Investigation of Interactions Between the Protein M^Pro and the Vanadium Complex VO(metf)₂∙H₂O: A Computational Approach for COVID-19 Treatment

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors Analysis on "Investigation of Interactions between the Protein M Pro and the
Vanadium Complex VO(metf)₂∙H₂O: A Computational Approach for COVID-19
Treatment" by Tavares and co-workers. The manuscript presents an interesting and timely investigation into the
potential therapeutic applications of vanadium complexes against COVID-19.
The authors have employed computational methods to explore the interactions
between the vanadium complex and the M Pro protein. While the study
demonstrates a solid grasp of computational chemistry techniques, several
critical issues need to be addressed to enhance the overall quality and rigor
of the work. The authors' justification for the choice of the B3LYP/6-31+G(d) level of
theory is unconvincing. The substantial differences between gas-phase
optimizations and experimental solid-state data should be acknowledged.
A higher-level theory, such as B3LYP/ZORA-def2-TZVP would be more appropriate
for this type of study. Consistency in the level of theory used throughout
the calculations is essential for reliable comparisons. The authors should
ensure that all calculations are performed at the same level of theory. The authors have developed a new AMBER force field for the vanadium complex.
While this is a commendable effort, a clear citation to the previous work
where this force field was validated is necessary. The definition of the binding site is crucial for the accuracy of the docking
and molecular dynamics simulations. It is need for clarification regarding
the cutoff radius used to define the binding site. A justification for the
chosen cutoff value should be provided. The use of the unit "kcal/mol" for a force constant is incorrect. Force
constants are typically expressed in units of energy per distance squared
(e.g., kcal/mol/Ų). The authors should verify all units used throughout
the manuscript. A major limitation of the study is the lack of experimental validation.
The authors' conclusions about the importance of interactions with residues
ARG188 and GLU166 would be significantly strengthened by experimental data,
such as mutagenesis studies or binding affinity measurements. The manuscript presents a valuable contribution to the field of computational
drug design. However, the issues raised in this review need to be addressed
to enhance the overall quality and reliability of the work. With the
recommended revisions, this manuscript has the potential to be a significant
contribution to the literature on COVID-19 therapeutics.

 

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors present a report on the molecular interactions between the main protease (M^Pro) of SARS-CoV-2 and two ligands: vanadium complex (VC) and Metformin (Mtef). These ligands, VC and Mtef, were docked into Mpro binding pocket, which has been established in a previous literature. Subsequently, all-atom molecular dynamics (MD) simulations were conducted on M^Pro, M^Pro+VC, and M^Pro+Mtef systems. Based on analyses of the root-mean-square deviation, root-mean-square fluctuation, conformational changes, and hydrogen bonds patterns. specific residues involved in Mpro-ligand interactions were identified. Overall, the manuscript is clearly written, and the findings are supported by the simulation data. However, given the following issues, I do not deem the manuscript suitable for publication at this time.

1.     The Introduction lacks sufficient background information and omits some crucial relevant references. On one hand, some included context is not closely aligned with this research, such as the statement, "The transmission of the coronavirus from animals to humans may have been facilitated by human intervention in nature…". On the other hand, the crucial reference (literature 27) used as a benchmark for molecular docking is missing.

2.     The Methods section has not been adequately described. For instance, while it mentions that "the energy criteria were not the only factor considered in selecting the best pose; the conformation of the molecules was also taken into account," it fails to elaborate on how the conformation was considered and what the specific criteria for conformation were. Additionally, it is unclear whether and how pressure was maintained during the MD simulations.

3.     The MD simulation results are heavily reliant on (and even determined by) the initial model. Since the initial docking model was built based on literature 27 ("the lowest energy pose that reproduced literature data was selected as the starting point for the next step, the Molecular Dynamics simulation"), the obtained MD simulation results are predictable, significantly limiting the novelty of this study.

4.     The techniques used in this study are relatively traditional, and no analysis was conducted on binding free energy, which is essential for understanding protein-ligand interactions.

5.     Considering that "The cutoff for non-136 bonded interactions was set at 10 Å", "a truncated octahedral box measuring 20 Å" is insufficient for MD simulation under periodic boundary conditions.

6.     The results have not been presented clearly. For example, while it mentions that "although the frequency of these interactions occurring was approximately 1%", it does not specify whether these interactions occurred only at the beginning of the MD simulations or throughout the entire duration.

7.     Typographical errors remain, such as "In Figure 7.a, as mentioned previously, the nitrogen N6" which should be corrected to "In Figure 8.a, as mentioned previously, the nitrogen N6."

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Alll reviewer queries have been adequately addressed by the authors. The manuscript is now ready for publication.

Author Response

Reviewer 1:

 

1. All reviewer queries have been adequately addressed by the authors. The manuscript is now ready for publication.

Author reply: We would like to thank the reviewer for your comment. From our manuscript, we hope to contribute to the search for new COVID-19 treatment, and to encourage new studies on the use of oxovanadium (IV) complexes for new drug design and optimization. Again, thank you for your great contribution for the manuscript improvement.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has undergone substantial revisions in response to previous comments. I believe it is suitable for publication after addressing the following issue:

1.      Regarding the previous comment that the cutoff for non-bonded interactions (10 Å) was too small for a truncated octahedral box measuring 20 Å, the authors' response that this setup is similar to a previous publication is not sufficiently convincing. Firstly, the cited literature was also authored by the same authors, which may introduce a bias. Secondly, the cutoff information has not been described in that literature.

Author Response

Dear Editor,

Biophysica

 

 

We very much appreciate the response e-mail containing the valuable and helpful reviewer evaluation of our manuscript. Please, find enclosed the new version including the suggestions made by Reviewers 1 and 2.

Accordingly, we carried out a complete revision of the manuscript with emphasis on the scientific content and on the formatting request. We hope that all the necessary improvements of the discussion were implemented. The list of changes and responses to the referees’ comments are listed below and modified parts of the paper are highlighted in yellow.

 

 

 

 

Reviewer Comments:

 

 

Reviewer 1:

 

1. All reviewer queries have been adequately addressed by the authors. The manuscript is now ready for publication.

Author reply: We would like to thank the reviewer for your comment. From our manuscript, we hope to contribute to the search for new COVID-19 treatment, and to encourage new studies on the use of oxovanadium (IV) complexes for new drug design and optimization. Again, thank you for your great contribution for the manuscript improvement.

 

 

 

Reviewer 2:

 

1. The manuscript has undergone substantial revisions in response to previous comments. I believe it is suitable for publication after addressing the following issue: Regarding the previous comment that the cutoff for non-bonded interactions (10 Å) was too small for a truncated octahedral box measuring 20 Å, the authors' response that this setup is similar to a previous publication is not sufficiently convincing. Firstly, the cited literature was also authored by the same authors, which may introduce a bias. Secondly, the cutoff information has not been described in that literature.

Author reply: We sincerely thank the reviewer for their valuable contribution to enhancing our manuscript. We believe their feedback has been instrumental in improving the reader's understanding and in highlighting the novelty of our work. Regarding the previous question, which the reviewer has reiterated, we fully agree with the comment and deeply appreciate this important observation.

            In this sense, we would like to mention that a mistake from our part has occurred in the written, and the sentence “These simulations were carried out with explicit solvent, adopting the OPC model, within a truncated octahedral box measuring 20 Å” is misleading, we apologize for that. From the current sentence, the reader is driven to believe that the simulation box measured 20 Å, which is not true.

            Therefore, after a careful revision of our simulation setup, we have ensured the accurate information is presented to the reader, as demonstrated highlighted in yellow in Section 2.4. The simulation setup was designed to create a solvated truncated octahedral box large enough to provide a 20 Å buffer distance between the edges of the protein and the edges of the simulation box. In this scenario, starting from a buffer distance of 20 Å, a truncated octahedral box with dimensions of 90–100 Å was generated, resulting in a simulation box significantly larger than 20 Å. Such dimensions are suitable for applying a non-bonded interaction cutoff of 10 Å, as they effectively prevent interactions between the protein and its periodic images. In addition to that, the simulation setup was carried out by centralizing the protein in the created simulation box, which again is of great importance for not allowing interactions between the protein and its periodic image.

            We sincerely apologize for the mistake in the writing of the MD simulation methodology section and thank the reviewer for raising this important concern. We believe that the modifications made have ensured accurate information, now provided for future readers.

 

 

 

We acknowledge again the referee’s comments, which have enabled us to significantly improve our paper.

 

Furthermore, we thank the editorial assistance and hope with the changes and clarifications implemented, the manuscript would be now acceptable for publication in Biophysica. Finally, we also remain at your disposal for any further inquiries.

 

With best regards,

Teodorico C. Ramalho

Department of Chemistry,

Federal University of Lavras,

Campus Universitário, C.P. 3037, 37200-000, Lavras,

Brazil.

e-mail:  teo@ufla.br

          https://orcid.org/0000-0002-7324-1353