From the journal RSC Chemical Biology Peer review history

30-Mar-2022

Dear Dr Strelkov:

Manuscript ID: CB-ART-02-2022-000052
TITLE: Discovery of novel druggable pockets on polyomavirus VP1 through crystallographic fragment-based screening to develop capsid assembly inhibitors

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Yours sincerely,

Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

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

Reviewer 1

The authors present a study on drug development targeting on polyomavirus capsid protein VP1 through fragment-based drug screening method. Except one peptide reported in 2020, they have identified several potential pockets on the VP1 pentamer, one of which could possibly block its pentamer assembly. The values of all these pockets are enhanced by the mutational experiment done by other groups. Several known compounds are shown in these pockets and worth further pursuing their potentiality. The manuscript is well written in general but required careful editing for errors, in addition to what should be addressed below:

Major points

These hits appear very preliminary for lacking biochemical/biophysical data to support.

Better compounds are expected to be built from these fragments, for example, tetrahydroquinoline acetamide and TFP at R92 site. It is also possible to link some hits from F50, R92 and M109 together because these two pockets appear close.

Supp Figure 5: No disassociation, and therefore Kd is not convincing. This experiment needs to be repeated using a series of concentrations of the compound. Could authors possibly use other two hits or the published peptide in the experiment for comparison?

Minor points

Figure 1
1. Color of red is confusing. Red in panel B looks as gold/orange.
2. Two rotamers of F50 in panel D is better to color differently.
3. Electron density of F50 in panel D was calculated only at 0.7σ, which is not reasonable for 1.44 Å resolution data.
4. What are roles for those residues marked in panel D? Not explained in its figure legend.
5. Legend is panel D also needs to be rephrased.

Figure 2.
1. Sialic acid pocket is colored in pink?

Figure 3
1. In panel B, how was polder difference map calculated?


Figure 4
1. In panel A, color residue labels accordingly.

Figure 5
1. Chemical property of the interactions are not clearly explained.

Figure 6
1. Could you compare pocket sizes of panel B with Figure 5 and figure 4 panel B?

Supplementary
Not understand why all legends in italic.
In table 1, remove “buffer”.
In table 2, truncate the resolution for BKVP1.26-299.C104S to meet the completeness >80% for the highest resolution shell.
Figure 1, sequence alignment was not explained in figure legend.
Figure 2. axis labels are not readable.
Figure 3. panel B, why are tetramers instead of pentamers in ASU? Not able to tell difference at all. What is RMSD?

Reviewer 2

This paper is very interesting which has described about model how to discover novel druggable pockets on polyomavirus VP1 through crystallographic fragment-based screening in order to develop capsid assembly inhibitors.
However, in order to be considered for publication in ChemBio, there are some points should be consider by authors:
(1) please elaborate how to select fragment used in this study and diversity of fragment interact with binding pocket.
(2) please provide the structure of fragment hits listed in Table 2 in supplementary material to help readers.
(3) how to confirm the validity of the protocol.
(4) based on pharmacophore of fragment hits which interact in each binding pocket it is better to elaborate more deeper about future drug structure targeting polyomavirus.

Dear Editor,

We are grateful to you and the Referees for a careful evaluation of our manuscript and many useful remarks and suggestions.

We have thoroughly revised the manuscript and most figures. We upload a separate Word document with all changes traced and marked.

Below we include a point-by-point response to the comments, and explain the changes that we have made to the manuscript. To this end, the Referees’ comments are cited one by one, followed by the tag “Response:” and our feedback.

Referee: 1

Comments to the Author
The authors present a study on drug development targeting on polyomavirus capsid protein VP1 through fragment-based drug screening method. Except one peptide reported in 2020, they have identified several potential pockets on the VP1 pentamer, one of which could possibly block its pentamer assembly. The values of all these pockets are enhanced by the mutational experiment done by other groups. Several known compounds are shown in these pockets and worth further pursuing their potentiality.

Response: We thank the Referee for these very supportive remarks!
The manuscript is well written in general but required careful editing for errors, in addition to what should be addressed below:

Major points

These hits appear very preliminary for lacking biochemical/biophysical data to support.

Response: In this work, we have established six novel binding pockets on VP1 and performed their detailed structural characterization. This became possible through a crystallography-based identification of as many as 144 drug-like fragment binders. While quantification of the binding strength of all these binders was not yet performed, we have collected invaluable high-resolution data on their exact binding poses. There is therefore a wealth of data reported. These data should enable the development of polyomavirus capsid assembly inhibitors, a task that could not be approached through SBDD until today. Importantly, three of the discovered pockets belong to the attachment interface for the C-terminal arms that are clearly relevant for capsid assembly. The remaining three pockets are at the interaction interface with the minor capsid protein VP2, which is also indispensable. These facts boost the impact of our findings.

Better compounds are expected to be built from these fragments, for example, tetrahydroquinoline acetamide and TFP at R92 site. It is also possible to link some hits from F50, R92 and M109 together because these two pockets appear close.

Response: We fully agree with the Referee that individual fragment hits can be grown into larger and more potent compounds. The possibility of linking fragments binding in adjacent pockets is certainly very attractive, as already indicated in the text. The current manuscript provides a solid basis for these future developments.

Supp Figure 5: No disassociation, and therefore Kd is not convincing. This experiment needs to be repeated using a series of concentrations of the compound. Could authors possibly use other two hits or the published peptide in the experiment for comparison?

Response: We apologize if the figure provided originally was not clear enough. The experiment had been originally performed using a concentration series, just as suggested by the Referee. To make it more clear, we have now added the raw SPR curves (Supplementary Fig. 5A).

Minor points

Figure 1
1. Color of red is confusing. Red in panel B looks as gold/orange.

Response: We have further optimized the colours of the figures.

2. Two rotamers of F50 in panel D is better to color differently.

Response: Thank you for this suggestion. The figure has been changed to highlight two distinct conformations of F50 in green and orange respectively.

3. Electron density of F50 in panel D was calculated only at 0.7σ, which is not reasonable for 1.44 Å resolution data.

Response: Indeed a typical cut-off for an electron density map is 1-1.5σ. However, specifically in this case we have deliberately chosen a lower cut-off to fully reveal two conformations of the side chain of residue F50, each at about 50% occupancy. As can be well seen in the figure, due to the excellent quality of the phases, even this lower cut-off produces a very clean map also for the rest of the structure.

4. What are roles for those residues marked in panel D? Not explained in its figure legend.

Response: Most residue labels were indeed not necessary. We removed them.

5. Legend is panel D also needs to be rephrased.

Response: The legend has been adjusted to explain this.

Figure 2.
1. Sialic acid pocket is colored in pink?

Response: Indeed the colour was not as mentioned in the legend; we have fixed this.

Figure 3
1. In panel B, how was polder difference map calculated?

Response: We have added a reference in every figure legend, and also a paragraph with extra detail in the Materials and Methods section.

Figure 4
1. In panel A, color residue labels accordingly.

Response: Thanks for this suggestion. We have labelled the residues of the C-terminal arm in red.

Figure 5
1. Chemical property of the interactions are not clearly explained.

Response: We have extended the legend with a detailed explanation of the interactions.

Figure 6
1. Could you compare pocket sizes of panel B with Figure 5 and figure 4 panel B?

Response: This is a fair question. The pockets F50 and M109 are each formed by 13 residues, while the pocket R92 is somewhat smaller and formed by 9 residues. We have also added an explanation that the binding pockets were defined through all protein residues that interacted with at least one bound fragment in our FBS-X experiments (within 4.5 Å cut-off). We have found out that counting the number of residues forming each pocket is the best way to characterize the pocket size. At the same time, various algorithms (Sitemap, fpocket, PyVoL) calculated the pocket volumes very differently. For example, Sitemap reports 60 Å3 for the F50 site, while PyVol indicates 120 Å3. This is why we avoided using geometrical volumes as a measure of pocket size.

Supplementary
Not understand why all legends in italic.

Response: We have changed the legends to regular font.

In table 1, remove “buffer”.
Response: Done.

In table 2, truncate the resolution for BKVP1.26-299.C104S to meet the completeness >80% for the highest resolution shell.

Response: Thank you for pointing this out. The originally reported statistics was indeed misleading for this dataset, since a very thin highest resolution shell had been used. In the revised manuscript we use the same highest resolution shell thickness (about 10%) as for other datasets. This resulted in an improved completeness and signal-to-noise ratio for the highest resolution shell of the BKVP1.26-299.C104S dataset.

Figure 1, sequence alignment was not explained in figure legend.

Response: Thank you for this remark. This was indeed overlooked originally. The sequence alignment is included to illustrate the design of the fusion proteins. Now we have extended the legend to explain this in full detail.

Figure 2. axis labels are not readable.

Response: This figure has been re-done, with more informative labels shown in increased font.

Figure 3. panel B, why are tetramers instead of pentamers in ASU? Not able to tell difference at all.

Response: There has been a typo in the legend, for which we apologize. The figure indeed shows the pentamers. We have corrected the legend.
What is (the) RMSD?

Response: Thank you for this question. In one of the clusters, one pentamer is shifted by 4 Å along the pentamer axis. This is the most appropriate description of the difference between the clusters. The structure of individual pentamers is not significantly changed (RMSD < 1 Å upon superposition). The shift of one pentamer is now shown in the figure and explained in the legend.

Referee: 2

Comments to the Author
This paper is very interesting which has described about model how to discover novel druggable pockets on polyomavirus VP1 through crystallographic fragment-based screening in order to develop capsid assembly inhibitors.

Response: We thank the Referee for this very positive evaluation.

However, in order to be considered for publication in ChemBio, there are some points should be consider by authors:
(1) please elaborate how to select fragment used in this study and diversity of fragment interact with binding pocket.

Response: This is indeed a very important question. As we explain in the manuscript (see Table 1), a vast majority of the established binders (124 of 144) belongs to the DSiP library. The design principles of this library were described in Cox et al, Chemical Science, 2016, 7, 2322- 2330. As can be seen in Fig. 4 of that article, the chosen fragments cover a large diversity of scaffolds and functional groups. A further discussion of optimization of fragment diversity can be found in a very recent preprint authored by some of us: https://www.biorxiv.org/content/10.1101/2022.03.18.484642v1. Indeed the ongoing accumulation of experimental data on fragment binders enables a further optimization of screening libraries.

(2) please provide the structure of fragment hits listed in Table 2 in supplementary material to help readers.

Response: Thank you for this suggestion. Structural formulas for sample fragments bound to each of the pockets F50, R92 and M109 have been added directly to Figs. 3B, 4B and 6B in the main text, since there was some space available.

(3) how to confirm the validity of the protocol.

Response: In this manuscript we provide a detailed account on the importance of optimization of the protocol. We report (see the first section of Results) that initially the crystallographic system was not sufficiently optimized. Because of this the initial screening using the XChem facility yielded a limited number of hits (1.7%). Due to multiple improvements including an adjustment of the protein fragment used for crystallization, the success rate of the ultimate XChem screening became much better (19.5%). We trust that this detailed description on how we could improve the protocol so drastically would be of interest to a wide community of researchers using FBS-X approach.

(4) based on pharmacophore of fragment hits which interact in each binding pocket it is better to elaborate more deeper about future drug structure targeting polyomavirus.

Response: This is a very valid remark. In the final section (“Conclusions and outlook”) we elaborate on the possible future directions. In particular, we discuss that through inhibition of the pockets on the core VP1 pentamer it becomes possible to prevent the normal attachment of either the C-terminal arms of other VP1 pentamers, or the attachment of VP2. This means that major mechanisms of polyomavirus capsid assembly would become blocked. We are currently testing this possibility experimentally.

Sincerely,
Sergei Strelkov, on behalf of the authors’ team.

24-Apr-2022

Dear Dr Strelkov:

Manuscript ID: CB-ART-02-2022-000052.R1
TITLE: Discovery of novel druggable pockets on polyomavirus VP1 through crystallographic fragment-based screening to develop capsid assembly inhibitors

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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Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

Reviewer 1

The authors have answered all my questions and it is therefore recommended for publication.