From the journal RSC Chemical Biology Peer review history

26-Jan-2022

Dear Prof Jung:

Manuscript ID: CB-ART-12-2021-000244
TITLE: Development of a NanoBRET assay to validate dual inhibitors of Sirt2-mediated lysine deacetylation and defatty-acylation that block prostate cancer cell migration

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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Yours sincerely,

Prof Gonçalo Bernardes

Associate Editor, RSC Chemical Biology

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

Reviewer 1

This manuscript by Jung and coworkers describes the development of novel small molecule SIRT2 inhibitors that are able to potently inhibit both deacetylation and demyristoylation activity of SIRT2. Since this has only been achieved previously with modified lysine-based, thioamide-containing chemotypes, the current inhibitors are highly interesting. Furthermore, the developed NanoBRET assay should find further use in the field and serve as inspiration for the investigation of other sirtuins.

After the authors address a few minor issues, I would certainly support acceptance of the work in RSC Chem. Biol.

1. The discovery of defatty-acylase activity of SIRT2 is mentioned in the introduction to date back to 2015. This was first reported by Feldman and Denu in 2013 (JBC, 2021, 288, 31350)

2. I do not agree with the use of the term "dual inhibitor", as introduced by the authors on page two, last paragraph. Because both substrates and inhibitors compete for the same active site of one enzyme, the term "dual" appears misleading. Whether a compound will inhibit both activities is simple down to potency, because it is competing against substrates with very different Km values.

3. In table 1, the authors should include a positive control compound that has previously been shown to inhibit both activities, for example TM or JH-T4.

4. In figure 6, I would suggest to reduce the image sizes, while keeping the same font size.

5. In figure 7, it would be informative if more explanation on the performed assays were added, to make the figure more self-explanatory.

6. For Fig 8, how many replicates were performed here and could quantification with statistics be added?

7. Reported Ki values based on Cheng-Prusoff calculations in Table 2 may be somewhat inappropriate unless the authors know the actual concentrations of tracer and inhibitor inside the cells.

8. In figure 9, it would be great to add JH-T4 or TM as well, since these have shown target engagement in cells previously.

9. Finally, there are no HRMS measurements for final compounds. However, whether that is a requirement, I will leave to the editor.

Reviewer 2

SIRT2 is a fascinating and relevant target for a variety of diseases, from cancer to neurological disease. SIRT2 appears to be able to deacylate both short and long chain acyl groups, with deacetylation being the more famous activity. However, as the authors point out in their introduction, there is much confusion on the role of SIRT2 in disease and the role of SIRT2-mediated long chain deacylation. Clearly, access to better chemical probes to interrogate these questions would be hugely valuable. This is one of the aims of this paper.

I found this manuscript to be a slightly curious read. It seems to be two papers crudely stuck together: a reasonably traditional SIRT2 inhibitor paper and a SIRT2 assay development short paper (nanoBRET). This is not an issue/criticism per se, I just don’t understand why the nanoBRET work was not more significantly integrated into the study. For example, there are plenty of cases in the cellular work reported where one would expect compound 2 to be significantly active, based on the in vitro IC50 values in Table 1. But it is not. This outcome is clearly explained by the (very nice!) nanoBRET work, but it comes rather late in the paper. I would suggest a rewrite, placing the nanoBRET work after the in vitro target inhibition work. Then the flow of the paper would be: compound design and synthesis -> in vitro functional target activity -> in vitro target binding affinity (the FP data) -> in cell target binding affinity -> target binding mode by docking. Then, the downstream activity of the inhibitors in cells. Following this change, the analysis of the downstream activity should be revisited, given the data from the nanoBRET work.

In addition to this, I have the following comments:

1) Perhaps my largest question is *why* compounds become dual active, which is left unanswered. From Table 1, dual activity simply seems to track with compound activity in general: the more active compounds are active against both (albeit more weakly active against long chain deacylation). Does this trend hold for data on other series in the literature? Or is this a feature of the SirReals? What is known about the different binding affinities of the acetyl versus long chain acyl substrate? Does the latter bind more tightly (hence need a more potent inhibitor to displace)? To publish in RSC Chem Bio, I think the authors need a more rigorous analysis of this outcome, which will ultimately inform future inhibitor design in this area.

2) The rationale for synthesis of compounds 8-12 is not clearly described, given that these regions of the molecule point towards the peptidic site/into solvent. The docking may partly explain what is going on, but this comes after the fact. The authors should update their design hypothesis to be clearer.

3) For convenience, the number of replicates (n = ?) needs to be stated in the relevant legends of biological data.

Reviewer 3

In this manuscript, Vogelmann et al. demonstrate the development of a series of new SirReal-based dual inhibitors of Sirt2-catalyzed deacetylation and defatty-acylation (demyristoylation). The effects of those developed inhibitors on cell viability and colony formation are cell type-dependent, with the metastatic androgen-independent prostate cancer cell line PC-3M-luc as the most sensitive cell line. Also, it has been revealed that the dual inhibitors of Sirt2 can induce downregulation of the oncogene c-Myc and lead to inhibition of cancer cell migration. More importantly, the authors developed a NanoBRET assay to profile the cellular targets of distinct Sirt2 inhibitors and revealed the unnecessary link between alpha-tubulin hyperacetylation and Sirt2 inhibition. The NanoBRET assay developed here can be of great significance in the field of Sirtuin family enzyme inhibitors development and cellular evaluation. Overall, the study in itself is solid and novel, and the experiments are all of high quality and clear. However, there are some concerns that need to be addressed before this is suitable for acceptance.
On page 3, “We based the design of these Sirt2 inhibitors on two approaches, reasoning that: a) targeting the selectivity pocket with lipophilic groups would mimic the binding of fatty acylated substrates and, thus inhibit Sirt2 defatty-acylation activity,”, as the authors mentioned in their previous work, “T. Rumpf, M. Schiedel and B. Karaman, et al., Selective Sirt2 inhibition by ligand-induced rearrangement of the active site, Nat Commun, 2015, 6, 6263.”, the SirReal2 compound will induce a conformational change upon binding to the enzyme and the dimethylmercaptopyrimidine part of the molecule is responsible for the formation of the selectivity pocket. In the design described here, if the dimethylmercaptopyrimidine was replaced with fatty acid mimics, the formation of the selectivity pocket will be disrupted, thus making the hypothesis “As the unique selectivity pocket of Sirt2 also accommodates the long-chain fatty acid of a myristoyl substrate, we tried to mimic the binding of fatty acid substrates by replacing the pyrimidine” really unlogic. This may also be the reason this design (compound 5 and 6) failed. I would suggest deleting this design and related compounds in the final manuscript for precise and concise purposes.
For Figure 7 and S7, improved pictures with high resolution should be provided. The pixelated image should be replaced with pictures with high-resolution image types such as Tiff or png.

For Figure 9, the emission peak of the energy donor and receptor for NanoBRET should be specified as the ratio value is calculated and employed as the indicator of binding affinity. More desirably, spectrums showing emission profile changes can be provided.

For Figure S4 and S5, the internal reference for Western Blot is not equal.

Dear Dr. Bernardes,

thank you very much for the opportunity to submit a revised version of our manuscript which you find attached.

Below you find our answers to the reviewers, structured point by point. We think that we have addressed all concerns adequately and hope that you now find the manuscript suitable for acceptance in the journal.

Sincerely

Manfred Jung



Response to REVIEWER REPORT(S):
Referee: 1

Comments to the Author
This manuscript by Jung and coworkers describes the development of novel small molecule SIRT2 inhibitors that are able to potently inhibit both deacetylation and demyristoylation activity of SIRT2. Since this has only been achieved previously with modified lysine-based, thioamide-containing chemotypes, the current inhibitors are highly interesting. Furthermore, the developed NanoBRET assay should find further use in the field and serve as inspiration for the investigation of other sirtuins.

After the authors address a few minor issues, I would certainly support acceptance of the work in RSC Chem. Biol.

We thank the reviewer for the overall very positive evaluation.

1. The discovery of defatty-acylase activity of SIRT2 is mentioned in the introduction to date back to 2015. This was first reported by Feldman and Denu in 2013 (JBC, 2021, 288, 31350)

Thank you very much for pointing this out. We have corrected the text and the corresponding reference.

2. I do not agree with the use of the term "dual inhibitor", as introduced by the authors on page two, last paragraph. Because both substrates and inhibitors compete for the same active site of one enzyme, the term "dual" appears misleading. Whether a compound will inhibit both activities is simple down to potency, because it is competing against substrates with very different Km values.

Our intention for the term “dual inhibition” was to use it more as a concept for Sirt2 inhibitor design in terms of the cellular activity. However, we understand the reviewer’s concern that this may be misleading and replaced the term “dual inhibition” with “simultaneous inhibition of both Sirt2 activities” or similar terms. We hope this makes it more clear for the reader.

3. In table 1, the authors should include a positive control compound that has previously been shown to inhibit both activities, for example TM or JH-T4.

We included JH-T4 as positive control for simultaneous inhibition of both Sirt2 activities in the in vitro assays and added the resulting data to table 1.

4. In figure 6, I would suggest to reduce the image sizes, while keeping the same font size.

We reduced the image size in Figure 6.

5. In figure 7, it would be informative if more explanation on the performed assays were added, to make the figure more self-explanatory.

We added more details to the figure description for better clarity.

6. For Fig 8, how many replicates were performed here and could quantification with statistics be added?

The number of replicates was included into the description of Figure 8 and an additional graph displaying the quantified fluorescence signal (including statistics) was added.

7. Reported Ki values based on Cheng-Prusoff calculations in Table 2 may be somewhat inappropriate unless the authors know the actual concentrations of tracer and inhibitor inside the cells.

The reviewer is definitely right on the limitations of the approach here. Still, the calculation of Ki values for compounds tested in the NanoBRET assay based on Cheng-Prusoff equations is a commonly used approach in the literature. (M. B. Robers, J.D: Vasta, C.R. Corona, et al., Methods Mol. Biol. 2019, 1888, 45-71., L. L. Ong, J. D. Vasta, L. Monereau, et al., SLAS Discov. 2020, 25, 176-185., L. Grätz, K. Tropmann, M. Bresinsky, et al. Sci. Rep. 2020, 10, 13288.) Therefore, we kept our calculated Ki values in Table 2 but added a footnote to table 2 addressing this issue.

8. In figure 9, it would be great to add JH-T4 or TM as well since these have shown target engagement in cells previously.

We tested the Sirt2 inhibitor JH-T4 in the NanoBRET assay and added the resulting data to figure 9 and table 2.

9. Finally, there are no HRMS measurements for final compounds. However, whether that is a requirement, I will leave to the editor.

It seems to us that this is not mandated by the journal and it has not been explicitly requested by the editor. Still, we would be willing to perform HRMS measurements if necessary.

Referee: 2

Comments to the Author
SIRT2 is a fascinating and relevant target for a variety of diseases, from cancer to neurological disease. SIRT2 appears to be able to deacylate both short and long chain acyl groups, with deacetylation being the more famous activity. However, as the authors point out in their introduction, there is much confusion on the role of SIRT2 in disease and the role of SIRT2-mediated long chain deacylation. Clearly, access to better chemical probes to interrogate these questions would be hugely valuable. This is one of the aims of this paper.

I found this manuscript to be a slightly curious read. It seems to be two papers crudely stuck together: a reasonably traditional SIRT2 inhibitor paper and a SIRT2 assay development short paper (nanoBRET). This is not an issue/criticism per se, I just don’t understand why the nanoBRET work was not more significantly integrated into the study. For example, there are plenty of cases in the cellular work reported where one would expect compound 2 to be significantly active, based on the in vitro IC50 values in Table 1. But it is not. This outcome is clearly explained by the (very nice!) nanoBRET work, but it comes rather late in the paper. I would suggest a rewrite, placing the nanoBRET work after the in vitro target inhibition work. Then the flow of the paper would be: compound design and synthesis -> in vitro functional target activity -> in vitro target binding affinity (the FP data) -> in cell target binding affinity -> target binding mode by docking. Then, the downstream activity of the inhibitors in cells. Following this change, the analysis of the downstream activity should be revisited, given the data from the nanoBRET work.

We thank the reviewer for the very positive feedback regarding the NanoBRET assay and agree that the paper could also be written in the suggested order. The other reviewers did not request a change of the outline and it was not specifically addressed by the editor. Weighing both options, we think that the NanoBRET work at the end of the paper nicely rounds up the story. Since we include structurally different Sirt2 inhibitors in the NanoBRET experiments we expand beyond the SirReal scaffold with more general data on Sirt2 target engagement and hence chose to keep the outline.

In addition to this, I have the following comments:

1) Perhaps my largest question is *why* compounds become dual active, which is left unanswered. From Table 1, dual activity simply seems to track with compound activity in general: the more active compounds are active against both (albeit more weakly active against long chain deacylation). Does this trend hold for data on other series in the literature? Or is this a feature of the SirReals? What is known about the different binding affinities of the acetyl versus long chain acyl substrate? Does the latter bind more tightly (hence need a more potent inhibitor to displace)? To publish in RSC Chem Bio, I think the authors need a more rigorous analysis of this outcome, which will ultimately inform future inhibitor design in this area.

The reviewer rightfully raises some very interesting questions here. First of all, in line with reviewer 1 we changed the wording from `dual´ to `simultaneous´ which is more precise from the biochemical point of view. Indeed, in our study the more potent compounds reveal simultaneous inhibition of both Sirt2 activities whereas less potent inhibitors only block Sirt2 deacetylase activity. As pointed out by the reviewer, this observation might raise the question whether this is caused by the general increase in potency of the inhibitors or by specific differences in the interactions of the inhibitors with the enzyme. Given the flexibility of the sirtuins both options (or a mix) are possible. Based on our data, we stick to the hypothesis that actually potency is decisive in the investigated inhibitor but mention shortly that other options are possible.

Does this trend hold for data on other series in the literature? Or is this a feature of the SirReals?

This correlation of increased potency and higher affinity leading to simultaneous Sirt2 inhibition has been reported for other compounds e.g., the thiomyristoyl peptides TM and JH-T4 (N. A. Spiegelman, et al. ChemMedChem 2019, 14, 744-748) as well as the thiourea- and thioamide containing Sirt2 inhibitors. (A. L. Nielsen, et al. RSC Chem Biol. 2021, 2, 612-626) and we addressed that in the manuscript.

What is known about the different binding affinities of the acetyl versus long chain acyl substrate? Does the latter bind more tightly (hence need a more potent inhibitor to displace)?

Regarding the affinity of the acetyl and long-chain acyl substrates, there are indeed differences. It has been shown that the myristoyl substrate binds stronger to Sirt2 compared to the acetyl substrate since KM values for the Sirt2 substrates decrease with increasing chain length. (J. L. Feldman, et al., Biochemistry 2015, 54, 3037-3050., I. Galleano, et al., J Med Chem. 2016, 59, 1021-1031). The myristoyl substrate leads to a different conformational state of Sirt2 compared to the acetyl substrate that enables the binding of the aliphatic substrate chain in the hydrophobic pocket of the enzyme.

We determined KM values for our acetylated (ZMAL) and myristoylated (ZMML) substrates of the Sirt2 activity assay and could confirm a higher binding affinity for ZMML (KM = 6.7 ± 1.0 µM) compared to ZMAL (KM = 510 ± 95 µM). Hence, the increased potency of compounds 8-12 presumably allows these inhibitors to better compete with the myristoylated substrate for Sirt2 binding. However, as Sirt2 conformations differ upon binding of the myristoylated or acetylated substrate, other reasons cannot be completely ruled out but so far we do not have data on this.

To explain the above mentioned observations, two models have been proposed in literature: a) a retention model, where the myristoyl-substrate and inhibitor could bind simultaneously to the enzyme with a further conformational change that allows the defatty-acylation reaction to occur or b) a kick-out model, where the inhibitor is kicked-out by the acyl-substrate due to additional hydrophobic interactions in the active site of the enzyme which lead to a stronger binding to the enzyme compared with the inhibitor. (N. Kudo, et al. Philos Trans R Soc Lond B Biol Sci. 2018, 373, 20170070). To our knowledge, none of the two models has been finally validated resp. ruled out, so far.

The kinetic analyses and resulting KM values and corresponding Michaelis-Menten plots have been added to the main text of the paper and the ESI, respectively.

2) The rationale for synthesis of compounds 8-12 is not clearly described, given that these regions of the molecule point towards the peptidic site/into solvent. The docking may partly explain what is going on, but this comes after the fact. The authors should update their design hypothesis to be clearer.

To be perfectly honest, we did an exploratory study extending through the lysine channel towards the entry of the active site which resulted in the highly potent morpholine derivative 12 and then indeed performed the docking afterwards which nicely provides an explanation.

3) For convenience, the number of replicates (n = ?) needs to be stated in the relevant legends of biological data.

We added this information to the respective Figures.


Referee: 3

Comments to the Author
In this manuscript, Vogelmann et al. demonstrate the development of a series of new SirReal-based dual inhibitors of Sirt2-catalyzed deacetylation and defatty-acylation (demyristoylation). The effects of those developed inhibitors on cell viability and colony formation are cell type-dependent, with the metastatic androgen-independent prostate cancer cell line PC-3M-luc as the most sensitive cell line. Also, it has been revealed that the dual inhibitors of Sirt2 can induce downregulation of the oncogene c-Myc and lead to inhibition of cancer cell migration. More importantly, the authors developed a NanoBRET assay to profile the cellular targets of distinct Sirt2 inhibitors and revealed the unnecessary link between alpha-tubulin hyperacetylation and Sirt2 inhibition. The NanoBRET assay developed here can be of great significance in the field of Sirtuin family enzyme inhibitors development and cellular evaluation. Overall, the study in itself is solid and novel, and the experiments are all of high quality and clear. However, there are some concerns that need to be addressed before this is suitable for acceptance.
On page 3, “We based the design of these Sirt2 inhibitors on two approaches, reasoning that: a) targeting the selectivity pocket with lipophilic groups would mimic the binding of fatty acylated substrates and, thus inhibit Sirt2 defatty-acylation activity,”, as the authors mentioned in their previous work, “T. Rumpf, M. Schiedel and B. Karaman, et al., Selective Sirt2 inhibition by ligand-induced rearrangement of the active site, Nat Commun, 2015, 6, 6263.”, the SirReal2 compound will induce a conformational change upon binding to the enzyme and the dimethylmercaptopyrimidine part of the molecule is responsible for the formation of the selectivity pocket. In the design described here, if the dimethylmercaptopyrimidine was replaced with fatty acid mimics, the formation of the selectivity pocket will be disrupted, thus making the hypothesis “As the unique selectivity pocket of Sirt2 also accommodates the long-chain fatty acid of a myristoyl substrate, we tried to mimic the binding of fatty acid substrates by replacing the pyrimidine” really unlogic. This may also be the reason this design (compound 5 and 6) failed. I would suggest deleting this design and related compounds in the final manuscript for precise and concise purposes.

We do not agree with this statement. The SirReal2 bound conformation of Sirt2 is indeed different to the acetyl-substrate or apo form. However, this rearranged conformation is also observed in other ligand-bound crystal structures where a bulky or hydrophobic moiety (esp. a myristoyl chain) is part of the ligand. This can be clearly recognized by the superimposition and comparison of SirReal bound crystal structure (e.g. PDB 5DY5 Triazolo-SirReal) with for example. We have added these examples for review purposes only but if desired we could still add some of the information to the Supplementary information:


5Y0Z Human SIRT2 in complex with a specific inhibitor NPD11033
Backbone RMSD 0.77 Å, heavy atoms binding pocket 1.90 Å

6L72 Sirtuin 2 with demyristoylation native final product (H3K18 myristoylated peptide)
Backbone RMSD 1.22 Å, heavy atoms binding pocket 1.76 Å

7BOT Human SIRT2 in complex with myristoyl thiourea inhibitor no. 23
Backbone RMSD 1.09 Å, heavy atoms binding pocket 1.68 Å

In all examples the backbone deviation as well as the heavy atom binding pocket deviation is low indicating high structural similarity. Therefore, we think that our taken design strategy is supported by the available structural data.

We included an additional sentence in the main text of the paper with corresponding references to clarify our strategy for the reader.

For Figure 7 and S7, improved pictures with high resolution should be provided. The pixelated image should be replaced with pictures with high-resolution image types such as Tiff or png.

We cannot provide images with a higher resolution due to the setup that was used for the experiment. As one can still distinguish changes of the overall tubulin acetylation of the cells between different compounds in our provided images and we are not aiming to show changes in subcellular compartments, we think that the images are of sufficient quality for this purpose. Given the time allotted for revision by the editor we could also not produce the data in that timeframe. However, if reviewer and/or editor still would mandate us to submit images with higher resolution, we could repeat the experiment with a different setup but would need a few weeks of time for this.

For Figure 9, the emission peak of the energy donor and receptor for NanoBRET should be specified as the ratio value is calculated and employed as the indicator of binding affinity. More desirably, spectrums showing emission profile changes can be provided.

We measured the emission spectrum of our NLuc-Sirt2 fusion protein and the excitation and emission spectra of our TAMRA-labeled tracer and added the information to the ESI (Figure S9) together with some illustration of the NanoBRET principle and the wavelengths that were used for measurement.


For Figure S4 and S5, the internal reference for Western Blot is not equal.

We agree that the GAPDH loading control of the Western Blot images is not of very high quality. As we have normalized c-Myc signals to signals of the loading control, we think that the Western Blots still display the effect of 48 hours of compound treatment and co-treatment with proteasome inhibitor. We apologize that we had not clarified the normalization in the description of the figure initially. We have added this information to the figures and hope this is sufficient.

28-Feb-2022

Dear Prof Jung:

Manuscript ID: CB-ART-12-2021-000244.R1
TITLE: Development of a NanoBRET assay to validate dual inhibitors of Sirt2-mediated lysine deacetylation and defatty-acylation that block prostate cancer cell migration

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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Prof Gonçalo Bernardes

Associate Editor, RSC Chemical Biology

Reviewer 1

The authors have done an excellent job at addressing the concerns of the reviewers and I recommend acceptance of the manuscript for publication in RSC Chem. Biol.