Discovery of Arylfuran and Carbohydrate Derivatives from the BraCoLi Library as Potential Zika Virus NS3^pro Inhibitors

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Serafim and coworkers reported “ Discovery of arylfuran and carbohydrate derivatives from the BraCoLi library as potential Zika virus NS3pro inhibitors.” In this manuscript, the authors employed a consensus docking protocol combining GOLD and DockThor scores to identify 28 potential inhibitors of NS3pro in ZIKV. Among these, three compounds were validated in vitro. After careful consideration, this manuscript needs major revision before publication in the Future Pharmacology Journal.

Comments:

1.     Authors have identified 28 potential inhibitors in their docking studies. However, only three compounds have been selected to test inhibition activities in vitro against NS3pro of Zika virus. The authors should clarify why they did not test all 28 compounds in vitro.

2.     They should mention in the abstract that they chose 3 compounds to test in vitro.

3.     BR020325’s Proton NMR is not matching with the structure. Authors should recheck their NMR spectrum and confirm whether they obtained the correct compound in their synthesis experiments.

4.     Authors should provide HRMS for the final compounds.

5.     The authors should provide NMR spectrums for all the compounds and HRMS spectrums for final compounds in the supporting information.

6.     The authors mentioned in line 590 that BR020113 presents a nitro group. However, I did not see any nitro group in the BR020113 structure. The authors should clarify this.

7.      There are some typos and grammatical-related errors in the manuscript.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

We thank the reviewer very much for taking the time to critically comment on our manuscript.

Comments:

1. Authors have identified 28 potential inhibitors in their docking studies. However, only three compounds have been selected to test inhibition activities in vitro against NS3pro of Zika virus. The authors should clarify why they did not test all 28 compounds in vitro.

We appreciate your recommendation. We have previously described the reason for the testing only three compounds in the results section. We have now reworked this part and further mentioned it in the Abstract. We hope that these changes will clarify the reasons to the readers.

2. They should mention in the abstract that they chose 3 compounds to test in vitro.

Thank you for your suggestion. We have added this information and the reason for the selection of the three compounds in the Abstract.

3. BR020325’s Proton NMR is not matching with the structure. Authors should recheck their NMR spectrum and confirm whether they obtained the correct compound in their synthesis experiments.

Thank you for pointing this out. We have reviewed the structure and confirmed it is correct and coherent with the NMR data. To facilitate analysis of these results, we included the expanded NMR spectra in the newly added Figures S3-S9. We hope that these changes have contributed to avoiding any misunderstanding with the compound structures.

4. Authors should provide HRMS for the final compounds.

We appreciate this recommendation. We have provided the complete NMR spectra of the two unpublished structures as the new Figures S3-S9. We have also added the HRMS for one of the compounds still available in the laboratory. With respect, we believe that based on the established synthesis routes regarding the two unpublished hits and having the expanded NMR of the structures would be enough for the characterization of the compounds, without changing the decision-making process of the hit results.

5. The authors should provide NMR spectrums for all the compounds and HRMS spectrums for final compounds in the supporting information.

We also do appreciate this recommendation. However, we respectfully disagree that performing NMR and MS of the other hit compounds from the docking will bring added value to the study, considering that only three compounds were available for testing, and therefore evaluated in the antiviral and enzymatic assays, validating the computational simulations. Nonetheless, regarding this and the previous comment, we have added the complete NMR spectra for the two unpublished structures and HRMS for the one still available.

6. The authors mentioned in line 590 that BR020113 presents a nitro group. However, I did not see any nitro group in the BR020113 structure. The authors should clarify this.

Thank you for pointing this out. We apologize for the confusion when mistakenly mentioning a nitro group twice in that paragraph. We have corrected the information regarding the presence of different nitrogen groups and the triazole in the hit compound. We hope that the changes have clarified the SAR discussion to the readers.

7. There are some typos and grammatical-related errors in the manuscript.

Thank you for highlighting this out. We have thoroughly reviewed the manuscript for any typos and English grammar and corrected them throughout the text. We hope that the updated version may be clearer to the readers.

Reviewer 2 Report

Comments and Suggestions for Authors

 

Zika virus (ZIKV) infection during pregnancy causes microcephaly in fetus, . ZIKV is known to associate with Guillain Barre syndrome. There are no effective vaccines and antivirals available. Authors conducted in silico screening by combining multiple approaches. These approach is considered as more promising measure compared to others.

 

1.     Line 59; Authors should use C. jejuni as example relating to GBS development in this sentence.

2.     Line 270 and others; SFB should read as FBS. Authors must check carefully.

3.     Line 273; “MEM 0% FBS” should be replaced as serum free MEM.

4.     Table S2; The selected 28 compounds should be indicated in bold so that readers can identify easily.

5.     Figure S1; Please provide the NS3 structure in the same angle with figure 1.

6.     Is it possible to argue that less anti viral activities of the tested compounds were due to difference of the viral strain using in silico screening with the strain used in the in vitro evaluation?

7.     Methods; Please provide the details to obtain NS2B-3 utilized in the protease activity inhibition assay.

Author Response

1. Line 59; Authors should use C. jejuni as example relating to GBS development in this sentence.

We appreciate the recommendation. Campylobacter jejuni was also cited as an example of a causative agent associated with GBS.

2. Line 270 and others; SFB should read as FBS. Authors must check carefully.

Thank you for pointing this typo. We have reviewed the manuscript and corrected all FBS typos.

3. Line 273; “MEM 0% FBS” should be replaced as serum free MEM.

We appreciate the suggestion and reworked “serum-free MEM” in the text.

4. Table S2; The selected 28 compounds should be indicated in bold so that readers can identify easily.

Table S2 had all selected compounds already written in bold. Nevertheless, we have increased the font size and hope that this improved readability.

5. Figure S1; Please provide the NS3 structure in the same angle with figure 1.

We have reworked Figure S1 with the protease structure in the same angle as Figure 1, as suggested. We have also reworked Figure 2 to the same angle. Thank you for your recommendation.

6. Is it possible to argue that less anti viral activities of the tested compounds were due to difference of the viral strain using in silico screening with the strain used in the in vitro evaluation?

Arguably, considering potential substitutions in the active site, especially near the catalytic triad (e.g., residues known for inhibitors’ binding), that possibility could be suggested. However, in our analysis, there is a high identity (> 98%) among the three NS3pro sequences used in the study (docking, antiviral, and enzymatic), having no notable substitutions that would impact binding to the active site. In support of this notion, we have included a sequence alignment (Figure S9) showing the conservation of the target sequences.

7. Methods; Please provide the details to obtain NS2B-3 utilized in the protease activity inhibition assay.

Thank you for your recommendation. We have included a detailed description of the ZIKV NS3pro expression and purification in the main text.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed all of my previous comments, leading to significant improvements in the manuscript. Additionally, in response to one of my comments, they have corrected the HNMR spectra of BR020325. However, they have not provided any explanation for the discrepancy in the data for the tested compound in the manuscript. This raises a crucial question: which compound was actually used for the biological evaluation—the one corresponding to the old HNMR data or the new HNMR data? The authors must clarify this.

Author Response

The authors have addressed all of my previous comments, leading to significant improvements in the manuscript. Additionally, in response to one of my comments, they have corrected the HNMR spectra of BR020325. However, they have not provided any explanation for the discrepancy in the data for the tested compound in the manuscript. This raises a crucial question: which compound was actually used for the biological evaluation—the one corresponding to the old HNMR data or the new HNMR data? The authors must clarify this.

We sincerely appreciate the reviewer taking time to review the revised version of the manuscript. We also appreciate the last comment regarding the NMR. To this regard, the compound that was tested is the same as before. What changed was the spectrometer that was used for the analysis. The spectrometer used in the first spectrum was a 200 MHz one, and the one used in this last one was a 600 MHz one. The conditions for making the spectrum (including the solvent used, CDCl₃) were the same, including the same original sample. The resolution of this spectrometer is now much higher, and the signals are better defined, which justifies the changes indicated in the text. In support of this notion, the expanded spectra was previously added in the supporting information. We hope that this has clarified the commentary. Thank you very much.

Reviewer 2 Report

Comments and Suggestions for Authors

Authors revised this manuscript accordingly. Thus it is now acceptable for publication in Future Pharmacology.

Author Response

We appreciate the reviewer reply and feedback. Thank you.