). So, please indicate which color refers to which channel or method of detection.
The colors in figure 1 have been adjusted and an extra clarification has been added. This was discussed in the response to reviewer 1.
- Please include the used concentrations and incubation/reaction times in Fig. 2.
We added this information to the figure 2 (now figure 4) and to the materials and methods section.
- "inhibit cathepsins with a specificity similar to that of its parent compound" > It would be helpful to provide numbers if possible (e.g., the results from competition experiments should allow - at least some - quantification; binding IC50). In addition, is the data really showing/confirming inhibition or "just" binding!?
We quantified the results of the competition experiment and added the apparent IC50 curves as Figure S2. As our inhibitors are likely covalent inhibitors, we feel reluctant to speak about IC50s as potency is strongly depended on the incubation time. We believe this data shows that our probe 5 acts as an inhibitor, as the targeted proteases are unable to react with known cathepsin activity-based probe TAMRA-DCG-04 after incubation with probe 5.
- Fig. 3: A competition experiment with the parent inhibitor would help to rule out any unspecific binding of the probe.
We included this experiment into the manuscript in figure 3. This shows no specific binding of the probe. I.e. all signal is competed out by the parent inhibitor. In figure 5 (microscopy) and 6 (flow cytometry) it can be seen that addition of the parent inhibitor also reduces the signal to that of the samples not treated with probe 5.
- It's certainly too much asked for, but it would have been great to see a non-substituted Tz in comparison. I agree with the authors' argument that such "Proton-tetrazines" (I'd suggest saying "H-tetrazines" or "mono-substituted tetrazines") are known to be rather unstable. However, I am not sure whether reference 30 is the correct one!? In addition, low stability has mainly been observed and reported for mono-substituted aryl-tetrazines, while in this case (replacing Ph in phenylalanine with HTz) it would be a mono-substituted alkyl-Tz, which can be quite stable compounds. Hence, I'd suggest double-checking the references and revising the discussion about MeTz vs. HTz.
We agree with the reviewer that H-Tz-Ala would be a great target. However, our efforts at making it have always resulted in very poor yields and this precluded the incorporation into the probe. We have changed the reference to this compound and have altered the discussion on the Me vs. H-Tz to express the desirability of the H-Tz variants of the compound.
- please correct "biorthogonal" in the title to "bioorthogonal"
We corrected the title. For some reason our version of word has a propensity to randomly apply auto-correct to this word.
- electrophillic > electrophilic
We have corrected this type-O.
"using a two step reaction, where it was first reacted with..." > please rephrase (e.g., using a two-step protocol, in which the starting material was first reacted with...)
We have rephrased this sentence.
- DIEA (manuscript) vs. DIPEA (SuppInfo) > I'd suggest using DIPEA.
We corrected this inconsistency and use DIPEA throughout now.
- "After incubation, the lysates were incubated with..." > please rephrase
We have rephrased this sentence.
- please show the structure of CF(c)500-TCO in Fig. 3 (e.g., bottom-right)
Unfortunately, this structure is patented and the supplier was unwilling to share structural information with us.
Round 2
Revised manuscript submitted on 29 Pha 2022![](https://www.rsc-cdn.org/pubs-core/2022.0.147/content/NewImages/icon-letter.png)
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07-Sep-2022
Dear Prof van Kasteren:
Manuscript ID: CB-ART-05-2022-000120.R1
TITLE: Methyltetrazine as a small live-cell compatible biorthogonal handle for imaging enzyme activities in situ
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Reviewer 2
For this resubmission, the authors have performed a series of additional experiments and changed the figures that were mentioned by the referees. These efforts greatly improved the quality of the manuscript. The revised version is suitable for publication.
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