From the journal RSC Chemical Biology Peer review history

21-May-2020

Dear Dr Bernardes:

Manuscript ID: CB-ART-03-2020-000033
TITLE: Structural and biophysical insights of the mode of covalent binding of rationally designed potent BMX inhibitors

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.

I have carefully evaluated your manuscript and the reviewers’ reports, and the reports indicate that major revisions are necessary.

Please submit a revised manuscript which addresses all of the reviewers’ comments. In particular you should address and provide experimental evidence for the question raised about testing the possibility of new compounds to induce BMX degradation in cells on their own as was reported for the BMX-IN-1. Further peer review of your revised manuscript may be needed. 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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Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

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

Reviewer 1

This paper reports the synthesis and testing of several compounds based on the modification of a tool compound published by the Gray lab (BMX-IN-1). The data are clearly presented and discussed. The conclusions are largely supported. However, before the paper can be accepted for publication, I suggest the authors address the following major concerns.

Major concerns:

1) The authors claimed that “JS24-JS27 possess topological pharmacophoric features not yet explored in the BMX inhibitors chemical space”.

This is not true, as JS24-JS27 have very similar structures as the tool compound BMX-IN-1 with only a minor difference. I suggest the authors revise or remove this sentence.

2) Table 2: the author presented the kinetic on and off rates and attempted to deduce K_D values from them. BMX-IN-1 has a Koff of 5.1 x 10-4, which is confusing, as it is also a covalent inhibitor. Overall, in my opionion the data and discussion are inappropriate given the nature of covalent inhibitors, and I suggest the authors remove them.

3) Table 3: there is no description as to how the data in Table 3 was obtained. The text “JS25 …with a binding affinity of 323 pM.” is incorrect, if the authors did the correct measurement of KI, as KI it is not a binding affinity.

4) Molecular dynamics simulations: the authors simulated BMX in the presence of covalent bonding with JS24 and JS27. However, the covalent bond needed to be represented by modification of the protein force field particularly the parameters of the Cys involved, which was not done. Moreover, how the complex structure with JS27 was modeled also not described either. Besides the incorrectness, the MD simulation also did not provide any additional support to the conclusions and seemed rather artificially forced into the paper. Thus, I suggest removing the simulation part.

5) Conclusion: The authors claimed “introduced a chemical handle that may be used for … PROTAC strategy”. This claim is not supported by the data or discussion earlier. It seems to me that the authors added this sentence in order to build in the PROTAC keyword. I suggest removing it.

Minor concerns:

1) Page 1: “five small molecule entities have recently been approved”
This an outdated information. Please look up the current number of approved covalent kinase inhibitors.

Reviewer 2

This is an interesting work reporting on improved covalent agents targeting BMX and related kinases. The manuscript reports most of the typical benchmark experiments required to characterize the new inhibitors (X-ray, denaturation thermal shifts, off rates measurements, IC50s, kinase panel, etc.). The manuscript could be split into two distinct main areas: a) SAR, medicinal chemistry and in vitro characterizations and b) cellular mechanistic studies.
I have the following moderate considerations/suggestions/revisions for the authors:
a) The first part, a relatively limited SAR study is reported with few agents reported in Figure S1. Although the final agents are different from BMX-IN-1 originally reported in 2013 by the Gray’s lab (dx.doi.org/10.1021/cb4000629 ACS Chem. Biol. 2013, 8, 1423−1428). 1).
- Intriguingly, the agents that are reported to be more potent than BMX-IN-1 have moved the ring containing the sulfonamide to point outside of the binding pocket, as the author then discover experimentally with the X-ray structure and confirmed by the MD simulations. One would expect that such a move would cost affinity, but the apparent IC50 in the assay of the authors improved compared to that found for BMX-IN-1 in the same assay. This would require some interpretation that is hidden the paper. Given the highly conjugated system, changes in any part of the structure may (or may not) also tune the reactivity of the Michael acceptor for the Cys residue. This seemed to have happened based on the data in Table 3. I would suggest adding more explicitly how the new agents present increased inhibitory properties compared to BMX-IN-1 in the discussion of the SAR and later in the conclusions.
b) The mechanistic studies characterizing the agents in cellular assays report cell proliferation assays and synergy with a few agents are also reported. Nonetheless, one property of BMX-IN-1 reported by the Gray’s lab was the ability of the agent to induce BMX degradation in cell. The authors seem to appreciate that protein degradation by PROTAC (see below) could render the molecules even more interesting. Yet, the current ms does not verifies whether the agents could induce such degradation like BMX-IN-1 on their own. I’d say that such simple WB analyses would provide a very direct-target based cellular SAR that seems missing and it could be very informative and would bring the paper to another level.
- Related to the above point: the authors mention as an important finding that the agent could be derivatized with a PROTAC for E3 induced degradation (see point above). It is not too difficult to derivatize JS24 (I suppose off the sulfonamide) and to test this important point. This could go hand in hand in the point raised above. Or if too difficult to do (or unnecessary if the agents induce degradation on their own) then I would not stress this point too much.
Minor. There are few typos and misplaced items, for example the legend to Table 2 is erroneously included in another Table 5 and it contains typos as well.

This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters.

Dear Prof. Nikolovska-Coleska,
We wish to submit our revised manuscript (Manuscript ID: CB-ART-03-2020-000033) for
consideration as an Article in RSC Chemical Biology. We are grateful to you and the reviewers
for insightful comments and suggestions. We agree with the points raised and considered these
during the preparation of this revised version of the manuscript. We have addressed the
concerns raised directly through the useful experiments and changes suggested, including the
successful demonstration of induction of BMX degradation in PC3 cells by JS25 and new MD
simulations that took into account the reviewers' suggestions. We hope that these are useful
comments and changes which are highlighted in the uploaded documents. Please find in the
next page a point-to-point response to each specific question raised.
Non-scientific changes:
1. A Table of Contents (TOC) graphic was placed on the last page the manuscript and
uploaded as well as a sentence highlighting the novelty of the work, as requested.
2. Additional data and changes were made to the ESI. A new PDF has been uploaded.
Finally, and has previously mentioned, a preliminary version of the manuscript has been
deposited in ChemRxiv
(https://chemrxiv.org/articles/Rationally_Designed_Potent_BMX_Inhibitors_Reveals_Mode_of_C
ovalent_Binding_at_the_Atomic_Level/11558310).
We look forward to your reply.
Yours sincerely,
Dr Gonçalo Bernardes, on behalf of all authors.

Referee#1
Comments:
This paper reports the synthesis and testing of several compounds based on the modification
of a tool compound published by the Gray lab (BMX-IN-1). The data are clearly presented and
discussed. The conclusions are largely supported. However, before the paper can be
accepted for publication, I suggest the authors address the following major concerns.
ACTION: We thank the referee for supporting publication of our work and for the suggested
improvements.
Major concerns:
1) The authors claimed that “JS24-JS27 possess topological pharmacophoric features not yet
explored in the BMX inhibitors chemical space”.
This is not true, as JS24-JS27 have very similar structures as the tool compound BMX-IN-1 with
only a minor difference. I suggest the authors revise or remove this sentence.
ACTION: We have now revised the manuscript with data from the latest ChEMBL27 version.
We computed ECFP4-like fingerprints for BMX inhibitors annotated in ChEMBL with a defined
IC50, KD or Ki value and JS24 (as a reference structure for our lead series). Computing the
Tanimoto coefficient between JS24 and each of the previously reported BMX inhibitors resulted
in the distribution shown in Figure S2.
The Tanimoto value ranges from 0 to 1. It assumes a value of 1 in the case of complete
fingerprint identity and 0 in the opposite case. It is accepted that molecules present apparent
similarity if the Tanimoto value is > 0.8 (cf. J Med Chem 1996, 39, 3049). In this particular case,
the Tanimoto value obtained was 0.258 ± 0.165, which indeed suggests structural dissimilarity
to the grand majority of known BMX inhibitors in ChEMBL. As the reviewer correctly points out,
there are however exceptions (e.g. CHEMBL4091991).
We have also reformulated Figure 1d, to include the most recent BTK and BMX data in
ChEMBL.
Color codes: Green: BTK inhibitors; Orange: BMX inhibitors; Grey: BMX-IN-1; Red: JS24–27.
Our data indeed shows that most BTK and BMX inhibitors display a different pharmacophore
arrangement to the JS compounds. A rare exception is BMX-IN-1 from which our molecules
were inspired. Together with the above detailed structural analyses, one can conclude the JS
chemical entities populate a region in chemical space only scarcely exploited by other
molecules. We have reformulated the manuscript accordingly: “JS24–JS27 possess
topological pharmacophoric features not exploited in the BMX inhibitors chemical space”.
2) Table 2: the author presented the kinetic on and off rates and attempted to deduce K_D
values from them. BMX-IN-1 has a Koff of 5.1 x 10-4, which is confusing, as it is also a covalent
inhibitor. Overall, in my opinion the data and discussion are inappropriate given the nature of
covalent inhibitors, and I suggest the authors remove them.
ACTION: Regarding this point, we understand the reviewers concern and agree with the raised
point. As mentioned in the manuscript, the inactivation of BMX occurs in a two-step process
that is governed by two parameters: the affinity of the initial non-covalent binding and the rate
of the subsequent covalent bond-forming reaction with the thiol of the cysteine residue.
Therefore, we modified the manuscript to address the reviewer’s point, highlighting that the
determined Koff may be linked with the initial non-covalent interaction that precedes the
irreversible covalent interaction. The SPR data presented in this manuscript is used as an
orthogonal method that confirms the DSF results which suggest that compounds JS24–JS27
have higher affinity/thermal stability to BMX in comparison to BMX-IN-1 compound.
Furthermore, the team believes that within drug target inhibitor development programmes,
kinetic interaction data is a powerful tool for compound ranking and selection, as can be
observed in this manuscript where the low-affinity binders were de-prioritized for structural
follow-up (Figure S3). We have revised the text accordingly.
3) Table 3: there is no description as to how the data in Table 3 was obtained. The text “JS25
…with a binding affinity of 323 pM.” is incorrect, if the authors did the correct measurement of
KI, as KI it is not a binding affinity.
ACTION: We have added a literature reference to the method used and changed “binding
affinity” to “inhibition rate constant”.
4) Molecular dynamics simulations: the authors simulated BMX in the presence of covalent
bonding with JS24 and JS27. However, the covalent bond needed to be represented by
modification of the protein force field particularly the parameters of the Cys involved, which
was not done.
UMAP 1
UMAP 2
ACTION: This is a good and valid point. Parameters for ligands JS24 and JS27 covalently
linked to Cys496 were produced with the antechamber module of AMBER 16 and using the
General Amber Force Field (GAFF) with partial charges set to fit the electrostatic potential
generated with HF/6-31G(d) by RESP. This is a standard protocol to simulate non-standard
residues in AMBER. The sentence ‘Parameters for ligands JS24 and JS27 covalently linked to
Cys496’ has been added to the MD simulations section in the ESI to clarify this idea.
Moreover, how the complex structure with JS27 was modeled also not described either.
ACTION: The referee is correct. Hence, we have indicated in the revised version of the paper
that the structure of JS27 covalently linked to Cys496 was superimpose on the pose found for JS24 in the X-ray structure and used as starting point in the simulations on the complex
BMX/JS27.
Besides the incorrectness, the MD simulation also did not provide any additional support to the
conclusions and seemed rather artificially forced into the paper. Thus, I suggest removing the
simulation part.
ACTION: We have re-written the MD simulations section in the main manuscript to describe
deeper the outcomes derived from this type of calculations, and we have compared the results
with those derived from the X-ray structure. We have also added new Figure 4 and Figure S5 in
the revised versions of the manuscript and the Supplementary information, respectively.
As we indicate in the main text, the results derived from the MD simulations for complex
BMX/JS24 are in line with those found in the X-ray structure, suggesting that the complex is
also stable in solution. On the other hand, the MD simulations performed on BMX/JS27 show
that the complex is stable and retains a similar pose than analogue JS24. Therefore, we
consider that these simulations provide additional support in solution to the conclusions
derived from the X-ray structure's analysis and allow us to propose a 3D model for the complex
of ligand JS27 covalently linked to BMX.
5) Conclusion: The authors claimed “introduced a chemical handle that may be used for …
PROTAC strategy”. This claim is not supported by the data or discussion earlier. It seems to
me that the authors added this sentence in order to build in the PROTAC keyword. I suggest
removing it.
ACTION: We have removed the suggestion to build a PROTAC as indicated by the reviewer.
Minor concerns:
1) Page 1: “five small molecule entities have recently been approved”
This an outdated information. Please look up the current number of approved covalent kinase
inhibitors.
ACTION: We have rephrased and included an updated literature reference.
Referee#2
Comments to the Author
This is an interesting work reporting on improved covalent agents targeting BMX and related
kinases. The manuscript reports most of the typical benchmark experiments required to
characterize the new inhibitors (X-ray, denaturation thermal shifts, off rates measurements,
IC50s, kinase panel, etc.). The manuscript could be split into two distinct main areas: a) SAR,
medicinal chemistry and in vitro characterizations and b) cellular mechanistic studies.
I have the following moderate considerations/suggestions/revisions for the authors:
ACTION: We thank the referee for the supportive words about our work and for the suggested
experiments, which in our view have improved the manuscript significantly.
5
a) The first part, a relatively limited SAR study is reported with few agents reported in Figure
S1. Although the final agents are different from BMX-IN-1 originally reported in 2013 by the
Gray’s lab (dx.doi.org/10.1021/cb4000629 ACS Chem. Biol. 2013, 8, 1423−1428). 1).
- Intriguingly, the agents that are reported to be more potent than BMX-IN-1 have moved the
ring containing the sulfonamide to point outside of the binding pocket, as the author then
discover experimentally with the X-ray structure and confirmed by the MD simulations. One
would expect that such a move would cost affinity, but the apparent IC50 in the assay of the
authors improved compared to that found for BMX-IN-1 in the same assay. This would require
some interpretation that is hidden the paper. Given the highly conjugated system, changes in
any part of the structure may (or may not) also tune the reactivity of the Michael acceptor for
the Cys residue. This seemed to have happened based on the data in Table 3. I would suggest
adding more explicitly how the new agents present increased inhibitory properties compared
to BMX-IN-1 in the discussion of the SAR and later in the conclusions.
ACTION: We have added a brief SAR analysis of key analogues described in Table S1 in order
to justify the results observed with analogues JS24-27. We believe that this explanation
complements that analysis provided for leads JS24-27 and helps the reader understand the
rational for this design and to justify the observed results.
ACTION: We have added a brief SAR analysis of key analogues described in Table S1 in order
to justify the results observed with analogues JS24–27. We believe that this explanation
complements that analysis provided for leads JS24–27 and helps the reader understand the
rational for this design and to justify the observed results.
b) The mechanistic studies characterizing the agents in cellular assays report cell proliferation
assays and synergy with a few agents are also reported. Nonetheless, one property of BMX-IN- 1 reported by the Gray’s lab was the ability of the agent to induce BMX degradation in cell. The
authors seem to appreciate that protein degradation by PROTAC (see below) could render the
molecules even more interesting. Yet, the current ms does not verifies whether the agents
could induce such degradation like BMX-IN-1 on their own. I’d say that such simple WB
analyses would provide a very direct-target based cellular SAR that seems missing and it
could be very informative and would bring the paper to another level.
ACTION: We thank the reviewer for this very useful suggestion, and we are delighted to include
data showing the successful degradation of BMX in PC3 cells after treatment with JS25 as
demonstrated by Western blot. We included in our experiments BMX-IN-1 as a control. New
text in the manuscript and a new ESI Figure (Figure S11) were included in this revision.
Figure S11. BMX degradation after treatment with JS25 and BMX-IN-1 in PC3 cells. (a) JS25
(10 µM) and BMX-IN-1 (10 µM) induce degradation of wild-type BMX in PC3 cells. Sampling
was taken after 24 h and 72 h of incubation with JS25. (b) Raw data from BMX degradation
assay. Protein band intensity normalized with α-Tubulin band intensity and quantified using
ImageJ. Data was obtained from at least three independent measurements (n=3).

- Related to the above point: the authors mention as an important finding that the agent could
be derivatized with a PROTAC for E3 induced degradation (see point above). It is not too
difficult to derivatize JS24 (I suppose off the sulfonamide) and to test this important point. This
could go hand in hand in the point raised above. Or if too difficult to do (or unnecessary if the
agents induce degradation on their own) then I would not stress this point too much.
ACTION: Indeed, our new data (previous point) shows that our new derivatives can
successfully induce degradation of BMX in cells. As a next step, one could derivative,
functionalize through the sulfonamide (for example) with different ligands including an E3
ligand in a PROTAC strategy. We feel that such a strategy and compound are outside the
scope of the current work.
Minor. There are few typos and misplaced items, for example the legend to Table 2 is
erroneously included in another Table 5 and it contains typos as well.
ACTION: We have corrected the typos and eliminated this additional sentence that was
erroneously included in Table 5.

21-Aug-2020

Dear Dr Bernardes:

Manuscript ID: CB-ART-03-2020-000033.R1
TITLE: Structural and biophysical insights of the mode of covalent binding of rationally designed potent BMX inhibitors

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, 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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With best wishes,

Professor Zaneta Nikolovska-Coleska
Associate Editor, RSC Chemical Biology

Reviewer 2

The authors did their best to address my previous concerns and improved the paper in several ways, including new data that suggests a similar mechanism of action as previously reported agents.

Reviewer 1

The authors have addressed my concerns.