From the journal RSC Chemical Biology Peer review history

22-Jul-2021

Dear Dr Knecht:

Manuscript ID: CB-ART-06-2021-000135
TITLE: Identification of fragments binding to SARS-CoV-2 nsp10 reveals ligand-binding sites in conserved interfaces between nsp10 and nsp14/nsp16

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Professor/Shanghai Institute of Materia Medica, CAS
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Reviewer 1

This manuscript described the fragment based screening of nsp10 ligands using a combination of binding assays and X-ray crystallization. The ligands discovered in the X-ray crystal structures might be interesting starting points for further lead optimization. The results are significant to justify publication. However, the interpretations need to be modified. Comments are:

1) The X-ray crystal structure of nsp10 was solved before. For example, the complex structure of nsp10-nsp16 was reported in Nat Commun 11, 3717 (2020). It is suggested that the authors should describe the current status of nsp10 structural biology in the introduction and acknowledge of prior work.
2) The TSA data in Table 2 is very confusing. The two temperatures listed are not △T. It should be revised.
3) It was found that fragments identified in the X-ray crystal structure did not stabilize nsp10, and the authors concluded that thermal shift assay is not a reliable assay. This statement needs to be revised. It is well known that ligand binding can either stabilize or de-stabilize a target protein.
4) It appears that the authors put more weight on the X-ray crystal structure than the binding assays. However, the authors should be cautious that promiscuous compounds can co-crystalize with the target protein at high drug concentration. In other words, X-ray crystallization does not suggest potent and specific binding.
5) The authors might need to consider reorganize the flow of this manuscript. Compounds that show stabilization of the protein but did not crystalize can put in the last section.
6) It will also be beneficial for the readers if the authors can provide a cartoon showing how nsp10, nsp14, and nsp16 fit in the whole RdRp complex.

Reviewer 2

Kozielski and colleagues describe in their manuscript the results of a fragment screening campaign on a SARS-CoV-2 target, nsp10, intended to show starting points for drug design of the vital interaction of nsp10 with the nsp14 or nsp16 protein.
The author made used of their previously established crystallization setup and performed crystallographic screening at the FragMAX facility in MAX IV as well as screening by nano differential scanning fluorometry, both using the FragLib library of about 100 compounds. In addition, they used MST as an additional technique to quantify fragment interactions as well as the protein-protein interaction of nsp14 and nsp10. While the authors report that much larger screening endeavors on the same target are still in progress, they intend to already share their results of the 100-compound screening as quick as possible, to help drive open development of antivirals against SARS-CoV-2 as a 2nd line of defense apart from vaccination.
I highly support this open science attitude of the authors and also publishing the results in RSC Chemical Biology. However, in my opinion, the manuscript requires some major and minor revisions before it can be accepted.

major points:
crystallographic screening:
- the authors evaluated the data with conventional crystallographic analysis, thus they might miss a number of hits that could be found with state-of-the-art analysis for such data (e.g. PanDDA approach or GlobalPhasing club-class). As far as the FragMAX-facility reports in their recent ActaD-publication, such methods would have been available. However, this is of course not obligatory. But their classical approach will only deliver the high-occupancy binders. Often, fragments bind only weakly and with low occupancy. Therefore, in order to foster their attitude of open science, they should provide the processed diffraction data (e.g. MTZ files) on a platform like Zenodo.org, or, alternatively the raw data (i.e. images) on a platform like proteindiffraction.org.
This way, others in the field with more time/computational/personal resources could re-evaluate the data carefully for low occupancy binding events that might also be helpful for future drug design on this target.
- the magnification of binding sites in figure 2 should be shown in 3D. (e.g. pictures from models in pymol, chimera or suchlike). The way they showed, the reader does not get a good impression of the special orientation of the fragments.
- the authors show four hits that, are involved in contacts to the symmetry mates in the crystal. This should be checked by the authors and also described in the text. Especially residues Met8 and Thr47 form hydrophobic interactions with the aromatic parts of the fragments, as far as one can see in the models. I suspect they are a main driving force for the binding events there, apart from the H-Bonds. These hydrophobically interacting residues should also be included in the refurbished figure 2, or an extra figure should describe this hydrophobic pocket created by the symmetry mates.
- still, the fragments perform good hydrophilic interactions that can give ideas/starting points in drug design, but the authors should factor in the situation in the crystal in their discussion, e.g. in reasoning why the fragments could hardly be validated by other methods or how relevant/stable these binding modes will be in solution.


The nano differential scanning fluorometry / TSA
- the spread of some of assay results for the two measurements performed compared to the control w/o fragment looks a bit large for a number of conditions. As the authors rightfully concluded, this setup seems not to be a suitable assay for fragment screening on nsp10. Anyway, it is useful that the authors report their difficulties, so other can refrain from TSA screening with nsp10.
major point here: some details in the methods about the TSA are missing, e.g. it cannot be found if the control experiments were done with our w/o DMSO present. And could this explain why the spread in the controls is much smaller, compared to most samples? If the controls were done w/o DMSO, the authors could repeat the controls in presence of DMSO to clarify the issue. It could be that the DMSO itself interferes (even indirectly via protein-stability) with the reproducibility of the measurements.


MST with fragments
- after less conclusive TSA, it is very positive that the authors also included another biophysical approach to verify fragment binding
- Although in the field of fragment-based drug design, higher affinity fragments to not necessarily lead to better final compounds, the authors could correlate their results in context of the binding poses observed by Xray. E.g. do the measured affinities correlate with the number of H-bonds/hydrophobic interactions revealed from the crystal structures?


MST of protein-protein interaction
- it would strengthen the manuscript if the authors could show that the assay would work, (i.e deliver reproducible and largely unchanged protein-protein affinity) if a certain amount of DMSO was included. As it also would be the case if the interaction were to be tested in presence of future compounds. Therefore, the assay could be termed fully establish to determine effects on the respective PPI.

Minor Points by paragraph

Introduction 3rd paragraph.
- many statements are given twice here (e.g. related long-term vaccine’s efficacy) this can be written more concise
- the comparison to HIV /hepC should be tuned down in my opinion as SARS-CoV-2 antivirals have limited application in the course of COVID-19, as they would have to be administered early, ideally in pre-symptomatic stage or early symptomatic stage to take effect. To my knowledge in the later stage of COVID-19, auto-immune effects play a larger role in the disease while the virus itself is only present at low levels. Such caveats should be mentioned.
- also the author compare antivirals to vaccines regarding transport, storage, cost, stability etc.
I think this is a very wide statement, if not met by appropriate references, should be omitted
(just think about the cost of a 5day treatment of Remdesivir (2500 USD) and it’s 6-9 month production time … any vaccine is cheaper and faster to produce in large quantities … also HIV medication is expensive.)

Introduction 5th paragraph
“As nsp10 is not found in host cells, targeting nsp10 and thereby indirectly nsp14 and nsp16, provides an exclusive and targeted strategy to prevent SARS-CoV-2 replication. This is also because the nsp14 and nsp16 interact largely with an overlapping area of nsp10.”
- the causality to the 2nd sentence not clear, why would it be exclusive/targeted in regard to the overlapping interaction area?
- plus, “with an overlapping area of nsp10” sounds misleading in my opinion and maybe should be rephrased

XFS.
- “mFo-Dfcalc” should be “mFo-DFc” or “mFobs-DFcalc” and should be consistent throughout, see also figure 1 legend etc.
- “refined with full occupancy” this point should be omitted, as refining partial occupancies with ligands is not good practice as shown by others.
( see https://doi.org/10.1107/S2059798317003412 )
Table 1
- it looks like high resolution data was omitted to a certain extent, as indicated by very high completeness and CC1/2 in the highest shell. Also the I/sigma ratio is far above 1 in the highest shell, therefore extremely conservative for the EIGER detector that is employed at the BioMAX beamline.
- The authors should commend on which criteria was used to determine the resolution cut-off, matched by the appropriate references to the literature. Especially if the gold standard (paired refinement) was not performed. If the cut-off comes from an automatic pipeline, this could also be stated incl. which criterion the pipeline uses.

Further characterisation of fragment hits found by XFS using microscale thermophoresis (MST) to determine affinities
- “suitable assay to predict binding” predict --> determine
(an assay is a real experiment not a in silico prediction)
- in last paragraph the authors connect solubility prediction of the fragments to the solubility of future to-be-designed larger compounds. I think this connection cannot be made and should be omitted if not underscored by appropriate literature. To my knowledge there is no clear-cut correlation of the solubility of a fragment, vs the solubility of the final drug candidates that came out of the design process.
Table 2
- clogS is wrongly reported, [mg/L] is not a logarithmic scale, usually clogS is in the range of 12 to 2 … these values are also reported by the tool that the authors used for clogS calculation, so they can be easily updated.

Nsp10 targeting fragment hits reveal novel binding sites located in nsp14 and nsp16 interfaces with nsp10
1st paragraph
- “Potentially these hits could be further developed into protein-protein interaction inhibitors preventing the formation or, on the contrary, could be exploited to stabilise the complex.”
statement seems contradictory, either they interfere with the interaction, or they stabilize (e.g. if they are on the rim of the interfaces) Thus this should be rephrased.

Acknowledgment
- could it be that a BioMAX beamline acknowledgement is required due to the measurements carried out at this beamline?

Point by point response to: CB-ART-06-2021-000135

REVIEWER REPORT(S):
Referee: 1

Comments to the Author
This manuscript described the fragment based screening of nsp10 ligands using a combination of binding assays and X-ray crystallization. The ligands discovered in the X-ray crystal structures might be interesting starting points for further lead optimization. The results are significant to justify publication. However, the interpretations need to be modified. Comments are:

We thank the reviewer for the constructive and valid points raised below. We have addressed them as outlined under each point.

1) The X-ray crystal structure of nsp10 was solved before. For example, the complex structure of nsp10-nsp16 was reported in Nat Commun 11, 3717 (2020). It is suggested that the authors should describe the current status of nsp10 structural biology in the introduction and acknowledge of prior work.

This is a very good suggestion that we gladly take up. We have added a paragraph in the introduction addressing the state of the art for nsp10 and 16, but now also nsp10 and 14 (ExoN domain) complexes. We have also deleted potential duplication of the later point in the results and discussion section.

2) The TSA data in Table 2 is very confusing. The two temperatures listed are not △T. It should be revised.

Thanks for spotting this mix-up. We have revised the numbers to the △Tm instead of the Tm, that were previously listed there.

3) It was found that fragments identified in the X-ray crystal structure did not stabilize nsp10, and the authors concluded that thermal shift assay is not a reliable assay. This statement needs to be revised. It is well known that ligand binding can either stabilize or de-stabilize a target protein.

See point 5) below.

4) It appears that the authors put more weight on the X-ray crystal structure than the binding assays. However, the authors should be cautious that promiscuous compounds can co-crystalize with the target protein at high drug concentration. In other words, X-ray crystallization does not suggest potent and specific binding.

We partially agree with this comment. X-ray crystal structure is often the last step in unambiguous validation of hits in a screening cascade. While we fully agree that crystallographic structure does not indicate potency, a well-defined binding pose revealed in the crystallographic model could be considered as a testament of specific molecular recognition event amongst potential non-specific interactions. A short section to elaborate on this was added to the Conclusion section of the manuscript.

5) The authors might need to consider reorganize the flow of this manuscript. Compounds that show stabilization of the protein but did not crystalize can put in the last section.

The flow of the results discussion section has been rearranged according to the suggestion by the reviewer. This was done together with revising TSA statements (destabilisation/stabilisation – point 3) and addressing the poor assay reproducibility (see comment from reviewer 2 and our answer to that). Additionally, we also added literature discussing the TSA and outcome in the context of fragment-based drug discovery.

6) It will also be beneficial for the readers if the authors can provide a cartoon showing how nsp10, nsp14, and nsp16 fit in the whole RdRp complex.

It is generally assumed that the nsp14-nsp10 and also the nsp16-nsp10 complexes are part of the Replication-Transcription complex (RTC). Schematic cartoons are sometimes included in previous papers on the SARS nsp14-nsp10 complex as a model to support this, by showing that nsp14 is somehow connected to the RdRp(nsp12)-nsp8-nsp7 complex. With the significant advances in cryo-electron microscopy, solving the structures of the RTC is now a possibility as has been employed for the determination of the RdRp-nsp8-nsp7 complex and the RdRp-nsp8-nsp7-helciase complex [Chen et al., Structural basis for helicase-polymerase coupling in the SARS-CoV-2 Replication-Transcription complex. 2020 Cell]. We would not be surprised if we would soon see the publication of a more complete CTR complex including the nsp14-nsp10 complex determined by cryo-EM. After careful discussion we therefore propose not to provide such a schematic cartoon, as it may be negated very quickly by a real EM structure.

Referee: 2

Comments to the Author
Kozielski and colleagues describe in their manuscript the results of a fragment screening campaign on a SARS-CoV-2 target, nsp10, intended to show starting points for drug design of the vital interaction of nsp10 with the nsp14 or nsp16 protein.
The author made used of their previously established crystallization setup and performed crystallographic screening at the FragMAX facility in MAX IV as well as screening by nano differential scanning fluorometry, both using the FragLib library of about 100 compounds. In addition, they used MST as an additional technique to quantify fragment interactions as well as the protein-protein interaction of nsp14 and nsp10. While the authors report that much larger screening endeavors on the same target are still in progress, they intend to already share their results of the 100-compound screening as quick as possible, to help drive open development of antivirals against SARS-CoV-2 as a 2nd line of defense apart from vaccination.
I highly support this open science attitude of the authors and also publishing the results in RSC Chemical Biology. However, in my opinion, the manuscript requires some major and minor revisions before it can be accepted.

We thank the reviewer for the constructive and valid points raised below. We have addressed them as outline under each point.

major points:
crystallographic screening:
- the authors evaluated the data with conventional crystallographic analysis, thus they might miss a number of hits that could be found with state-of-the-art analysis for such data (e.g. PanDDA approach or GlobalPhasing club-class). As far as the FragMAX-facility reports in their recent ActaD-publication, such methods would have been available. However, this is of course not obligatory. But their classical approach will only deliver the high-occupancy binders. Often, fragments bind only weakly and with low occupancy. Therefore, in order to foster their attitude of open science, they should provide the processed diffraction data (e.g. MTZ files) on a platform like Zenodo.org, or, alternatively the raw data (i.e. images) on a platform like proteindiffraction.org.
This way, others in the field with more time/computational/personal resources could re-evaluate the data carefully for low occupancy binding events that might also be helpful for future drug design on this target.

We fully agree with this comment and indeed, we tried data analysis with PanDDA software as implemented within the screening platform. Unfortunately, it was not successful and did not reveal more crystallographic hits – possibly because of poor soaking tolerance of the system and deterioration of diffraction for soaked crystals. However, we fully support the idea to share as much data as possible with the scientific community. We have now uploaded all processed diffraction datasets, data reduction log files, PDB and MTZ files after initial refinement, and fragment PDB and restraint files at the ZENODO data repository. Moreover, we have deposited raw diffraction images of the deposited datasets at proteindiffraction.org. Material and Methods and Results sections are modified accordingly.

- the magnification of binding sites in figure 2 should be shown in 3D. (e.g. pictures from models in pymol, chimera or suchlike). The way they showed, the reader does not get a good impression of the special orientation of the fragments.

Figure 2 has been re-done. Please see below.

- the authors show four hits that, are involved in contacts to the symmetry mates in the crystal. This should be checked by the authors and also described in the text. Especially residues Met8 and Thr47 form hydrophobic interactions with the aromatic parts of the fragments, as far as one can see in the models. I suspect they are a main driving force for the binding events there, apart from the H-Bonds. These hydrophobically interacting residues should also be included in the refurbished figure 2, or an extra figure should describe this hydrophobic pocket created by the symmetry mates.

Again, we fully agree with the reviewer and investigated the possible interaction of fragments with residues from their symmetry mates in the crystal using the same default conditions in Ligplot+. All fragments also interact with at least one residue from a symmetry mate and these interactions will contribute to the overall binding, as mentioned by the reviewer. As requested by the reviewer, we refurbished figure 2 and updated the individual subfigures, including interaction to the symmetry mates, providing a more complete view of the detailed interactions.

- still, the fragments perform good hydrophilic interactions that can give ideas/starting points in drug design, but the authors should factor in the situation in the crystal in their discussion, e.g. in reasoning why the fragments could hardly be validated by other methods or how relevant/stable these binding modes will be in solution.

We have updated the section on the detailed interactions between the fragment hits and residues of nsp10, including their symmetry mates in the crystal. We were also able to correlate the situation in the crystal with data obtained from MST. We also added a new sentence “The default cut-off values in Ligplot+ were employed to identify hydrogen bond and hydrophobic interactions between residues of nsp10 and fragments.” so that the reported interactions and calculated distances can be verified.


The nano differential scanning fluorometry / TSA
- the spread of some of assay results for the two measurements performed compared to the control w/o fragment looks a bit large for a number of conditions. As the authors rightfully concluded, this setup seems not to be a suitable assay for fragment screening on nsp10. Anyway, it is useful that the authors report their difficulties, so other can refrain from TSA screening with nsp10.
major point here: some details in the methods about the TSA are missing, e.g. it cannot be found if the control experiments were done with our w/o DMSO present. And could this explain why the spread in the controls is much smaller, compared to most samples? If the controls were done w/o DMSO, the authors could repeat the controls in presence of DMSO to clarify the issue. It could be that the DMSO itself interferes (even indirectly via protein-stability) with the reproducibility of the measurements.

Yes, we agree with the reviewer, that in some cases for example only one curve was usable and this gives the impression of a poor assay. However, this illustrates the real-life challenges when doing screening in larger numbers and therefore is important to be pointed out. It is also an additional reason why we do not recommend this assay setup for nsp10, on top of the uncertainty if screening should be done for destabilization or stabilization (see comment from reviewer 1) and moved on to MST.
The nsp10 control was run in DMSO to match the fragment conditions. This has been added to the method description as an explicit statement.


MST with fragments
- after less conclusive TSA, it is very positive that the authors also included another biophysical approach to verify fragment binding
- Although in the field of fragment-based drug design, higher affinity fragments to not necessarily lead to better final compounds, the authors could correlate their results in context of the binding poses observed by Xray. E.g. do the measured affinities correlate with the number of H-bonds/hydrophobic interactions revealed from the crystal structures?

This is a very interesting question raised by the reviewer. As suggested we have therefore looked at the number of hydrogen bond and hydrophobic interactions between residues of nsp10 and the fragments hits. However, we did not observe any correlation as for example fragment 00022 with the highest number of hydrophobic interactions (3) shows the lowest measured Kd value. Fragment 00239 with the best Kd value has the lowest number of hydrogen bond interactions (1). We also added a sentence at the end of page describing the nsp10 fragment interactions “It is also noteworthy that the number of hydrogen bonds and hydrophobic interactions between residues of nsp10 and the fragment hits does not correlate with their measured Kd values.”, as well as added a sentence commenting on the in solution nature of the MST assay in the MST section: “ Because MST is conducted in solution when the protein is monomeric and does not have the extra interactions with symmetry mates present in the crystal, the measured Kd values proof that fragment binding does not require the interactions from the symmetry mates.”

MST of protein-protein interaction
- it would strengthen the manuscript if the authors could show that the assay would work, (i.e deliver reproducible and largely unchanged protein-protein affinity) if a certain amount of DMSO was included. As it also would be the case if the interaction were to be tested in presence of future compounds. Therefore, the assay could be termed fully establish to determine effects on the respective PPI.

We agree with the reviewer and conducted more MST experiments on the quantification of the nsp14-nsp10 interaction, also in the presence of up to 3% DMSO. The data have been added, including to figure 4B showing that the presence of 3% DMSO does not have an impact on the nsp14-nsp10 interaction now allowing us to conduct the assay in the presence of inhibitor stocks (those are usually prepared in DMSO or deuterated DMSO). We also updated the experimental section and the result section with the new data.

Minor Points by paragraph

Introduction 3rd paragraph.
- many statements are given twice here (e.g. related long-term vaccine’s efficacy) this can be written more concise
- the comparison to HIV /hepC should be tuned down in my opinion as SARS-CoV-2 antivirals have limited application in the course of COVID-19, as they would have to be administered early, ideally in pre-symptomatic stage or early symptomatic stage to take effect. To my knowledge in the later stage of COVID-19, auto-immune effects play a larger role in the disease while the virus itself is only present at low levels. Such caveats should be mentioned.
- also the author compare antivirals to vaccines regarding transport, storage, cost, stability etc.
I think this is a very wide statement, if not met by appropriate references, should be omitted (just think about the cost of a 5day treatment of Remdesivir (2500 USD) and it’s 6-9 month production time … any vaccine is cheaper and faster to produce in large quantities … also HIV medication is expensive.)

The comparison with HCV and HIV was primarily meant to show examples of viral diseases that can be managed with small molecule drugs. However, we recognize from the reviewers` comments, that we try here to capture a topic with many aspects e.g. with pricing of drugs, their intended profile of use etc., that would need a much more comprehensive and in-depth discussion with many more facets, outside the scope of an introduction of a research paper. We have therefore simplified and straight-lined this paragraph in the introduction, to merely point out that additional possibilities of small drug intervention, would also be desirable in the case of SARS-CoV 2 and as readiness towards future new variants of SARS.

Introduction 5th paragraph
“As nsp10 is not found in host cells, targeting nsp10 and thereby indirectly nsp14 and nsp16, provides an exclusive and targeted strategy to prevent SARS-CoV-2 replication. This is also because the nsp14 and nsp16 interact largely with an overlapping area of nsp10.”
- the causality to the 2nd sentence not clear, why would it be exclusive/targeted in regard to the overlapping interaction area?
- plus, “with an overlapping area of nsp10” sounds misleading in my opinion and maybe should be rephrased

Thank you for putting this forward. The 2nd sentence has been deleted in connection to the discussion of state of the art of structural biology with nsp10 – nsp16 and – nsp14 ExoN domain complexes and the fact that much of the binding interface of nsp10 to nsp14 or 16 is identical.


XFS.
- “mFo-Dfcalc” should be “mFo-DFc” or “mFobs-DFcalc” and should be consistent throughout, see also figure 1 legend etc.

The manuscript was revised accordingly.

- “refined with full occupancy” this point should be omitted, as refining partial occupancies with ligands is not good practice as shown by others.
( see https://doi.org/10.1107/S2059798317003412 ) Table 1

We agree with this comment and the corresponding subsection in Material and Methods part was modified.

- it looks like high resolution data was omitted to a certain extent, as indicated by very high completeness and CC1/2 in the highest shell. Also the I/sigma ratio is far above 1 in the highest shell, therefore extremely conservative for the EIGER detector that is employed at the BioMAX beamline.
- The authors should commend on which criteria was used to determine the resolution cut-off, matched by the appropriate references to the literature. Especially if the gold standard (paired refinement) was not performed. If the cut-off comes from an automatic pipeline, this could also be stated incl. which criterion the pipeline uses.

We agree that the resolution cut-offs could be slightly higher, and the data quality allows to increase resolution by 0.02-0.05 Å. In this study, resolution cut-off was determined as CC1/2 > 0.5, trying to keep mean signal-to-noise ratio above 1.3, being indeed too conservative. Corresponding section in the Material and Methods part was supplemented with the used criteria.

Further characterisation of fragment hits found by XFS using microscale thermophoresis (MST) to determine affinities
- “suitable assay to predict binding” predict --> determine
(an assay is a real experiment not a in silico prediction)

This has been deleted in the re-arranging and rewriting of the TSA section.

- in last paragraph the authors connect solubility prediction of the fragments to the solubility of future to-be-designed larger compounds. I think this connection cannot be made and should be omitted if not underscored by appropriate literature. To my knowledge there is no clear-cut correlation of the solubility of a fragment, vs the solubility of the final drug candidates that came out of the design process.

Thanks for pointing this out, we have deleted this statement in the revised manuscript.

Table 2
- clogS is wrongly reported, [mg/L] is not a logarithmic scale, usually clogS is in the range of 12 to 2 … these values are also reported by the tool that the authors used for clogS calculation, so they can be easily updated.

As requested by the reviewer, we updated the MolLogS values in Table 2 and now provide logarithmic values. Accordingly, we also changed the units to “Log(moles/L)”.

Nsp10 targeting fragment hits reveal novel binding sites located in nsp14 and nsp16 interfaces with nsp10 1st paragraph
- “Potentially these hits could be further developed into protein-protein interaction inhibitors preventing the formation or, on the contrary, could be exploited to stabilise the complex.”
statement seems contradictory, either they interfere with the interaction, or they stabilize (e.g. if they are on the rim of the interfaces) Thus this should be rephrased.

We agree with reviewer 2 as this statement was contradictory and we have now updated this sentence by removing the second part of the sentence referring to the stabilization.

Acknowledgment
- could it be that a BioMAX beamline acknowledgement is required due to the measurements carried out at this beamline?
The BioMAX beamline is explicitly mentioned in the methods and it is as LP3 included in the FragMAX project and two beamline scientists of BioMAX are co-authoring the paper, but this comment remined us that we forgot to thank all BioMAX staff and have added this as done for LP3 staff. We have also added the generic acknowledgement for finance support to the whole synchrotron facility MAX IV laboratory.

03-Oct-2021

Dear Dr Knecht:

Manuscript ID: CB-ART-06-2021-000135.R1
TITLE: Identification of fragments binding to SARS-CoV-2 nsp10 reveals ligand-binding sites in conserved interfaces between nsp10 and nsp14/nsp16

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.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after revisions.

Please revise your manuscript to fully address the reviewers’ comments. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

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

Reviewer 1

Comments from the previous round of review were properly addressed. I therefore recommend acceptance.

Reviewer 2

The manuscript of Kozielski and colleagues was largely improved, addressing in-depth all the comments raised by the reviewers. It is a very nice read and I look forward to its publiciation. It is noteworthy that the authors share their crystallographic data in various forms, helping the community to compare and drive further investigations on this SARS-CoV-2 target.

I have only very few minor points spotted upon reading:

in Figures 1,2,3 some cartoon parts of the structure are referred to as "blue" in the figure legend, but appear purple in the PDF version I got (both on screen and printed). It should be checked if this is just an issue of RGB-CMYK conversion or vice versa, i.e. a technical issue of this online portal for submission, or if "purple" would be a more appropriate description.

in conclusions, first sentence, the authors use the term "fragment-like ligands" although they used "fragments" (rightfully) in the whole manuscript, they could do the same here for consistancy. It would be fully justified as they used a fragment library. "fragment-like" sounds like these compounds would not be fully agreed on as fragments, e.g. Rule-of-3-compliend etc

in the methods section, the Crystal Shifter device is mentioned, altough it is also a commercial product, there is an describtion of the device published in Acta D, which the authors could consider to cite:
https://doi.org/10.1107/S2059798320014114
also the Integrated Resource for Reproducibility in Macromolecular
Crystallography has a signiture publication, which might be cited:
https://doi.org/10.1107/S2059798316014716

I wish the authors good luck and many succes with their research on antivirals to fight the current and future pandemics.

Point by point response to: CB-ART-06-2021-000135.R1


REVIEWER REPORT(S):
Referee: 1

Comments to the Author
Comments from the previous round of review were properly addressed. I therefore recommend acceptance.

We thank the reviewer again for the constructive and valid points raised in the first round of revision that helped us to improve the manuscript.

Referee: 2

Comments to the Author
The manuscript of Kozielski and colleagues was largely improved, addressing in-depth all the comments raised by the reviewers. It is a very nice read and I look forward to its publiciation. It is noteworthy that the authors share their crystallographic data in various forms, helping the community to compare and drive further investigations on this SARS-CoV-2 target.

We thank the reviewer again for the constructive and valid points raised in the first round of revision that helped us to improve the manuscript. We have addressed the remaining points below as follows.

I have only very few minor points spotted upon reading:

in Figures 1,2,3 some cartoon parts of the structure are referred to as "blue" in the figure legend, but appear purple in the PDF version I got (both on screen and printed). It should be checked if this is just an issue of RGB-CMYK conversion or vice versa, i.e. a technical issue of this online portal for submission, or if "purple" would be a more appropriate description.

Thanks for pointing this out and the reviewer is of course right, it is a certain shade of purple. We did choose a simplification blue (technically it is called "slate blue" in Pymol) in the first place because the color impression, lilac is not often used, and the color could also stand as lavender (both lavender and lilac are pale shades of purple but lilac has a pink tint to it, while lavender has a blue tint). We changed the blue to lilac in the figure legends (after consulting a few persons around if they would rather see it as lavender or lilac….).

in conclusions, first sentence, the authors use the term "fragment-like ligands" although they used "fragments" (rightfully) in the whole manuscript, they could do the same here for consistancy. It would be fully justified as they used a fragment library. "fragment-like" sounds like these compounds would not be fully agreed on as fragments, e.g. Rule-of-3-compliend etc

We agree with the reviewer and have changed this to fragments.

in the methods section, the Crystal Shifter device is mentioned, altough it is also a commercial product, there is an describtion of the device published in Acta D, which the authors could consider to cite:
https://doi.org/10.1107/S2059798320014114
also the Integrated Resource for Reproducibility in Macromolecular Crystallography has a signiture publication, which might be cited:
https://doi.org/10.1107/S2059798316014716

We also agree here with the reviewer that adding these citations is appropriate and we have done so.

I wish the authors good luck and many succes with their research on antivirals to fight the current and future pandemics.

06-Oct-2021

Dear Dr Knecht:

Manuscript ID: CB-ART-06-2021-000135.R2
TITLE: Identification of fragments binding to SARS-CoV-2 nsp10 reveals ligand-binding sites in conserved interfaces between nsp10 and nsp14/nsp16

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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