Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript reports an in silico investigation of phytocompounds from Cistus incanus as potential inhibitors of the Ebola virus VP40 protein. Using molecular docking, MM-GBSA binding free energy calculations, 100 ns molecular dynamics simulations, and ADME prediction, the authors identify methyl gallate, catechin, and quercetin as promising candidates. The study addresses a relevant biological target and applies a standard computational workflow appropriate for exploratory screening studies. While the topic is suitable for Biophysica, several methodological and interpretational issues should be addressed before publication.

 

1. The manuscript suggests that the compounds inhibit RNA binding and abort viral budding. However, no competitive docking with RNA, mutational validation, or functional modeling is performed. The simulations only demonstrate stable binding near the RNA-binding region.

Recommendation:

Rephrase conclusions to state that the compounds bind within the RNA-binding region and may potentially interfere with VP40 function, rather than asserting functional inhibition.

 

2. There is a likely typo in the MD section (“6800 ns”).

Several grammatical issues and tense inconsistencies require language editing.

Improve consistency in formatting of ΔG_bind and SI units.

 

3. Overinterpretation of MM-GBSA Results

The reported MM-GBSA binding energies (−51 to −48 kcal/mol) are interpreted as evidence of “strong affinity.” However, MM-GBSA is an approximate endpoint method and often overestimates absolute binding energies. Entropic contributions are not discussed, and the thermodynamic equation provided is incomplete.

Recommendation:

Clarify whether entropy was included and emphasize that MM-GBSA values should be interpreted comparatively rather than quantitatively. Avoid overstatement of affinity.

 

4. Some sentences are unclear and need clarification or grammatical correction:

Line 22: These complexes had good stability over the 100 ns (molecular dynamics simulation?) time.

Line 54: EBOV is an enveloped, negative-sense ribonucleic acid (RNA) genome consists of different structural proteins having crucial roles in the viral life cycle:

Line 55: The seven different structural proteins in EBOV such as VP24, VP30, VP35, VP40, a glycoprotein, a nucleoprotein, and a polymerase (L) that could be used as promising the therapeutic targets as potential inhibitors of the virus [14].

Line 76: The lack of preventive vaccines and medicines against Ebola virus disease (EVD) that the major reason that it could become a pandemic threat and also case of fatality rates can be increased up to 90% [23].

Line 117: Prime MM/GBSA and MDS (are?) performed to examine the binding free energy and binding stability of the complexes. Several preclinical compounds are getting failure to enter into clinical trials because of poor pharmacokinetic properties of the lead compounds [50].

There are more sentences I did not list here, but the authors should go through the manuscript and revise them.

Author Response

This manuscript reports an in silico investigation of phytocompounds from Cistus incanus as potential inhibitors of the Ebola virus VP40 protein. Using molecular docking, MM-GBSA binding free energy calculations, 100 ns molecular dynamics simulations, and ADME prediction, the authors identify methyl gallate, catechin, and quercetin as promising candidates. The study addresses a relevant biological target and applies a standard computational workflow appropriate for exploratory screening studies. While the topic is suitable for Biophysica, several methodological and interpretational issues should be addressed before publication.

Comment 1: The manuscript suggests that the compounds inhibit RNA binding and abort viral budding. However, no competitive docking with RNA, mutational validation, or functional modeling is performed. The simulations only demonstrate stable binding near the RNA-binding region. Recommendation: Rephrase conclusions to state that the compounds bind within the RNA-binding region and may potentially interfere with VP40 function, rather than asserting functional inhibition.

Response 1: Thank you for the positive recommendation of our manuscript. We agree with the expert reviewer’s comment. Earlier statement mentioning ‘RNA-binding inhibition or viral budding abortion’ has been rephrased according to the recommendation of the reviewer. The rephrased sentence has been highlighted in the discussion section of the revised manuscript (Page 11), accordingly.

 

Comment 2: There is a likely typo in the MD section (6800 ns”). Several grammatical issues and tense inconsistencies require language editing. Improve consistency in formatting of ΔG_bind and SI units.

Response 2: The typographical error in section 3.3. [Molecular Dynamics Simulation] has been corrected and highlighted, accordingly, on Page 7. The “6800 ns” has been revised to reflect the correct simulation timescale within the 100 ns trajectory. In addition, the entire manuscript has undergone comprehensive language editing to correct grammatical errors, improve clarity, and ensure consistency in verb tense and scientific style. The formatting of ΔG_bind, SI units, symbols, and notations has also been standardized throughout the manuscript to maintain uniformity and compliance with journal guidelines. All the corrections were highlighted accordingly in this revised version of the manuscript

 

Comment 3: Over interpretation of MM-GBSA Results: The reported MM-GBSA binding energies (−51 to −48 kcal/mol) are interpreted as evidence of “strong affinity.” However, MM-GBSA is an approximate endpoint method and often overestimates absolute binding energies. Entropic contributions are not discussed, and the thermodynamic equation provided is incomplete.

Recommendation: Clarify whether entropy was included and emphasize that MM-GBSA values should be interpreted comparatively rather than quantitatively. Avoid overstatement of affinity.

Response 3: Thank you for this valuable comment. We agree with the expert reviewer’s point. Hence, the manuscript has been revised to clarify that Prime MM-GBSA does not include entropic contributions (TΔS) and provides endpoint free energy estimates. Accordingly, the thermodynamic equation has been corrected, and the ΔG_bind values are now interpreted comparatively rather than quantitatively, for relative ranking and stability assessment only. All these changes are highlighted on page 4 of section 2.4 of the revised manuscript. All overstatements regarding “strong affinity” and definitive binding have been removed in the revised manuscript for better clarity and understanding.

 

Comment 4: Some sentences are unclear and need clarification or grammatical correction:

Line 22: These complexes had good stability over the 100 ns (molecular dynamics simulation?) time.

Line 54: EBOV is an enveloped, negative-sense ribonucleic acid (RNA) genome consists of different structural proteins having crucial roles in the viral life cycle:

Line 55: The seven different structural proteins in EBOV such as VP24, VP30, VP35, VP40, a glycoprotein, a nucleoprotein, and a polymerase (L) that could be used as promising the therapeutic targets as potential inhibitors of the virus [14].

Line 76: The lack of preventive vaccines and medicines against Ebola virus disease (EVD) that the major reason that it could become a pandemic threat and also case of fatality rates can be increased up to 90% [23].

Line 117: Prime MM/GBSA and MDS (are?) performed to examine the binding free energy and binding stability of the complexes. Several preclinical compounds are getting failure to enter into clinical trials because of poor pharmacokinetic properties of the lead compounds [50].

There are more sentences I did not list here, but the authors should go through the manuscript and revise them.

Response: Thank you for the comment. All the specified sentences have been corrected, and the entire manuscript has been revised for grammar, clarity, and consistency in language and tense. All changes are highlighted in the revised manuscript accordingly.

Line 22 is corrected to ‘These complexes showed good stability over the 100 ns MD simulation time.’

Lines 54 & 55 were corrected to “EBOV is an enveloped, negative-sense RNA virus consisting of multiple structural proteins that play crucial roles in the viral life cycle [13]. The seven structural proteins of EBOV, namely VP24, VP30, VP35, VP40, glycoprotein (GP), nucleoprotein (NP), and polymerase (L) represent promising therapeutic targets for the development of antiviral compounds [14].”

Line 76 is corrected to ‘The lack of effective preventive vaccines and approved therapeutics for Ebola virus disease (EVD) represents a major concern, as outbreaks can pose pandemic threats and case fatality rates may reach up to 90% [23].’

Line 117 is corrected to ‘Prime MM/GBSA and molecular dynamics simulations were performed to examine the binding free energy and binding stability of the complexes. Several preclinical compounds fail to progress into clinical trials due to poor pharmacokinetic properties of lead candidates [50].’

Reviewer 2 Report

Comments and Suggestions for Authors

The authors presented an in silico study that evaluated the binding potential of several phytocompounds of the mediterranean herb cistus incantus against the VP40 protein of the Ebola virus. The study could be of interest for scientists working on drug design against Ebola, but several methodological issues should be improved before considering publication. The authors do not mention multiple replicas of the MD simulations of the docked complexes. The MD results should be replicated at least three times, as there are a lot of cases where one complex happens to be steady in the short simulation time of 100ns but if the run is repeated with different initial velocities (i.e., in different replicas) the complex is no longer stable. Therefore, in order for the results to have any statistical meaning, at least three replicas of the complexes that are reported as steady in the manuscript should be presented. Moreover, the authors do not mention which thermostat and barostat they used to generate the NVT and NPT ensembles of the simulations. In lines 233-234 they mention "In the VP40– Quercetin complex, RMSD 233 values gradually increase until 6800 ns." What are these 6800ns since they mention runs of 100ns?   The authors present RMSD and RMSF of the protein which reflect the conformational stability of the protein but not RMSD of the ligand inside the binding cavity in order to be able to assess the binding stability of the ligand. The presented h-bond plots would be easier to interpret if they would be accompanied with distance over time plots with the relevant aminoacids with which the putative hbonds are formed.

Comments on the Quality of English Language

The manuscript would benefit from an improvement in the level of written english.

Author Response

The authors presented an in silico study that evaluated the binding potential of several phytocompounds of the mediterranean herb cistus incantus against the VP40 protein of the Ebola virus. The study could be of interest for scientists working on drug design against Ebola, but several methodological issues should be improved before considering publication.

Comment 1: The authors do not mention multiple replicas of the MD simulations of the docked complexes. The MD results should be replicated at least three times, as there are a lot of cases where one complex happens to be steady in the short simulation time of 100ns but if the run is repeated with different initial velocities (i.e., in different replicas) the complex is no longer stable. Therefore, in order for the results to have any statistical meaning, at least three replicas of the complexes that are reported as steady in the manuscript should be presented.

Response 1: Thank you. We acknowledge the Reviewer’s point regarding the reproducibility of MD simulations. The current study was conducted using single 100 ns MD trajectories per complex, which has now been clearly stated as a limitation of the study at the end of the discussion section (Page 12). We agree that multiple replicas improve statistical robustness; however, due to computational constraints, replicated simulations were not performed at this time. Considering this, our future studies will include different replicas.

 

Comment 2: Moreover, the authors do not mention which thermostat and barostat they used to generate the NVT and NPT ensembles of the simulations. In lines 233-234 they mention "In the VP40– Quercetin complex, RMSD 233 values gradually increase until 6800 ns." What are these 6800ns since they mention runs of 100ns?  

Response 2: Thank you. The MD methodology (Section 2.5; Page 4) has been revised explicitly to specify the thermostat and barostat used for the NVT and NPT ensembles. Moreover, the typographical error “6800 ns” has been corrected to reflect the 100 ns simulation timeframe.

 

Comment 3: The authors present RMSD and RMSF of the protein which reflect the conformational stability of the protein but not RMSD of the ligand inside the binding cavity in order to be able to assess the binding stability of the ligand.

Response 3: We agree with the Reviewer’s comment. In addition to the protein–ligand complex RMSD, ligand-specific RMSD within the VP40 binding cavity has now been included to directly assess ligand binding stability. The manuscript has now been revised to clearly distinguish global complex RMSD from ligand RMSD inside the binding pocket, and the corresponding results and discussion have been added on Page 7. Accordingly, Figure S1 has been included in the supplementary material (revised file).

 

Comment 4: The presented h-bond plots would be easier to interpret if they would be accompanied by distance overtime plots with the relevant aminoacids with which the putative hbonds are formed.

Response 4: We appreciate the Reviewer’s comment. To improve the interpretation of the hydrogen bond analyses, an enhanced hydrogen bond interaction/occupancy profile with a clear legend has been added to the revised manuscript (Figure 3; Page 9). In addition, this limitation has been acknowledged, and future work will incorporate distance-over-time analyses of specific ligand–residue hydrogen bonds (Page 12).

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed many of the comments that were proposed during the first round of revisions. However, I insits on the matter of independent replicas. The results of just one replica hold little value in terms of statistical significance. The authors agree that the independent replicas are imprtant, however they claim that they cannot conduct them due to computational constraints. With nowadays computers, even if the authors do not have access into supercomputing systems, which is something acceptable and absolutely understandable, even a usual laptop could achieve 100ns in a reasonable time. The software used (GROMACS) is open source and could be downloaded and isntalled via a conda environment even in a conventional laptop using WSL. For these reasons, I must insist that additional replicas are required at l;east for their three best systems.

Comments on the Quality of English Language

The manuscript would benefit from an improvement in the level of written english.

Author Response

Comment: The authors have addressed many of the comments that were proposed during the first round of revisions. However, I insist on the matter of independent replicas. The results of just one replica hold little value in terms of statistical significance. The authors agree that the independent replicas are important, however they claim that they cannot conduct them due to computational constraints. With nowadays computers, even if the authors do not have access into supercomputing systems, which is something acceptable and absolutely understandable, even a usual laptop could achieve 100ns in a reasonable time. The software used (GROMACS) is open source and could be downloaded and installed via a conda environment even in a conventional laptop using WSL. For these reasons, I must insist that additional replicas are required at least for their three best systems.

Response: Thank you for your positive feedback and good appreciation regarding our first round of revisions. We acknowledge the Reviewer’s point regarding the importance of conducting the replicas. Hence, we have now performed replicas successfully and revised the manuscript accordingly in this second round. All new changes are highlighted in yellow in the revised version of the manuscript, and the updated figure has been included in the supplementary material.

Author Response File: Author Response.pdf

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for addressing my comments. I believe that now the article is ready for publication.

Comments on the Quality of English Language

The manuscript would benefit from an improvement in the level of written english.