Dichlororesorcinols Produced by a Rhizospheric Fungi of Panax notoginseng as Potential ERK2 Inhibitors
Round 1
Reviewer 1 Report
The following major revisions are required:1- The GenBank accession number provided by authors does not belong to the named fungal strain in the study. For that please visit the following URL (https://www.ncbi.nlm.nih.gov/nuccore/JX076958.1).
2- Actually the fungus must be identified by beta tubulin primer because it is very risky to identify this genus using ITS-primer.
3- Phylogenetic trees must be generated to confirm identification and taxonomy of the fungus.
4-It is known that ECD is a tentative tool to determine AC. Several isolated metabolites contain a secondary hydroxyl group and Mosher’s derivatization must be carried out.
5-ECD curves are not smooth and sceptical and chiral HPLC must be provided for all chiral metabolites.
6- Cartesian coordinates for ECD must be also provided.
7- It is not OK for natural product chemists to mention doublets in NMR tables without coupling constants.
8- Extensive English editing is highly needed with a certificate.
9- All dose response curves of bioassay must be provided in supporting information.
10- Safety index of isolated compounds must be evaluated by comparing their cytotoxicity on normal cells.
11- Detailed supporting information containing all spectroscopic data must be provided.
8- Extensive English editing is highly needed with a certificate.
Author Response
Point-to-Point Responses to Reviewers’ Comments:
Reviewer #1:
- Comment 1: The GenBank accession number provided by authors does not belong to the named fungal strain in the study. For that please visit the following URL (https://www.ncbi.nlm.nih.gov/nuccore/JX076958.1).
Response: We are sorry for our mistake. The correct accession number is OQ945315. We have made modification to the content of the manuscript in the part of “2.2. Fungal Material and Fermentation”. Please check them in page 6 line 116.
- Comment 2:Actually the fungus must be identified by beta tubulin primer because it is very risky to identify this genus using ITS-primer.
Response: Thank you for your suggestion. Our manuscript is centered on the separation, identification, and activity research of secondary metabolites. We did not examine the strain’s taxonomy, thus we compared the sequence similarity (ITSI-5.8S-ITS2-28S) with valid published strains, and found the strain SYP-F6997 had the highest similarity (99.24 %) with Chaetomium udagawae. This part has been revised in the “2.2. Fungal Material and Fermentation” section. We appreciate your understanding.
- Comment 3: Phylogenetic trees must be generated to confirm identification and taxonomy of the fungus.
Response: Thank you for your suggestion. Our manuscript does not focus on the taxonomy of the strain. As mentioned in question 2, we found that the ITS sequence of SYP-F6997 has the highest similarity (99.24%) with Chaetomium udagawae. We have also added the morphological characteristics of the strain, which can be found in the supporting documents. The colony grown on PDA medium is mainly grayish brown, with edges gradually turning white, and the mycelium is fluffy, with transparent leachate produced (Figure S 1A). The back is black brown, the edge is white, and there are radioactive wheel patterns emanating from the middle to the surrounding area (Figure S 1B). Observing its microstructure through an optical microscope, as shown in Figures S 1C and 1D. The mycelium is colorless, with a smoothly surface and mainly unbranched; The sporopodium has a septum, with terminal or lateral spores, and ascospores are round to oval, brown, smooth, without septum, about 9.7-20.2 μm. We appreciate your understanding.
- Comment 4: It is known that ECD is a tentative tool to determine AC. Several isolated metabolites contain a secondary hydroxyl group and Mosher’s derivatization must be carried out.
Response: Thank you very much for your valuable advice. However, I am sorry to say that the activity tests and chiral separation experiments have basically exhausted all of the compound 5, leaving insufficient material for the Mosher test. Therefore, under these circumstances, we calculated the ECD and optical rotation, although this method may not be as precise as Mosher's response. We hope that our perspective will be understood.
- Comment 5：ECD curves are not smooth and sceptical and chiral HPLC must be provided for all chiral metabolites.
Response: Thank you for your kind suggestions. These suggestions are very useful and help us to identify errors in the original annotation of Figure 3A and Figure 3B, where the colors of the calculated curve and the experimental curve were reversed. We have corrected the non-smooth curves as experimental data and the smooth curves as computational data. The newly revised figure and legends are shown below. Additionally, we have included all the chiral HPLC chromatograms for new chiral metabolites, except for compound 3, which could not be separated by our current chiral HPLC. We appreciate your understanding.
- Comment 6: Cartesian coordinates for ECD must be also provided.
Response: Thank you for your kind suggestions. We have added ordinate units in the ECD results in Figure 3A and Figure 3B.
- Comment 7: It is not OK for natural product chemists to mention doublets in NMR tables without coupling constants.
Response: Thank you for your valuable advice. We have added the coupling constants in Table 1.
- Comment 8:Extensive English editing is highly needed with a certificate.
Response: Thank you for your valuable advice. We have engaged a company to polish and edit our revised manuscript, and have received a certificate as shown below. Please check them in the “Revised Manuscript with Changes Marked”.
- Comment 9: All dose response curves of bioassay must be provided in supporting information.
Response: Thank you for your valuable advice. Our collaborative lab at Yale University commissioned the activity test of our compounds, and we received the IC50 directly without dose curves. The positive control was effective, and the results were credible. We have expressed our gratitude to Prof. Yung-Chi Cheng by adding acknowledgments on Page 20, Line 417, please check it.
- Comment 10: Safety index of isolated compounds must be evaluated by comparing their cytotoxicity on normal cells.
Response: Thank you for your valuable advice. In this study, we solely assessed the cytotoxicity of the isolated compounds to identify potential bioactivities, without considering safety concerns as drug development was not our focus. However, if future research reveals medicinal properties of a compound, we will investigate its biosafety. We have revised the topic headings in Table 3 to reflect the activity results and included relevant remarks. We appreciate your comprehension.
- Comment 11: Detailed supporting information containing all spectroscopic data must be provided.
Response: Thank you for your valuable advice. We have included spectroscopic data for the majority of compounds in the Supporting files. Unfortunately, due to a damaged computer hard disk and some lost information, a small portion of the data was lost. However, this does not impact the overall article. Please refer to the Supporting file for the available data. We apologize for any inconvenience this may cause.
Author Response File: 
Author Response.docx
Reviewer 2 Report
In the manuscript the authors describe new compounds isolated from a fungus and their biological activity test results. The the effort invested in obtaining the results is clear.
With the provided data alone I am unable to verify the constitution of 1, as positions 1 and 4 are interchangable. I see why the authors use Cosmochlorin D to justify the suggested constitution, but I miss the HMBC correlation from the Proton attached to 7 to the carbons 3 and 5. Actually this also is a problem within the citet literature for Cosmochlorin D. It would be nice to include some more information for the choice of this constitution.
To facilitate future use of the authors work, I suggest that the structure are provided in NMReDATA format, that will contain all the assignment information.
Minor language improvements should be made, example line 317.
Author Response
Point-to-Point Responses to Reviewers’ Comments:
Reviewer #2:
- Comment 1: With the provided data alone I am unable to verify the constitution of 1, as positions 1 and 4 are interchangable. I see why the authors use Cosmochlorin D to justify the suggested constitution, but I miss the HMBC correlation from the Proton attached to 7 to the carbons 3 and 5. Actually this also is a problem within the cited literature for Cosmochlorin D. It would be nice to include some more information for the choice of this constitution.
Response: Thank you for your valuable advice. We have added H-H COSY information to verify the constitution of 1 for the positions 1 and 4. In the spectrum of H-H COSY (as below), there had been correlations between H-1 (δH 6.84) and 2,6-OCH3(δH 3.90 d) which confirmed the side chain is not connected at the position of 1 but the position of 4. We have also included the results of the H-HCOSY experiment for compound 1 in the revised manuscript on Page 12, Line 241, please check it.
- Comment 2: To facilitate future use of the authors work, I suggest that the structures are provided in NMReDATA format, that will contain all the assignment information.
Response: Thank you for your valuable advice. Due to computer hard disk damage and some original NMR files being corrupted, we added parts of the NMR DATA format using professional Topspin software in the supporting documents. This does not affect the overall content of the article. We are sorry for the inconvenience. Please check them in the Supporting file.
Author Response File: Author Response.docx
Reviewer 3 Report
1- The full spectral chromatograms are needed for each new compound and at least proton and carbon for the known compounds.
2- The stereodescriptors are not shown in the drawn structures (Figures 1 and 2) especially, the author claims the use of chiral column to isolate the enantiomer of 4 and 5.
3- Details on the positive control in testing are needed.
4- The hypothesis of biosynthetic pathway of compounds 1-6 can be moved to the supporting information and could just be highlighted in the text.
5- In table 3, the activity measuring unit needs to be mentioned
6- The compounds are showing very weak activity compared to the standard and there is no measured activity towards ERK2 but rather molecular docking studies.
7- Figure 5 is of low quality and some symbols cannot be read.
Moderate editing of English language
Author Response
Point-to-Point Responses to Reviewers’ Comments:
Reviewer #3:
- Comment 1: The full spectral chromatograms are needed for each new compound and at least proton and carbon for the known compounds.
Response: Thank you for your valuable advice. We have added the spectroscopic data for most of the compounds 1-10 in Supporting file. Due to computer hard disk damage and some information lost, a little part of the data has not been added, which does not affect the whole article. Please check them in the Supporting file. We are sorry for the inconvenience.
- Comment 2: The stereodescriptors are not shown in the drawn structures (Figures 1 and 2) especially, the author claims the use of chiral column to isolate the enantiomer of 4 and 5.
Response: Thank you for your valuable advice. We added stereo descriptors in Figures 1 and 2. Please check them in Figures 1 and 2.
- Comment 3: Details on the positive control in testing are needed.
Response: Thank you for your valuable advice. We have added the details on the positive control and relevant informations marked in red in Table 3. Please check it.
- Comment 4: The hypothesis of biosynthetic pathway of compounds 1-6 can be moved to the supporting informationand could just be highlighted in the text.
Response: Thank you for your valuable advice. We have moved the hypothesis of biosynthetic pathway of compounds 1-6 to the supporting information, and highlighted in the text on Page 18, Line 381, please check it.
- Comment 5: In table 3, the activity measuring unit needs to be mentioned.
Response: Thank you for your valuable advice. We have added the details in Table 3. Please check it.
- Comment 6: The compounds are showing very weak activity compared to the standard and there is no measured activity towards ERK2 but rather molecular docking studies.
Response: Thank you for your comments. New compounds 1 and 2 showed cytotoxicities to H9 and CEM with IC50 lower than 10 μM. Although the activities are not strong compared with the positive control, new compounds 1 and 2 contain special Cl element, providing reference data for subsequent researchers to carry out structural modification to find more active compounds. ERK is a popular target in current research, we used molecular docking to try the possible mode of action initially, hoping to provide some reference research for subsequent research.
- Comment 7: Figure 5 is of low quality and some symbols cannot be read.
Response: Thank you for your valuable advice. We enlarged the font and adjusted the position as much as possible to make more symbols visible in Figure 5, also improved the quality of Figure 5, please check it. We hope to get your approval.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
I accept the manuscript in the present form to be published in fermentation
