Dauricine Mitigates Hypoxia Through Targeting ESR1, PIK3CA, and MTOR: A Network Pharmacology and Molecular Dynamics Simulation Investigation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Title: Dauricine mitigates hypoxia through targeting ESR1, PIK3CA, and MTOR: A network pharmacology and molecular dynamics simulation investigation

 

This manuscript presents a network pharmacology and computational study investigating the anti-hypoxic mechanism of dauricine, an alkaloid from Menispermum dauricum DC. Using an integrated pipeline of target prediction, PPI network analysis, GO/KEGG enrichment, molecular docking, and 100ns MD simulation, the authors identify ESR1, PIK3CA, and MTOR as core targets and propose involvement of the PI3K-Akt, MAPK, and mTOR signaling pathways. While the study addresses a relevant topic and follows a well-established computational workflow, it suffers several significant methodological, interpretive, and presentation deficiencies that must be addressed before it can be considered for publication.

1. The identification of ESR1 (estrogen receptor alpha) as a primary anti-hypoxia target of dauricine requires substantial justification. The authors provide no biological rationale for why dauricine with no obvious structural resemblance to estrogens would meaningfully engage ERalpha in the context of hypoxia adaptation, particularly in the setting of chronic mountain sickness, which affects both men and women. The molecular docking pose shown in Figure 9A is localized near GLU-423, which lies adjacent to the ligand-binding domain, but the docking grid box definition and the specific PDB structure used for ESR1 are not reported. This omission makes the result unreproducible.
2. The SwissTargetPrediction platform was used to predict 100 candidate targets from the dauricine SMILES string. The authors do not report the probability score threshold applied, nor do they justify retaining all 100 predictions regardless of confidence level.
3. The GeneCards relevance score cutoff of >= 0.55 is mentioned but not justified.
4. In core target selection, AKT1 is absent from the 56-target list (only AKT2 appears in Table 1), which is unexpected given AKT1’s well-established centrality in PI3K-Akt signaling and hypoxia. The author should clarify whether this reflects a database retrieval artifact or a genuine absence in the prediction output.
5. The PDB accession codes and resolution of the three crystal structures used for docking are not provided, and the docking grid box coordinates and dimensions are not reported.
6. The hydrogen bond analysis (Figure 10G-I) reveals that dauricine forms only 0-1 hydrogen bonds with MTOR, suggesting potential weak or transient interaction. This finding is inconsistent with the claim of high dynamic stability and warrants explicit discussion.
7. MM/GBSA and MM/PBSA inconsistency in the Method section and Results section.
8. Absence of experimental validation

Author Response

Thank you very much for your careful review and constructive comments. We have revised the manuscript point by point according to your comments, as well as the comments from the editor and the other reviewers.

Because the manuscript structure, figure numbering, table numbering, and several supplementary materials were revised during this round of revision, we have provided a complete point-by-point response to the editor’s and all reviewers’ comments in the attached response file. The revised manuscript and supplementary materials have also been updated accordingly.

Please see the attached files for the detailed responses and corresponding revisions.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1. The authors are suggested to present Pi3k, mtor , and MAPK pathways in the supplementary file.
2. The authors need to validate the docking procedure. For this, authors can redock the bound inhibitor into the binding pocket and check the docking pose.
3. The authors must report the intra-molecular interactions formed between the dauricine and the active site residues in the binding pocket of the identified target proteins. The authors may refer to : https://doi.org/10.3390/ijms26125445
   The authors must describe the ADME results for dauricine before describing Docking results.
4. Table 4 must be removed from the discussion section and should be moved to the results section before the docking results.
5. The authors must provide a clear conclusion.

Author Response

Thank you very much for your careful review and constructive comments. We have revised the manuscript point by point according to your comments, as well as the comments from the editor and the other reviewers.

Because the manuscript structure, figure numbering, table numbering, and several supplementary materials were revised during this round of revision, we have provided a complete point-by-point response to the editor’s and all reviewers’ comments in the attached response file. The revised manuscript and supplementary materials have also been updated accordingly.

Please see the attached files for the detailed responses and corresponding revisions.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript by Zengxun Ni and co-authors describes a computational study of the potential mechanisms of dauricine against hypoxia. The integration of network pharmacology, molecular docking, and molecular dynamics (MD) simulations is a logical and modern approach for multi-target mechanism elucidation. The identification of ESR1, PIK3CA, and MTOR as core targets and the PI3K-Akt/MAPK/mTOR pathways is consistent with known hypoxia biology. The 100 ns MD simulations and detailed energy decomposition analysis are important for the in silico validation. The study provides a valuable theoretical basis for future experimental work. However, several points should be addressed to warrant publication.

Specific comments:

(1) Abstract:  The phrase "multi-target molecular mechanism... was systematically investigated" is slightly misleading. The study investigates the mechanism, but its nature is predictive. Softer phrasing like "a multi-target anti-hypoxic mechanism for dauricine is proposed" would be more accurate for a purely computational work.

(2) The identification of Estrogen Receptor 1 (ESR1) as a top core target is an intriguing finding that requires more discussion. The link between estrogen signaling and hypoxia adaptation is not immediately obvious and seems disconnected from the other two core targets (PIK3CA, MTOR) which are directly in the hypoxia-sensing kinase cascade. The authors should pay more attention to the potential biological connection between ESR1 and the PI3K/mTOR axis in the specific context of hypoxia. Is this a known crosstalk, or a novel prediction? A brief literature-supported mechanistic hypothesis would be valuable.

(3) The authors mention that the three core targets were "successfully validated in the iHypoxia database." The results of this validation (e.g., are they up/downregulated in specific tissues under hypoxia?) should be briefly discussed, not just stated.

(4) The choice of a "relevance score ≥ 0.55" for GeneCards is a critical filter that significantly reduces the number of hypoxia-related genes. The authors should explan a choice of this specific cut-off. Without justification, it looks arbitrary, though it's obvious that a filter is needed.

(5) Methods section:  The PDB codes of the structures chosen for molecular docking study should be reported in the manuscript. Also, locations of the docking areas, grid box dimensions and spacing should be indicated.

(6) Figure 3:  The visibility of symbols in some panels is low and should be enhanced. The same applies to Figure 5.

Summarizing, I recommend major revision of the manuscript before acceptance.

Author Response

Thank you very much for your careful review and constructive comments. We have revised the manuscript point by point according to your comments, as well as the comments from the editor and the other reviewers.

Because the manuscript structure, figure numbering, table numbering, and several supplementary materials were revised during this round of revision, we have provided a complete point-by-point response to the editor’s and all reviewers’ comments in the attached response file. The revised manuscript and supplementary materials have also been updated accordingly.

Please see the attached files for the detailed responses and corresponding revisions.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed concerns raised during the previous round of review. The revisions made are satisfactory and the manuscript has been improved as result. Therefore, I recommend the manuscript for acceptance and publication in CIMB.

Reviewer 3 Report

Comments and Suggestions for Authors

The revised version of the manuscript was significantly improved by the authors. The comments were addressed properly. I recommend acceptance of the manuscript for publication in the revised form.