Deep-Sea Marine Metabolites as Promising Anti-Tubercular Agents: CADD-Guided Targeting of the F₄₂₀-Dependent Oxidoreductase

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript Title: Deep-Sea Marine Metabolites as Promising Anti-Tubercular Agents: CADD-Guided Targeting of the F420-Dependent Oxidoreductase

The authors have carried out computational screening of 2,773 deep-sea marine metabolites and identified three potential inhibitors of Rv1155 through molecular docking, ADMET profiling, and molecular dynamics simulations. The study focuses on an important drug target and presents a valuable dataset on marine-derived scaffolds with possible anti-tubercular activity. The overall workflow is relevant, and the topic aligns with current interest in novel mechanisms for combating MDR/XDR tuberculosis.

However, the manuscript requires major revision to improve scientific clarity, structure, and language quality. The presentation of the work can be strengthened significantly. The manuscript can be improved by addressing the following suggestions:

Comments:

1. The abstract does not read like the summary of a research article. Instead, it comes across more like a general review of the field, with too much background and very little emphasis on the actual findings of the study. The work reports docking, ADMET, and MD results, but these outcomes are mentioned only in broad terms, without any specific values or clear statements on what was achieved.
2. In introduction the first several paragraphs discuss TB epidemiology, deep-sea environments, and examples of marine metabolites in great detail. Much of this is standard information and can be shortened without affecting the scientific context.
3. The introduction summarising previous marine metabolites with anti-TB activity reads like a review article. Only two or three representative examples are needed to justify why marine scaffolds are relevant.
4. Although MDR/XDR-TB is mentioned, the introduction does not clearly explain why Rv1155 is an important target or what gap exists in current drug discovery related to F₄₂₀-dependent enzymes. This should be stated earlier and more directly.
5. Sections describing CADD methods, their advantages, and limitations are too detailed for an introduction. These topics belong in Methods or Discussion.
6. Figure 2 (the crystal structure of Rv1155 with the highlighted binding pocket) should not appear in the Methods section. Structural visualisations are part of the study’s findings and therefore belong in the Results section, where they can be introduced alongside the docking analysis. At present, the figure is not sufficiently annotated. The yellow box is shown, but the caption does not explain.
7. The Results and Discussion section reads more like a technical report than a focused scientific narrative; please simplify and highlight the main findings more clearly.
8. The opening of the docking section repeats background about Rv1155 and F₄₂₀-dependent enzymes that was already given in the Introduction; this should be shortened or moved to avoid redundancy.
9. The paragraph explaining what molecular docking is (general definition and theory) is not needed in Results and should be removed or reduced to one short sentence.
10. When you report the docking scores (−0.59 to −15.95 kcal/mol), it would help to explicitly state the docking score of F₄₂₀₂ and clearly say how many compounds were better than this reference.
11. The description of the binding pocket (residues Arg30, His27, Asn60, Arg55, Lys57, Trp77, Tyr79, etc.) is useful, but it would be stronger if you directly link these residues to specific interactions of Upenamide, Aspyronol, and Fiscpropionate F.
12. The RMSF results are very similar across systems; instead of listing all values, you can summarise that overall flexibility is comparable and only highlight regions (residues 1–2) that differ.
13. The PCA description is quite dense; try to state the main conclusion in simple terms: which complexes explore a limited conformational space and which show major shifts.
14. Across the Results, there is frequent switching between “Compound_1749 / Compound_1796 / CMNPD_22964” and “Aspyronol / Fiscpropionate F / Upenamide”; for clarity, choose one naming style and keep it consistent (for example, “Aspyronol (Compound_1749)” the first time, then only the common name).
15. The Results and Discussion are currently mostly “Results”; there is very little integration or higher-level interpretation; consider adding a short final paragraph in each subsection that interprets what the results mean for inhibitor design.
16. The RMSD, RMSF, and Rg plots need improvement in visual quality; the current y-axis ranges are inconsistent and make it hard to compare systems. Please standardise the y-axis window (for example, 0–4, 0–5, or 0–6 Å as appropriate) across panels so that differences between complexes can be visually compared at a glance.
17. The PCA figure is very difficult to interpret in its current form; the x- and y-axes are almost not visible, and axis labels and tick values cannot be read. Please regenerate the PCA plots with larger fonts, clear axis labels (PC1, PC2), and a readable scale.
18. The hydrogen-bond analysis currently reports only overall contact percentages; for completeness, a time-series hydrogen bond profile should be added to show how each key interaction behaves throughout the 200 ns simulation.
19. A time-resolved HBond plot for each complex (Residue vs. Time or Number of H-Bonds vs. Time) will help readers visualise stability, formation, and disruption patterns of critical interactions in the active site.
20. The conclusion is too long and repeats background material; please shorten it to focus only on the key findings and the main take-home message of the study.
21. Avoid re-describing methods and detailed results in the conclusion; highlight only the final outcome (the three lead compounds) and the future direction of validating and extending this work.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The manuscript requires substantial editing to improve clarity, grammar, and scientific expression. Several sentences are overly long or unclear, sections contain redundant background information, and the scientific terminology is not always used precisely. The writing would benefit from thorough revision by a fluent English speaker or professional language-editing service to ensure clarity, coherence, and proper scientific style.

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Desai et al. offers a cost-effective and generalized computational approach for investigating the wide range of marine metabolites in the discovery of new inhibitors targeting the F₄₂₀-dependent redox system of Mycobacterium tuberculosis

I have a few comments for polishing the manuscript.

1. The authors should emphasize the significance of marine-derived compounds as effective antimicrobial, antitumor agents and anti-helminth agents in the Introduction Section, followed by their significance as effective anti-mycobacterial agents. The authors should review the following articles in this regard.

https://doi.org/10.3390/ijtm4040051

https://doi.org/10.1039/D3NP00019B
https://doi.org/10.1021/acs.jnatprod.6b00235

2. The authors should perform wet lab experiments, validating the effect of Upenamide (CMNPD_22964), Aspyronol 23 and (Compound_1749), and Fiscpropionate F (Compound_1796) as effective anti-mycobacterial agents.
3. The molecular mechanism through which the agents work is not clearly defined. The authors should try to dissect the molecular mechanism.
4. The authors should state the major limitations associated with the study.
5. The authors should clearly define the novelty of their study.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents a computational search exploring deep-sea marine metabolites as potential inhibitors of the F₄₂₀-dependent oxidoreductase Rv1155 from Mycobacterium tuberculosis. The topic is timely, relevant, and clearly aligned with the scope of Marine Drugs. The authors successfully integrate molecular docking, ADMET filtering, MD simulations, and MM-GBSA calculations into a coherent workflow. The iThenticate score is low and the authors have chosen the proper Special Issue. However, the manuscript contains some errors that need revision before it can be considered for publication.

Detailed comments:

Line 37, “ According to the World Health Organisation (WHO, 2023), an estimated 37 1.25 million deaths were attributed to TB in 2024 alone” – I don’t know how the report from 2023 can describe the number of deaths in 2024?

Line 57, what is  “Formatting…” ?

Line 63-65, “Guanidine alkaloids such as batzelladine L and N have also demonstrated inhibitory effects against M. tuberculosis H37Rv fused[9], [10](Figure 1).” – I don’t understand what do you mean by “fused”?

Line 75-83, this should be supported by adding the references to the recent works https://doi.org/10.56782/pps.490 , https://doi.org/10.56782/pps.261 , https://doi.org/10.56782/pps.412

Lines 98-99, what do you mean by “scoring functions” in molecular dynamics? There are no scoring functions in molecular dynamics, solely in the molecular docking. Do you mean the MM/GBSA? This is not a scoring function…

Grid box dimensions and centre coordinates are provided, but no justification is given for box dimensions.

Docking such large ligands with AutoDock may be unrealistic. Why have you chosen this software instead of the one designed for studying the peptide-protein interactions? I..e. Glide?

Please specify precisely how stereochemistry, tautomers, and protonation states were treated. Have you generated all possible states? How?

The MD simulation description is very long but still misses essential details: Was any restraint applied during equilibration? Was periodic boundary fully described?

AutoDockVina values below –12 kcal/mol are often “false positives” and obtained for very large molecules; the authors should discuss this.

The PCA interpretation is overestimated; PCA plots do not imply “distinct conformational states” without clustering analysis.

MM-GBSA values are not directly comparable to F4202 because F4202 carries multiple charged phosphate groups and is structurally dissimilar.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Most of my previous comments have been adequately addressed; I have only a few minor suggestions that are primarily related to presentation and clarity.

• The redocking RMSD of 2.77 Å exceeds the commonly cited 2.0 Å threshold; please justify whether this value is acceptable for validating the docking protocol used.
• The hydrogen-bond time-series is provided as a visual persistence map. Could the authors briefly explain how this plot should be interpreted and what information it conveys regarding hydrogen-bond stability across the simulation time?
• The Conclusion is unnecessarily long and split into three paragraphs. Please condense it into a single, focused paragraph that summarises the key results, the identified lead compounds, and the future direction, without repeating background or methods.
• In Table 2, the reported ‘Proportion of frames (%)’ values should be presented in a consistent format (uniform decimal precision).
• The PCA plots remain difficult to interpret, as the axis labels and tick values are not clearly visible even at 100% zoom. Please regenerate the PCA figures with larger fonts, clearly readable axis values (PC1, PC2), and improved resolution.

Comments on the Quality of English Language

The English language is generally understandable; however, minor grammatical and stylistic issues remain. A light revision by a fluent English speaker or professional editing service is recommended.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed all the previous comments. The manuscript can be accepted in the present form.

Author Response

Comment: The authors have addressed all the previous comments. The manuscript can be accepted in the present form.

Response: We sincerely thank the reviewer for their positive evaluation and recommendation for acceptance. We greatly appreciate the time and effort invested in reviewing our manuscript and are delighted that the revisions have met the reviewer’s expectations.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have revised and improved their work, current version can be accepted.

Author Response

Comment: The authors have revised and improved their work, current version can be accepted.

Response: We sincerely thank the reviewer for their positive evaluation and recommendation for acceptance. We greatly appreciate the time and effort invested in reviewing our manuscript and are delighted that the revisions have met the reviewer’s expectations.