Review Reports

Round 1

Reviewer 1 Report

The paper is engaged in a highly actual and relevant topic, as Az űrlap teteje

Mammalian arachidonic acid lipoxygenases play crucial role maping the entire mechanism of the pathogenesis of inflammatory diseases. My questions and remarks are as follows:

• What is the current knowledge on the actual action mechanism of ALOX in terms of neurodegenerative diseases?
• Why have you selected in particular just Nordihydroguaiaretic acid as lipoxygenase inhibitor? Among several such substances why exactly NDGA proves to be the most appropriate model molecule?
• As far as in silico docking studies are concerned: how have you featured in detail all the plausible binding sites of the 2 molecules, when the molecular dynamics simulation of NDGA binding to human ALOX15 occured?
• Charecterisation of binding behavior of the enzyme ought to be based on the involvement of several methodologies in order to imply more variable parameters prevailing in the complex system oh human metabolism.
• In case of in silico mutagenesis studies comparative assessment with relevant research results of much more in vitro model studies should hve been considered.
• Why have not you made attempts to describe the antioxidant activities and changing such potencial of the studied model systems?
• Why have not you implied more tools the characterise human ALOX15 and enzyme mutants interactions, justby means of he IC50-values. Other functional properties could also be taken into consideration.

My comments have been detailed above.

Author Response

Please see attachemnt.

Author Response File: Author Response.pdf

Reviewer 2 Report

This paper systematically investigates the interaction mechanism between the natural antioxidant NDGA and the human ALOX15 enzyme, focusing on the crucial role of the Gln595 residue in binding. The study design is reasonable, the methods are comprehensive, and the results are significant for understanding the inhibitory mechanism of NDGA and the ligand-binding characteristics of ALOX15. However, the article has significant shortcomings in logical coherence, mechanistic depth, and discussion depth.

1. The background introduction of NDGA as an ALOX inhibitor in the introduction could be further simplified, focusing on the research gaps in its interaction with ALOX15.

2. The parameter settings for molecular docking and kinetic simulations should be more clearly explained (e.g., force field selection, solvent model, etc.) to enhance reproducibility.

3. The structural diagrams in Figures 2, 3, and 6 are not clearly labeled. It is recommended to use color to distinguish different residues or ligand parts and add explanatory captions.

4. The discussion of the functional diversity of Gln595 in the discussion section is somewhat scattered. It is recommended to integrate it into a systematic explanation from three dimensions: substrate binding, allosteric regulation, and inhibitor binding.

5. The binding constant (Kd) of NDGA to wild-type and mutant ALOX15 was directly determined using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to verify the reliability of the IC₅₀ results.

6. The inhibitory effect of NDGA on ALOX15 activity and its impact on downstream lipid mediators (such as 15-HETE) were verified in cell models (e.g., HEK293 cells overexpressing ALOX15) to enhance physiological relevance.

7. Several computational articles have been published on the screening of ALOX15 inhibitors, such as DOI: 10.1080/14756366.2024.2301756. Authors are advised to discuss the differences in their analyses in the discussion section.

 

This paper systematically investigates the interaction mechanism between the natural antioxidant NDGA and the human ALOX15 enzyme, focusing on the crucial role of the Gln595 residue in binding. The study design is reasonable, the methods are comprehensive, and the results are significant for understanding the inhibitory mechanism of NDGA and the ligand-binding characteristics of ALOX15. However, the article has significant shortcomings in logical coherence, mechanistic depth, and discussion depth.

1. The background introduction of NDGA as an ALOX inhibitor in the introduction could be further simplified, focusing on the research gaps in its interaction with ALOX15.

2. The parameter settings for molecular docking and kinetic simulations should be more clearly explained (e.g., force field selection, solvent model, etc.) to enhance reproducibility.

3. The structural diagrams in Figures 2, 3, and 6 are not clearly labeled. It is recommended to use color to distinguish different residues or ligand parts and add explanatory captions.

4. The discussion of the functional diversity of Gln595 in the discussion section is somewhat scattered. It is recommended to integrate it into a systematic explanation from three dimensions: substrate binding, allosteric regulation, and inhibitor binding.

5. The binding constant (Kd) of NDGA to wild-type and mutant ALOX15 was directly determined using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to verify the reliability of the IC₅₀ results.

6. The inhibitory effect of NDGA on ALOX15 activity and its impact on downstream lipid mediators (such as 15-HETE) were verified in cell models (e.g., HEK293 cells overexpressing ALOX15) to enhance physiological relevance.

7. Several computational articles have been published on the screening of ALOX15 inhibitors, such as DOI: 10.1080/14756366.2024.2301756. Authors are advised to discuss the differences in their analyses in the discussion section.

 

Author Response

Please see attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

I am attaching comments below.

Please see attached.

Comments for author File: Comments.pdf

Author Response

Please see attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I have no further comments.

I have no further comments.