Identification of Filovirus Entry Inhibitors from Marine Fungus-Derived Indole Alkaloids
, Michael R. Holbrook 3, Wen-Jian Lan 5,* and Shi-Hua Xiang 1,*Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The study shows the antiviral activities of three marine indole alkaloids against filovirus infections such as EBOV and MARV. Also, the assay was supported by molecular docking and structural analysis. However, for the manuscript to be accepted, the following major revisions are necessary.
1. Lines 305-336 of the conclusion are identical to lines 190-230 of discussion. Please revise to avoid repetition and ensure the conclusion adds unique value to the manuscript.
2. The study claims that the three indole alkaloids exhibit ‘strong’ antiviral effects against the viruses. However, W12, W26 and W27 seem significantly weaker compared to remdesivir. Can they be considered 'strong'?
3. In '2.5. Inhibition specificity analysis', why was W27 excluded for the analysis?
4. The resolution of the figures is low and needs to be improved. Also, in Fig. 8 and Fig. 9, please remove the red underline below ‘Trp’.
5. In the legend of Fig. 7, ‘W26’ should be added.
6. In Table 1., the compounds are W, not WJL. Please revise the explanation 'WJL(or W)'. Also, why is there no data on the activity of N-Ac-l- and d-tryptophan against MARV?
7. There are some errors in the text. Please correct.
- The letters 'L' and 'D' used to indicate configuration should be written in small capitals (l and d)
- Lines 199, 235, 305. The scientific names should be written in italics.
- In the legend of Fig. 3, CC50 should be revised to CC50.
- In the supporting information, there are two structures in W11.
Author Response
To Reviewer_1
1. Lines 305-336 of the conclusion are identical to lines 190-230 of discussion. Please revise to avoid repetition and ensure the conclusion adds unique value to the manuscript.
We delete the repeated Discussion section.
2. The study claims that the three indole alkaloids exhibit ‘strong’ antiviral effects against the viruses. However, W12, W26 and W27 seem significantly weaker compared to remdesivir. Can they be considered 'strong'?
Yes, we change the wording strong to modest.
3. In '2.5. Inhibition specificity analysis', why was W27 excluded for the analysis?
Yes, we only chosen two (W12 and W26) for testing which showed better inhibition and less toxicity.
4. The resolution of the figures is low and needs to be improved.
Yes, we remade the blurry figures in Fig. 6.
Also, in Fig. 8 and Fig. 9, please remove the red underline below ‘Trp’.
Yes, we did.
5. In the legend of Fig. 7, ‘W26’ should be added.
corrected
6. In Table 1., the compounds are W, not WJL. Please revise the explanation 'WJL(or W)'.
Yes, corrected.
Also, why is there no data on the activity of N-Ac-l- and d-tryptophan against MARV?
These IC50 values were estimated based on the preliminary tests since the activities are weak and the tested concentrations were not concentrated enough to determine the IC50s.
7. There are some errors in the text. Please correct.- The letters 'L' and 'D' used to indicate configuration should be written in small capitals (l and d).
corrected
- Lines 199, 235, 305. The scientific names should be written in italics.
Yes, we changed.
- In the legend of Fig. 3, CC50 should be revised to CC50.
Corrected
- In the supporting information, there are two structures in W11.
Corrected.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
The authors tested 31 alkaloid-like compounds from a marine fungus for activity against two filoviruses, Ebola and MARV. They performed tests with pseudotyped and infectious viruses, as well as cytotoxicity assays. They identified three compounds capable of inhibiting the viruses without causing cytotoxicity in human cells.
Although the work presents interesting results, I believe it should be revised before being accepted for publication.
1. The conclusions section is more aligned with the discussion, and point 3 of the discussion is entirely repeated in point 5.
2. The discussion of the results is limited and unclear. Which receptor shows a 70% difference? Later, they mention that paradoxically the NPC1 is conserved, so where is the difference they are referring to? How does this affect the results?
3. The authors change the nomenclature of the compounds. Initially, they are referred to by number, and later as W or WJL followed by the number. Please standardize the naming convention.
4. Although the authors mention that the compounds were provided by Dr. Wen-Jian Lan, they should include a citation with the source of the compounds and details on their extraction and characterization.
5. The methodology is not sufficiently detailed. For example: a. Point 4.1 refers to the compounds used (as mentioned in the previous point). b. Point 4.4: Where is the Integrated Research Facility of NIH at Fort Detrick, MD located, and how did they determine toxicity? c. Point 4.6: The receptor used for docking is not specified. How did they define the grid for the active site? d. Point 4.8: What statistical methods were used? How many replicates were performed in the experiments?
6. The authors mention docking scores to refer to affinity energies. However, each docking program typically has its own methodology for scoring poses, which is different from energy values. Additionally, they report values for tryptophan as a control. Where do these values come from? If the experiments were conducted, why are the results not included, and only the values appear in the tables?
7. Many of the figures are blurry.
Others:
L36-41 Why is it essential to search for small molecules, it seems that only size determines urgency, the vaccine and approved antibodies do not work?
Figure 1 caption: concertation for concentration
L85 lack of space 10um
Figure 9 legend, wording and citation. docking scores in kcal/mol…
Fig 10 legend space pH6.1
Comments on the Quality of English Language
There are some confusing sections and the wording needs to be revised.
Author Response
To Reviewr_2
1. The conclusions section is more aligned with the discussion, and point 3 of the discussion is entirely repeated in point 5.
We deleted the repeated Discussion section-5.
2. The discussion of the results is limited and unclear. Paradoxically the NPC1 is conserved, so where is the difference they are referring to? How does this affect the results?
Although so different in their sequence both EBOV and MARV can share the same receptor NPC1 for entry which implicates that the binding sites are similar structurally. Thus, the same compound showed difference in their neutralization activity which must be due to sequence differences in their binding sites.
3. The authors change the nomenclature of the compounds. Initially, they are referred to by number, and later as W or WJL followed by the number. Please standardize the naming convention.
Yes, we corrected and Just use W named compounds.
4. Although the authors mention that the compounds were provided by Dr. Wen-Jian Lan, they should include a citation with the source of the compounds and details on their extraction and characterization.
We have added the source of the compounds, and the compound extraction information can be found in their sources.
5. The methodology is not sufficiently detailed. For example: a. Point 4.1 refers to the compounds used (as mentioned in the previous point). The compounds listed was in the Supplementary table 1.
The information was corrected and Table _S1 was fixed.
6. Point 4.4: Where is the Integrated Research Facility of NIH at Fort Detrick, MD located.
The detailed address: 8200 Research Plaza, Ft. Detrick, Frederick, MD, US, 2170
We added it in the method section.
how did they determine toxicity?
This is the answer from the Co-authors in NIH: In the case of the W compounds, toxicity at each drug concentration was determined by cell loss compared to the infected control. We simply counted Hoechst-stained nuclei and compared them. There were 4 replicates per plate at each condition plus 24 infected, untreated controls and 24 uninfected, untreated controls. Plates were scored by Z’ as a check of assay fidelity. I believe the MARV plate scored 0.80 and EBOV was 0.58, both of which are good indications of an acceptable assay window and standard deviation.
We also added the information in the method section.
7. Point 4.6: The receptor used for docking is not specified. How did they define the grid for the active site?
We have revised Section 4.6 to clarify details about the receptor and the method used for defining grid for the active site.
8. Point 4.8: What statistical methods were used? How many replicates were performed in the experiments?
In GraphPad Prism, the statistical method used to calculate IC50 is nonlinear regression. The data was from three experiments and each sample was in triplicates.
9. The authors mention docking scores to refer to affinity energies. However, each docking program typically has its own methodology for scoring poses, which is different from energy values.
The docking scores reported were obtained using Autodock Vina, which empirically weights superficially physics-based terms (e.g., the 6–12 van der Waals interactions and Coulomb energies) to account for the difference between energies and free energies. For clarification, we have added more details in Section 4.6.
Additionally, they report values for tryptophan as a control. Where do these values come from? If the experiments were conducted, why are the results not included, and only the values appear in the tables?
These IC50 values were estimated based on the preliminary tests since the activities are weak and the tested concentrations were not concentrated enough to determine the IC50s.
10. Many of the figures are blurry.
We remade the figures which are much clear now.
Others:
L36-41 Why is it essential to search for small molecules, it seems that only size determines urgency, the vaccine and approved antibodies do not work?
The vaccine only restricted used for 12 older persons, the antibody regimes are consisted of two or three antibodies. These biologics-based medicine are heat-sensitive and difficult for distributions in the Arica countries. The small molecules-based drugs are required for use in the lower economy countries,
Figure 1 caption: concertation for concentration.
corrected
L85 lack of space 10um.
corrected
Figure 9 legend, wording and citation. docking scores in kcal/mol…
Checked and corrected.
Fig 10 legend space pH6.1.
corrected
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors
Filovirus especially the genera of Ebolavirus and Marburgvirus, can infect humans and cause severe illness. The manuscript found three marine indole alkaloids had anti-filovirus potential and the result would be helpful for the development of antiviral drugs. The manuscipt is suitable for the journal Marine Drugs and could be accept after minor revisions.
1 Please provoid the chemical name of compounds W12, W26 and W27.
2 Line 99. Supply the positive control data against pseudotyped filoviruses.
Author Response
To Reviewer_3
1 Please provide the chemical name of compounds W12, W26 and W27.
Their chemical names are:
W12 (Fumiquinazoline J), W26 (Emindole SB) and W27 (Fusaindoterpene B)
We added the chemical names in the Figure 8.
2 Line 99. Supply the positive control data against pseudotyped filoviruses.
For this pseudotyped virus experiment, which is tested on viral entry, the virus only sample is the positive control and cells only sample is the negative control. This platform has been established in the lab for several years to test viral entry inhibitors. For testing small molecule compounds, a paper was published in Antiviral Research 189 (2021) 105059.
For infectious virus inhibition, it is associated with viral replication beside viral entry, adding the remdesivir as a positive control which can test the new platform and also test the compound’s role in inhibiting viral replication as the remdesivir is RdRp (RNA-dependent RNA polymerase) inhibitor.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
The author stated in the response to the report that the revisions were made, but they have not been updated. Please make the necessary revisions.
For example,
2. The study claims that the three indole alkaloids exhibit ‘strong’ antiviral effects against the viruses. However, W12, W26 and W27 seem significantly weaker compared to remdesivir. Can they be considered 'strong'?
Yes, we change the wording strong to modest.
Also, in Fig. 8 and Fig. 9, please remove the red underline below ‘Trp’.
Yes, we did.
7. There are some errors in the text. Please correct
- The scientific names should be written in italics.
Yes, we changed.
- In the supporting information, there are two structures in W11.
Corrected.
Author Response
To Reviewer _2_v2
Sorry for some missed up changes, here we corrected.
2. The study claims that the three indole alkaloids exhibit ‘strong’ antiviral effects against the viruses. However, W12, W26 and W27 seem significantly weaker compared to remdesivir. Can they be considered 'strong'?
Yes, we have changed the wording from strong to modest.
Also, in Fig. 8 and Fig. 9, please remove the red underline below ‘Trp’.
Yes, we did, which were spelling check signs.
7. There are some errors in the text. Please correct
- The scientific names should be written in italics.
Yes, we have checked and changed.
- In the supporting information, there are two structures in W11.
Deleted the unwanted.
Round 3
Reviewer 1 Report
Comments and Suggestions for Authors
Most of the issues noted in the previous review seem to have been addressed, and there are no serious disqualifiers for this report to be published.
