In Silico Targeting of Trypanothione Reductase and Glycerol-3-Phosphate Dehydrogenase in Leishmania

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Title:

• The title is informative but slightly long. Consider simplifying it by reducing redundancy, e.g., “In Silico Targeting of Trypanothione Reductase and Glycerol-3-Phosphate Dehydrogenase in Leishmania”.

Abstract:

• The abstract contains excessive methodological detail (software names and parameters). Consider reducing these and emphasizing biological relevance and translational significance.
• Claims regarding “high selectivity” should be toned down, as docking scores alone may not justify strong selectivity assertions.

Introduction:

• Background on leishmaniasis is clear; however, some epidemiological statements require recent references.
• The importance of Trypanothione Reductase (TryR) is explained multiple times; consider condensing to avoid redundancy.
• The rationale for selecting GPDH as a second target is interesting but should be better linked to metabolic synergy.
• The novelty of the study should be highlighted more explicitly, clarifying how it advances beyond previous single-target in silico studies.

Materials and Methods:

• The manuscript uses TryR from L. braziliensis and GPDH from L. mexicana. A justification for cross-species protein selection should be provided.
• Ligand filtering criteria are generally appropriate, but threshold values need justification with references.
• Some compounds predicted as non-toxic are known to have clinical toxicity; this discrepancy should be acknowledged.
• Docking grid parameters are clearly described; however, the choice of binding affinity cut-off (−1 kcal/mol difference) appears arbitrary and should be justified.
• Interpretation of docking scores should be more cautious, considering limitations of docking algorithms.
• MD simulation protocol is appropriate and sufficiently detailed.
• RMSD and RMSF trends are described qualitatively; including mean values or ranges would improve clarity.

Results:

• Table 1 requires formatting improvements for readability and consistency.
• Statements regarding BBB permeability and toxicity predictions should be interpreted cautiously and clearly labeled as computational predictions.
• Binding affinity differences of ~1–1.7 kcal/mol are discussed as strong selectivity; this interpretation should be toned down.
• Results involving human GPDH docking should be discussed in greater depth to strengthen selectivity claims.
• Interaction descriptions are detailed but largely descriptive; linking key residues to catalytic or functional relevance would improve biological insight.
• Stability conclusions are reasonable, but comparisons with apo-protein simulations should be more explicit.
• PCA and covariance analyses are well presented but could benefit from brief biological interpretation.
• Discussion summarizes results well but could better integrate findings with existing TryR and GPDH inhibitor literature.
• Public health relevance of the findings, particularly for endemic regions, should be emphasized more clearly.
• A short paragraph outlining experimental validation strategies would significantly strengthen the manuscript.

Conclusion:

• Avoid repeating methodological details. Focus on biological significance and translational potential.

 

Author Response

Comments and Suggestions for Authors

Title:

• The title is informative but slightly long. Consider simplifying it by reducing redundancy, e.g., “In Silico Targeting of Trypanothione Reductase and Glycerol-3-Phosphate Dehydrogenase in Leishmania”.

We thank the reviewer for the valuable suggestion. We have simplified the title by removing redundancy and shortening it while retaining the key targets and organism. The title has been revised to: “In Silico Targeting of Trypanothione Reductase and Glycerol-3-Phosphate Dehydrogenase in Leishmania.”

Abstract:

• The abstract contains excessive methodological detail (software names and parameters). Consider reducing these and emphasizing biological relevance and translational significance.
• Claims regarding “high selectivity” should be toned down, as docking scores alone may not justify strong selectivity assertions.

We thank the reviewer for the constructive suggestion. We revised the abstract to reduce excessive methodological detail by removing parameter-level descriptions and limiting the methodology to a single concise sentence (retaining only essential tool mentions). We also strengthened the abstract by emphasizing the biological rationale for targeting TryR and GPDH and the translational significance of the prioritized lead compounds, and we clarified that experimental validation is required to confirm potency and selectivity.

Introduction:

• Background on leishmaniasis is clear; however, some epidemiological statements require recent references.

Reviewer 1 – Introduction, Comment 1:
We thank the reviewer for the suggestion. We have revised the Introduction by updating the epidemiological statements and adding recent references to support the key claims regarding the global burden of leishmaniasis, the population at risk, and estimated annual incidence.

• The importance of Trypanothione Reductase (TryR) is explained multiple times; consider condensing to avoid redundancy.

We agree with the reviewer. We removed redundant statements describing TryR’s biological role and retained a single concise mechanistic explanation, while keeping subsequent references to TryR focused on the study rationale and prior literature.

• The rationale for selecting GPDH as a second target is interesting but should be better linked to metabolic synergy.

We thank the reviewer for this valuable suggestion. We strengthened the rationale for selecting GPDH by explicitly linking it to metabolic synergy with TryR inhibition. The revised Introduction now explains that simultaneous disruption of redox homeostasis (DoT/thiol system via TryR) and energy/redox-linked metabolism (via GPDH) can impose a compounded metabolic burden on Leishmania, potentially increasing vulnerability compared with single-target inhibition.

• The novelty of the study should be highlighted more explicitly, clarifying how it advances beyond previous single-target in silico studies.

We agree with the reviewer. We revised the Introduction to state the novelty more explicitly and to clarify how this work advances beyond previous single-target in silico studies. Specifically, we now emphasize that our study applies a dual-target strategy (TryR and GPDH) within a single integrated workflow and includes the human GPDH homolog as an off-target filter to support early selectivity assessment, thereby extending beyond prior studies that typically evaluate TryR or GPDH in isolation.

Materials and Methods:

• The manuscript uses TryR from L. braziliensis and GPDH from L. mexicana. A justification for cross-species protein selection should be provided.

We thank the reviewer for this important observation. We have revised the “Protein Selection” subsection to justify the cross-species choice of targets. Specifically, we clarified that the selected TryR (L. braziliensis) and GPDH (L. mexicana) structures were used because they provide high-quality structural templates and because key catalytic/binding residues are conserved across Leishmania orthologs, supporting their use for structure-based screening.

• Ligand filtering criteria are generally appropriate, but threshold values need justification with references.

We thank the reviewer for the suggestion. We have revised the ligand filtering/ADMET subsection to justify the threshold values used during screening and added appropriate references for the drug-likeness and permeability cutoffs, as well as for the interpretation of the ADMET prediction outputs.

• Some compounds predicted as non-toxic are known to have clinical toxicity; this discrepancy should be acknowledged.

We thank the reviewer for this important observation. We revised the manuscript to acknowledge that in silico toxicity predictions are limited to the modeled endpoints and may not capture clinically reported, dose-dependent, or metabolism-mediated toxicities for certain compounds. Accordingly, we have toned down definitive “non-toxic” wording and clarified that the ADMET results are intended for early lead prioritization, and that experimental toxicity and cytotoxicity assays are required to confirm safety.

• Docking grid parameters are clearly described; however, the choice of binding affinity cut-off (−1 kcal/mol difference) appears arbitrary and should be justified.

We thank the reviewer for the comment. In Section 3.4, we revised the text to clarify that the ≥1.0 kcal/mol docking-score difference was used as a pragmatic screening/enrichment threshold rather than definitive evidence of selectivity. We also replaced “significant” selectivity wording with more cautious language and added a statement that docking-based prioritization was subsequently evaluated using interaction analysis and molecular dynamics stability.

• Interpretation of docking scores should be more cautious, considering limitations of docking algorithms.

We thank the reviewer for this important point. We have revised the manuscript to interpret docking scores more cautiously by clarifying that docking energies provide approximate, model-dependent rankings rather than quantitative binding free energies. We also emphasized that docking results were used for prioritization only and were complemented by interaction analysis and molecular dynamics simulations, and that experimental validation is required to confirm binding and activity.

• MD simulation protocol is appropriate and sufficiently detailed.
• RMSD and RMSF trends are described qualitatively; including mean values or ranges would improve clarity.

Thank you for this helpful suggestion. While the MD simulation protocol is already described in sufficient detail, we agree that the RMSD and RMSF results were previously presented mainly in qualitative terms. To improve clarity, we have revised the manuscript to include quantitative summaries of the RMSD and RMSF profiles by reporting the ranges (min–max) (and, where applicable, average/representative values) for each complex in the Results section corresponding to Figures 4 and 5. These additions provide a clearer numerical comparison of structural stability and residue flexibility across the simulated systems and strengthen the interpretation of the trends discussed in the text.

 

Results:

• Table 1 requires formatting improvements for readability and consistency.

Thank you for the comment. We have reformatted Table 1 to improve readability and consistency by standardizing fonts and alignment, refining the header layout, and correcting minor typographical and formatting inconsistencies across all entries.

• Statements regarding BBB permeability and toxicity predictions should be interpreted cautiously and clearly labeled as computational predictions.

Thank you for this important comment. We have revised the manuscript to interpret BBB permeability and toxicity outcomes more cautiously and to clearly label them as in silico/computational predictions rather than experimental findings

• Binding affinity differences of ~1–1.7 kcal/mol are discussed as strong selectivity; this interpretation should be toned down.

Thank you for the constructive feedback. We have revised the manuscript accordingly by improving clarity and consistency, strengthening quantitative reporting where needed, and toning down interpretations to ensure all conclusions are appropriately cautious and supported by the presented computational results.

• Results involving human GPDH docking should be discussed in greater depth to strengthen selectivity claims.

Thank you for this suggestion. We have expanded the discussion of the human GPDH docking results by explicitly comparing human vs. Leishmania GPDH docking scores for the lead compounds and discussing these differences as computationally predicted selectivity trends (rather than definitive selectivity), along with their limitations and the need for experimental validation.

 

• Interaction descriptions are detailed but largely descriptive; linking key residues to catalytic or functional relevance would improve biological insight.

Thank you for this helpful comment. We have revised the interaction analysis to highlight key binding-site residues with known catalytic or functional relevance and to explain how their interactions with the lead compounds may influence enzyme activity and binding-site dynamics, thereby strengthening the biological interpretation beyond descriptive contacts alone.

• Stability conclusions are reasonable, but comparisons with apo-protein simulations should be more explicit.

Thank you for the comment. We have revised the text to clarify that our stability conclusions are based on relative comparisons among the ligand-bound complexes using standard MD metrics (RMSD/RMSF and related analyses), and we explicitly note that apo–holo comparisons would further strengthen interpretation and are recommended for future validation.

• PCA and covariance analyses are well presented but could benefit from brief biological interpretation.

Thank you for this suggestion. We have added a brief biological interpretation to the PCA and covariance analyses by explaining what the dominant collective motions represent (e.g., breathing/loop-gating near the binding site) and how the observed correlated/anti-correlated residue movements relate to ligand-induced stabilization and binding-site dynamics.

 

• Discussion summarizes results well but could better integrate findings with existing TryR and GPDH inhibitor literature.

Thank you for the suggestion. We have added the previous paper findings and also discussed them in the discussion.

• Public health relevance of the findings, particularly for endemic regions, should be emphasized more clearly.

Thank you for this suggestion. We have revised the manuscript to more clearly emphasize the public health relevance of the findings, particularly for leishmaniasis-endemic regions, by highlighting the unmet need for safer, affordable treatments and the translational value of the prioritized candidates as starting points for future drug development and experimental validation.

 

• A short paragraph outlining experimental validation strategies would significantly strengthen the manuscript.

Thank you for the suggestion. We have added a short paragraph outlining feasible experimental validation strategies, including enzyme inhibition assays against Leishmania GPDH with a human GPDH counter-screen, confirmatory binding assays, and follow-up cell-based anti-leishmanial and cytotoxicity evaluations to support the computational findings.

Conclusion:

• Avoid repeating methodological details. Focus on biological significance and translational potential.

Thank you for this comment. We have revised the Conclusion to reduce methodological repetition and instead emphasize the biological significance of targeting TryR and GPDH, the translational relevance of the prioritized leads, and the potential public health impact for endemic regions, while keeping experimental validation and optimization as the key next steps.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled ‘In Silico Strategies Against Leishmania: Targeting Trypanothione Reductase and Glycerol-3-Phosphate Dehydrogenase for Synergistic Inhibition’ applies an integrated in silico pipeline to identify potential dual-target inhibitors against two key Leishmania enzymes, TryR and GPDH. Eupatorin emerged as leading candidates, demonstrating strong dual-target potential with minimal off-target risks. Overall, the manuscript is well written and well structured, and the methodological approach is described in a consistent and essentially reproducible manner. However, some technical aspects deserve clarification to strengthen the robustness of the interpretation: Species inconsistency between targets. TryR refers to L. braziliensis while GPDH is from L. mexicana. This may weaken the ‘dual-target’ conclusion as species-specific differences, especially at the pocket level, can influence docking and molecular dynamics. is recommended to use targets from the same species or to include an analysis of homology and conservation of the active site/pocket to support the transferability of the results. Furthermore, the human GPDH used as a control is a mutant (R269A). Since a mutation can alter the architecture and/or dynamics of the pocket, the estimated selectivity may not be representative of the wild type. It is recommended to justify the use of the mutant (e.g., mutation far from the site with no impact) and/or to include a wild-type structure for systematic comparison. In conclusion, the manuscript is well written and well structured, with a clear and largely reproducible in silico pipeline. The critical issues highlighted mainly concern aspects of consistency and methodological clarification (species alignment and appropriateness of human control), rather than the overall design of the study. Once these points have been addressed and the discussion strengthened, the interpretation and prioritisation of the leads will be more robust. Therefore, a minor revision is recommended.

Author Response

Comments and Suggestions for Authors

The manuscript entitled ‘In Silico Strategies Against Leishmania: Targeting Trypanothione Reductase and Glycerol-3-Phosphate Dehydrogenase for Synergistic Inhibition’ applies an integrated in silico pipeline to identify potential dual-target inhibitors against two key Leishmania enzymes, TryR and GPDH. Eupatorin emerged as leading candidates, demonstrating strong dual-target potential with minimal off-target risks. Overall, the manuscript is well written and well structured, and the methodological approach is described in a consistent and essentially reproducible manner. However, some technical aspects deserve clarification to strengthen the robustness of the interpretation: Species inconsistency between targets. TryR refers to L. braziliensis while GPDH is from L. mexicana. This may weaken the ‘dual-target’ conclusion as species-specific differences, especially at the pocket level, can influence docking and molecular dynamics. is recommended to use targets from the same species or to include an analysis of homology and conservation of the active site/pocket to support the transferability of the results. Furthermore, the human GPDH used as a control is a mutant (R269A). Since a mutation can alter the architecture and/or dynamics of the pocket, the estimated selectivity may not be representative of the wild type. It is recommended to justify the use of the mutant (e.g., mutation far from the site with no impact) and/or to include a wild-type structure for systematic comparison. In conclusion, the manuscript is well written and well structured, with a clear and largely reproducible in silico pipeline. The critical issues highlighted mainly concern aspects of consistency and methodological clarification (species alignment and appropriateness of human control), rather than the overall design of the study. Once these points have been addressed and the discussion strengthened, the interpretation and prioritisation of the leads will be more robust. Therefore, a minor revision is recommended.

Response: Thank you for your thorough and constructive evaluation. We appreciate the positive assessment of the manuscript’s structure and reproducibility. In response to your key concerns, we have strengthened the robustness of the interpretation by addressing target consistency and control selection. Specifically, we clarified the use of TryR and GPDH from different Leishmania species by adding discussion supporting cross-species transferability based on conserved active-site and pocket characteristics, and we moderated the dual-target interpretation accordingly. We also justified the use of the human GPDH R269A structure by verifying in PyMOL that the mutation is distal to the defined docking pocket and does not involve pocket-lining residues, and we revised the text to present the human comparison as an off-target trend rather than definitive selectivity. In addition, we expanded the biological interpretation where requested, improved clarity and formatting across the manuscript, and incorporated concise statements on public health relevance and experimental validation. We believe these revisions address the points raised and strengthen the manuscript’s conclusions.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been improved as per reviewer comments