Rocio Virus Encephalitis: In Silico Evidence for Drug Repurposing
Round 1
Reviewer 1 Report
This paper is publishable subject to minor revisions noted. Further review is not needed. In-silico virtual screening of four different drugs against Rocio virus was studied by authors. According to their molecular simulation results Itraconazole showed best properties as it can interacts with structural as well as non-structural proteins of virus. They claimed that itraconazole is the strong binder to the NS1 protein and can be used for rapid drug repurposing whenever there is emergency due to ROCV. Manuscript is very well written. The paper contains a lot of significant information. I have few suggestions and comments;
- Authors have cited a lot of articles on previous studies. I suggest authors to cite this recent review on ‘Rocio virus: An updated view on an elusive Flavivirus, Viruses, 2021, 13, 2293, (DOI: 3390/v13112293).
- Authors have performed molecular dynamics simulation over docked structure but they have not considered the effect of counterions. Can they explain how the results will change if counterion is included?
- In Table 1, is the data provided on four drugs already reported, if so, then I would suggest to include particular references in the Table 1. Can they also include specific pharmacore features of their four drugs given in the Table?
- In lines 330-333, authors wrote ‘Molecular dynamic simulations of the NS1-itraconazole docked structure revealed great stability of the proposed complex, while the ligand-binding affinity estimation highlighted their favorable interaction.’ What are those favorable interactions, they only wrote Replica 1 till Replica 5 in Table 2. Are these polar or non-polar interactions? I would suggest to name those interactions.
- Similarly, I noticed that in lines 334 – 335 they wrote ‘Such interactions seem to disturb protein packing contacts…..’. It will be good if authors can explain what are those such interactions in particular, are these pi-pi stacking, electrostatic, non-polar or polar? Which of these interactions are favorable and which interactions are unfavorable? Moreover, in Table 2, they have written only binding energy of different interactions, Replica 1 – Replica 5. Can authors describe what are different contributions (Vander Waals, electrostatic, polar, non-polar, etc) to the binding energy?
- Finally, I would suggest authors to provide also Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values of ITC-NS1 simulated interaction.
- Besides that I noticed some typos and suggest minor corrections;
- I suggest authors to change four digits after decimal to either two or three digits in Table 2.
- Reference 47, line 449, year number is wrong.
Author Response
Dear Reviewer,
We thank you for your time and consideration.
We have now addressed each question raised by the reviewers and the manuscript has been modified accordingly. We are sure that the recommended changes have enhanced our work.
Below you can find our replies to your comments.
Yours,
Rodrigo Ligabue Braun, DSc
Reviewer(s)' Comments to Author:
Reviewer: 1
Comments:
"This paper is publishable subject to minor revisions noted. Further review is not needed. In-silico virtual screening of four different drugs against Rocio virus was studied by authors. According to their molecular simulation results Itraconazole showed best properties as it can interacts with structural as well as non-structural proteins of virus. They claimed that itraconazole is the strong binder to the NS1 protein and can be used for rapid drug repurposing whenever there is emergency due to ROCV. Manuscript is very well written. The paper contains a lot of significant information. I have few suggestions and comments;"
Authors have cited a lot of articles on previous studies. I suggest authors to cite this recent review on ‘Rocio virus: An updated view on an elusive Flavivirus, Viruses, 2021, 13, 2293, (DOI: 3390/v13112293).
-- Our reply: We have added the suggested reference (it is now Reference #6).
Authors have performed molecular dynamics simulation over docked structure but they have not considered the effect of counterions. Can they explain how the results will change if counterion is included?
-- Our reply: We appreciate the reviewer's suggestion, however all of our simulations in the present work include counterions. In the Material and Methods, subtopic Molecular Simulations, we state that: "The two systems were embedded in explicit waters, while Cl- and Na+ ions were added to neutralize and reach the molar concentration of 0.15 M, leading to an orthorhombic periodic cell."
In Table 1, is the data provided on four drugs already reported, if so, then I would suggest to include particular references in the Table 1. Can they also include specific pharmacore features of their four drugs given in the Table?
-- Our reply: We have added the database references for the data. It reads “Available on Pubchem (https://pubchem.ncbi.nlm.nih.gov/) and DrugBank (https://go.drugbank.com/) databases under the numbers 25154714, 5311181, 55283, 24873435 and DB09102, DB01088, DB01167, DB06290, respectively”. Since we are proposing drug repositioning, pharmacophore features previously described for other actions would be inadequate in this instance.
In lines 330-333, authors wrote ‘Molecular dynamic simulations of the NS1-itraconazole docked structure revealed great stability of the proposed complex, while the ligand-binding affinity estimation highlighted their favorable interaction.’ What are those favorable interactions, they only wrote Replica 1 till Replica 5 in Table 2. Are these polar or non-polar interactions? I would suggest to name those interactions.
-- Our reply: The authors agree with the reviewer’s comment on this. We have included the name of those interactions in the manuscript. The following lines were included in the main text of the manuscript: "Molecular dynamics simulations of the NS1-itraconazole docked structure revealed great stability of the proposed complex (Figure 5 A,B; Suppl. Figure 1C), mainly through van der Waals (Figure 5B) and hydrophobic interactions (Figure 5C), while the ligand-binding free energy estimation (Table 2) has confirmed these favorable interactions."
Similarly, I noticed that in lines 334 – 335 they wrote ‘Such interactions seem to disturb protein packing contacts…..’. It will be good if authors can explain what are those such interactions in particular, are these pi-pi stacking, electrostatic, non-polar or polar? Which of these interactions are favorable and which interactions are unfavorable? Moreover, in Table 2, they have written only binding energy of different interactions, Replica 1 – Replica 5. Can authors describe what are different contributions (Vander Waals, electrostatic, polar, non-polar, etc) to the binding energy?
-- Our reply: The authors agree with the reviewer’s keen eye on this. As present in Figure 5C, there are a series of interactions between ITC and NS1, mainly hydrophobic and pi-pi stacking interactions. All of these interactions seem to be important and favorable to keep the ligand binding to the protein. We have included the interactions type in the main text of the manuscript.
The MM-GBSA method present in the paper calculates the estimated binding free energy between ITC and NS1. We calculated the interaction energy (Electrostatic and van der Waals interactions) and solvation free energy for the complex, receptor and ligand and averaged the results to obtain an estimate of the binding free energy. We changed the Material and Methods part in the main text of the manuscript to make it clearer. The following lines were included in the main text of the manuscript:
"This method calculated the interaction energy and solvation free energy for the complex, receptor and ligand and averaged the results to obtain an estimate of the binding free energy."
"Additional residue-ligand interactions are established during simulation (Figure 5C), mainly hydrophobic and pi-pi stacking interactions, when compared to the original docking pose (Figure 4). All of these interactions seem to be important and favorable to keep the ligand binding to the protein."
Finally, I would suggest authors to provide also Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values of ITC-NS1 simulated interaction.
-- Our reply: The authors appreciate the reviewer’s consideration on that point. We have shown the RMSF of ITC-NS1 systems in the Supplementary Material in the first version of the manuscript. Additionally, we have included the RMSD of the systems in the Supplementary Materials.
Besides that I noticed some typos and suggest minor corrections;
-- Our reply: The text has been revised by two native English speakers in other to remove all typos and mistakes found in its previous version.
I suggest authors to change four digits after decimal to either two or three digits in Table 2.
-- Our reply: Table 2 has been altered according to this request.
Reference 47, line 449, year number is wrong.
-- Our reply: The year in reference 47 (now 48) has been corrected.
Reviewer 2 Report
Authors wrote a computational study: "Rocio virus encephalitis: In silico evidence for drug repurposing".
Introduction is well written and covered with proper references.
Page 3. Materials and Methods
Authors state: "being additionally filtered only for drugs available in the National List of Essential Medicines"
Why the analysis was restricted only to the National List of Essential Medicines?
Page 6.
"To investigate the ''druggability'' of selected targets, the subset covered only drugs deposited in the DrugBank approved by the FDA, containing 1657 molecules."
This is only repetiton from Materials and Methods and should be rephrased.
Page 7
"Four molecules were selected (simeprevir, daclatasvir, iloprost, and itraconazole)"
It is not very clear why only these 4 molecules were selected and the criteria that were used. Could authors provide a more detailed information?
Page 12
"This molecule demonstrated interactions with most of its proteins, which may point to an interesting multitarget drug"
It may, but it also may not. This conclusion should be changed and made more specific considering the nature of the interactions.
Author Response
Dear Reviewer,
We thank you for your time and consideration.
We have now addressed each question raised by the reviewers and the manuscript has been modified accordingly. We are sure that the recommended changes have enhanced our work.
Below you can find our replies to your comments.
Yours,
Rodrigo Ligabue Braun, DSc
------
Reviewer: 2
Comments:
"Authors wrote a computational study: "Rocio virus encephalitis: In silico evidence for drug repurposing".
Introduction is well written and covered with proper references."
Page 3. Materials and Methods
Authors state: "being additionally filtered only for drugs available in the National List of Essential Medicines"
Why the analysis was restricted only to the National List of Essential Medicines?
-- Our reply: We have further explained this rationale. The segment now reads “The selected subset contained only molecules approved by the Food and Drug Administration (FDA) and consisted of 1657 molecules, being additionally filtered only for drugs available in the National List of Essential Medicines (RENAME) of Brazilian Unified Health System (SUS), one of the widest public health care systems in the world [31], and of particular interest to the authors due to the potential immediate application of identified drugs in case of infection reemergence in the country.”.
Page 6.
"To investigate the ''druggability'' of selected targets, the subset covered only drugs deposited in the DrugBank approved by the FDA, containing 1657 molecules."
This is only repetition from Materials and Methods and should be rephrased.
-- Our reply: The segment has been rephrased as suggested. It now reads “The assessment of potential druggability of the selected targets was performed over a subset encompassing only FDA-approved drugs deposited in the DrugBank, containing 1657 molecules.”
Page 7
"Four molecules were selected (simeprevir, daclatasvir, iloprost, and itraconazole)"
It is not very clear why only these 4 molecules were selected and the criteria that were used. Could authors provide more detailed information?
--Our reply: We have added further information regarding selection criteria. This segment now reads: “Four molecules were selected (simeprevir, daclatasvir, iloprost, and itraconazole) based on the following criteria: best score, different drug classes, route of administration and possibility of use in specific groups (children, pregnant women and the elderly), in order to evaluate and screen on the DrugBank database considering the various factors as use, dosage, contraindications, and molecular aspects (Table 1).”
Page 12
"This molecule demonstrated interactions with most of its proteins, which may point to an interesting multitarget drug"
It may, but it also may not. This conclusion should be changed and made more specific considering the nature of the interactions.
-- Our reply: We have changed this Conclusion segment, in accordance with the reviewer’s comment. It now reads “This molecule demonstrated interactions with most of its proteins, which may point to an interesting multitarget drug. However, the binding profile with NS1, via hydrophobic and pi-pi stacking interactions, seem to be most favored in this protein, pointing to its greater potential as a target candidate.”.
