From the journal RSC Chemical Biology Peer review history

28-Feb-2022

Dear Dr Wilson:

Manuscript ID: CB-COM-01-2022-000025
TITLE: Towards Identification of Protein-Protein Interaction Stabilizers via Inhibitory Peptide-Fragment Hybrids Using Templated Fragment Ligation

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Prof Seung Bum Park

Associate Editor, RSC Chemical Biology
Professor, Chemistry Department, Seoul National University, Korea

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Reviewer 1

In this manuscript Srdanovic et al. report a new approach for the identification of weak binding 14-3-3 fragments that when combined with an hDMX peptide can enhance its 14-3-3 binding. Further, when separated from the peptide and used as the fragment alone, some identified fragments show evidence of stabilisation of hDMX peptide/14-3-3 binding suggesting this approach could be used to identify starting points for PPI stabilisers. Overall, the screening concept is an elegant one and the experiments appear well designed and executed and are accompanied by a very detailed and useful experimental. I believe the described approach would be of great interest to the readers of RSC Chemical Biology and warrants publication subject to the following minor comments/queries:

- It would be helpful either in Fig 1 or in the SI to have a figure illustrating the location of the Fusicoccin binding site relative to the novel ligandable site identified by FTMap
- What was the rationale for selecting the two boxed hydrazones in Fig. 2a for follow up and not what looks to be the most potent fragment (38?)? I imagine there was some sort of practical reason for this, but it would be helpful if this was justified in the manuscript
- I am a little confused by the discordance between the single shot data in Fig 2a and the titrations in Fig 2b. What is the rationale for the selected fragments having reduced the aniosotropy by around a half in the initial screen but then having essentially no effect on the potency of competition in the follow up? These fragments were not then followed up as non-linked fragments – is the authors hypothesis that to have potential for stabilising a PPI the peptide-fragment hybrids would need to show enhanced binding relative to the peptide alone?
- Is the IC50 for NL_10 in Fig S6 correct? Of course, it is difficult to approximate values by eye, but I’m surprised that the IC50 is the same as for FC_50 when the curve appears to almost overlap with the peptide alone which has an IC50 closer to 10 µM

Reviewer 2

The manuscript by Srdanovic et. al. describes a strategy for discovering protein-protein interaction (PPI) stabilizers using a fragment ligation method. While PPI stabilizers have great potential as promising therapeutic candidates and probe molecules, it is a daunting task to identify them. For example, the fragment-based approach is quite successful in obtaining PPI inhibitors, but it is challenging to find PPI stabilizers. The authors developed a peptide-fragment hybrid method that can yield PPI stabilizing fragments that could serve as starting points to develop more potent PPI stabilizers. I think that the workflow is well implemented. They successfully demonstrate that their approach is able to efficiently discover fragment hits. Overall, this paper is well written, and this approach developed in this study could be useful in discovering PPI stabilizers. This reviewer recommends this manuscript for publication in RSC Chem Biol after addressing the following issues.

• In Figure 2a, two hydrazones showing low anisotropy values were selected as hits. However, there was another hydrazone even with the lowest value, but this one was not selected, and there is no explanation for this.
• Figure 2b, the hits (peptide hydrazones) exhibited higher IC50 values (10.4 and 13.7 uM), compared to the original peptide (hDMX361-374pSer367) and peptide hydrazide (hDMX361-369pSer367Gly-hydrazide) with IC50 values of 8.5 and 8.2 uM, respectively. This result is not consistent with the observation in the dynamic ligation screening depicted in Figure 2a. Whilst these compounds were selected in the initial screen as hits based on their significantly decreased anisotropy compared to the original peptide and peptide hydrazide, they displayed even higher anisotropy. This should be explained.
• The screening approach resulted in the successful discovery of several fragment hits that could be serving as starting points for further development of PPI stabilizer. But, in theory, this approach could generate PPI inhibiting fragments rather than stabilizing ones, as the assay system selects compounds with increased inhibitory activity, but not based on the stabilizing activity (though targeting Fusicoccin (a 14-3-3 PPI stabilizer) binding pocket). This possibility cannot be ruled out due to the nature of the screening, and the authors should mention this possibility.

Minor Comments:

• Page 1, line 3: “Reliant, on hydrazine exchange this approach is advantageous…” should be corrected to “Reliant on hydrazine exchange, this approach is advantageous…”
• In Figure 1a, the bonds between two N atoms are not visible.
• In Figure S2 caption: “1 μM 14 3 3η in 50 mM NH4OAc” should be “1 μM 14-3-3η in 50 mM NH4OAc”
• SI Figure S3 caption: “1 μM 14 3 3η” should be “1 μM 14-3-3η”

Please also see attached cover letter.

Below are the changes and clarifications made in response to the reviewer comments:
Referee: 1
I believe the described approach would be of great interest to the readers of RSC Chemical Biology and warrants publication subject to the following minor comments/queries:
It would be helpful either in Fig 1 or in the SI to have a figure illustrating the location of the Fusicoccin binding site relative to the novel ligandable site identified by FTMap
The Fusicoccin (FC) binding site is located in the same region as the ligandable site identified by FT-Map. Fusicoccin does not however stabilise the interaction between 14-3-3 and hDMX361-374pSer367 as the hDMX peptide protrudes into the FC binding site (we have added an additional panel to Fig S1 to make this clear). The FTMap suggests that despite the hDMX peptide partially occupying the FC binding site, suitably sized fragments may still fit into the remaining space. To maximise the possibility of identifying hits, we felt it was appropriate to screen using a series of peptide truncations (rather than a specific sequence) for the purposes of demonstrating the concept that identification of an inhibitory peptide-fragment can serve as a starting point for stabilizer identification.
What was the rationale for selecting the two boxed hydrazones in Fig. 2a for follow up and not what looks to be the most potent fragment (38?)? I imagine there was some sort of practical reason for this, but it would be helpful if this was justified in the manuscript.
For the hDMX361-369pSer367Gly-hydrazide screen there were several hits other than the two selected for dose response analyses and fragment 38 to which the reviewer refers. Our purpose in this preliminary study was to assess the extent to which selected hits may have modified inhibitory potency. We prioritised fragments bearing reactive handles (on the basis that these may be readily modified in future optimization efforts). The two selected bear such handles whereas 38 (an indole) was less promising. As the screening with hDMX361-370pSer367Gly-hydrazide and hDMX361-371pSer367Gly-hydrazide proceeded in parallel and led to a higher number of hits that did modify inhibitory potency, we selected more to characterize. We have modified the narrative to account for our selection as follows:
“A number of hydrazones exhibiting lower anisotropy than hDMX361-374pSer367 were identified as hits, and we selected two bearing handles for further future functionalization to be assessed: hDMX361-369pSer367Gly-hydrazone-FC45 and hDMX361-369pSer367Gly-hydrazone-SIG17 (Fig. 2a, orange boxes).”
I am a little confused by the discordance between the single shot data in Fig 2a and the titrations in Fig 2b. What is the rationale for the selected fragments having reduced the aniosotropy by around a half in the initial screen but then having essentially no effect on the potency of competition in the follow up? These fragments were not then followed up as non-linked fragments – is the authors hypothesis that to have potential for stabilising a PPI the peptide-fragment hybrids would need to show enhanced binding relative to the peptide alone?
The nature of the screen and because we are looking for an enhancement means that we needed to perform the screen under conditions where there is a already an inhibitory effect on the anisotropy so this is in a sensitive region of the IC50 curve where small differences in concentration have a significant effect on the observed anisotropy value. A single point concentration anisotropy value, whilst useful for identifying potentially interesting hits is therefore a poor guide to inhibitory potency which requires a full dose response curve.
Is the IC50 for NL_10 in Fig S6 correct? Of course, it is difficult to approximate values by eye, but I’m surprised that the IC50 is the same as for FC_50 when the curve appears to almost overlap with the peptide alone which has an IC50 closer to 10 µM
We apologize – in organizing the data into a single figure, annotating with IC50 values and formatting colour schemes for consistency, we did not give the correct IC50 for the peptide-NL_10 hybrid which has now been corrected (IC50 = 7.3 ± 0.7)
Referee: 2
This reviewer recommends this manuscript for publication in RSC Chem Biol after addressing the following issues.
In Figure 2a, two hydrazones showing low anisotropy values were selected as hits. However, there was another hydrazone even with the lowest value, but this one was not selected, and there is no explanation for this.
Please see response to reviewer 1
Figure 2b, the hits (peptide hydrazones) exhibited higher IC50 values (10.4 and 13.7 uM), compared to the original peptide (hDMX361-374pSer367) and peptide hydrazide (hDMX361-369pSer367Gly-hydrazide) with IC50 values of 8.5 and 8.2 uM, respectively. This result is not consistent with the observation in the dynamic ligation screening depicted in Figure 2a. Whilst these compounds were selected in the initial screen as hits based on their significantly decreased anisotropy compared to the original peptide and peptide hydrazide, they displayed even higher anisotropy. This should be explained.
Please see response to reviewer 2
The screening approach resulted in the successful discovery of several fragment hits that could be serving as starting points for further development of PPI stabilizer. But, in theory, this approach could generate PPI inhibiting fragments rather than stabilizing ones, as the assay system selects compounds with increased inhibitory activity, but not based on the stabilizing activity (though targeting Fusicoccin (a 14-3-3 PPI stabilizer) binding pocket). This possibility cannot be ruled out due to the nature of the screening, and the authors should mention this possibility.
The reviewer is correct in this statement and this was implicit in our original description; we apologize that this was not sufficiently clear and have added a comment to the conclusion to make this clearer as follows:
“Although here, the screen was directed towards a known stabilizer pocket on 14-3-3, we note that inhibitory peptide-fragment hybrids could also reveal fragments that inhibit when separated from the peptide anchor. ”
Minor Comments:
Page 1, line 3: “Reliant, on hydrazine exchange this approach is advantageous…” should be corrected to “Reliant on hydrazine exchange, this approach is advantageous…”
We corrected the sentence as follows: “Reliant on hydrazone exchange,24, 25 this approach is advantageous in….”
In Figure 1a, the bonds between two N atoms are not visible.
We have revised the figure to make the bond clearer
In Figure S2 caption: “1 μM 14 3 3η in 50 mM NH4OAc” should be “1 μM 14-3-3η in 50 mM NH4OAc”
We corrected this formatting error.
SI Figure S3 caption: “1 μM 14 3 3η” should be “1 μM 14-3-3η”
We corrected this formatting error.

25-Mar-2022

Dear Dr Wilson:

Manuscript ID: CB-COM-01-2022-000025.R1
TITLE: Towards Identification of Protein-Protein Interaction Stabilizers via Inhibitory Peptide-Fragment Hybrids Using Templated Fragment Ligation

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 Seung Bum Park

Associate Editor, RSC Chemical Biology
Professor, Chemistry Department, Seoul National University, Korea

Reviewer 1

I am happy that the authors have addressed my previous comments appropriately. Congratulations on putting together this nice piece of work!