Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a well-designed and high-quality structural and biophysical study combining surface plasmon resonance (SPR) and X-ray crystallography to elucidate the binding mode of saracatinib to Fyn kinase, a biologically and clinically relevant target in Tau-related neurodegenerative disorders. The work is scientifically sound and methodologically rigorous, and it provides a valuable structural contribution by reporting a high-resolution crystal structure of the Fyn kinase domain captured in an active-like (αC-in/DFG-in) conformation. This represents a meaningful advance for the field of Src-family kinase inhibition and structure-guided drug design.

A major strength of the study lies in the integration of kinetic and structural data, including a direct comparison between saracatinib and dasatinib, which convincingly highlights differences in affinity and residence time. The structural analysis is clearly presented and well supported by figures, and the discussion appropriately places the findings within the context of Tau biology, Fyn signaling, and previous clinical efforts to repurpose saracatinib for Alzheimer’s disease.

That said, several points would benefit from clarification. First, while the proposed “selectivity handle” emerging from the active-state Fyn conformation is an interesting and potentially important concept, its presentation remains largely qualitative. Providing additional structural or sequence-based comparisons with other Src-family kinases would strengthen the claim and help clarify whether this pocket is unique to Fyn or reflects a broader conformational preference. Second, there is an inconsistency in the reported crystallographic resolution (1.72 Å versus 2.22 Å) that should be corrected and unified throughout the manuscript. Third, the interpretation of the SPR data for full-length Fyn would benefit from a more explicit discussion of how SH2/SH3-mediated intramolecular regulation may influence inhibitor binding kinetics, even if presented as a mechanistic hypothesis supported by prior literature.

Several tables cited throughout the manuscript are missing from the submitted version. This omission prevents proper evaluation of key experimental results and should be corrected prior to further consideration.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In the article, the authors discuss the crystal structure of saracatinib with the Fyn kinase domain. I have reviewed the authors' publication list and believe that the authors obtained this complex. However, it would be advisable for the reviewer or the journal editor to provide an opportunity to verify the existence of the obtained complex via a link to a repository or another method.

The following questions also arise.

1. The choice of two molecules as low-molecular-weight inhibitors should be mentioned not only in the abstract but also in the introduction. The authors describe biological effects in the introduction, but the article itself is devoted to structural biology. I would recommend focusing on the complexes of low-molecular-weight inhibitors with the selected kinase, rather than providing a general overview of this type of kinase.

2. The SPR results section. The data should be presented in a table or as a diagram for each case (full or truncated length, time, etc.), including error margins. In one instance, the authors have 2 significant figures, while at the end of the range there are already three. The text should analyze the data, not just list constants.

3. Figure 1 does not carry significant meaning in the context of the complex and could be moved to supplementary materials.

4. What conclusion do the authors draw at the end of section 2.1? Why was this kinetics measured? In my opinion, it would be more logical to present it after the complex. The issue is that the complex was obtained for only one inhibitor, so it is not entirely clear how to analyze and compare the kinetics and structural data. The authors need to reconsider this.

5. Obtaining and characterization of the complex. The abstract states a resolution of 1.72 Å. In line 99, the resolution is already 2.22 Å. In line 100, the authors refer to Table 1, which is not present in the article at all. It is unclear what can be reviewed in this section.

6. Section 4.3 also lacks complete data on the crystal structure, detector, etc.

7. Furthermore, in section 3, the authors need to conduct an analysis with existing complexes. Why the authors again discuss biochemical pathways instead of structural biology is completely unclear. The authors should compare available complexes with the obtained one, conduct an analysis, and identify patterns. 5 out of 8 paragraphs in section 3 are not directly relevant to the work.

Due to the aforementioned serious errors, I recommend rejecting the material for publication.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I am satisfied with the authors responses to all comments.

Author Response

Thank you!

Reviewer 2 Report

Comments and Suggestions for Authors

I thank the authors for the thorough revision. In its current form, the manuscript can be accepted. I recommend checking the style of Figures 1 and 2; specifically, the use of the letter “A” in the first figure and “a” in the second. It would be better to use a consistent style. I wish the authors good luck with their future results.

Author Response

We appreciate the comments and we have made the letter formatting the same for all figures.