Novel Marine Fungus-Derived Mycophenolic Acids That Inhibit Acute Myeloid Leukemia Cell Proliferation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article reports isolation and identification of nine previously-undescribed mycophenolic acid derivatives from a marine-derived fungus. Two first time from nature and five known compounds were also isolated. Those compounds were evaluated their biological activities, revealing that some of them had potent inhibition activity against acute myeloid leukemia cells.  The methods of structure elucidation include standard spectroscopic techniques, 1D/2D NMR, HRESIMS, ECD spectra, which were appropriately utilized and described. Therefore, the reviewer would recommend publication in the journal, under conditions that the authors consider the following minor points.
(1) In Abstract, there is a line about ORD spectra, but discussion about stereochemistry in the manuscript is all based on ECD spectra. Do you need ORD?
(2) In line 43 on page 2, "withe" should be "white". The same errors are found in lines 78, 89, and 101.
(3) In lines 70 on page 3, the sentence "The latter showed...for1." is not clear to the reviewer. What the latter and high similarity mean are not obvious. Please improve. The same ambiguities are found in lines 98 and 107.
(4) In line 80 on page 4, "was" should be "be".
(5) In lines 126-128 on page 5, HMBC correlations are not precisely described. Please be helpful for readers.
(6) The format of Table 2 is not complete. 13C should be well-shaped. Proton chemical shifts for C1' of compound 6 can be well aligned (not with two-columns). "Recorded in CDCl3" can put in a better place.
(7) In line 134 on page 6, "as a yellow oil" should be "as yellow oil". The same errors are found in lines 196 and 202.
(8) Figure 5 on page 6 should have plots for 5 and 6 on left and those for 8 on right.
(9) In line 155 on page 7, important HMBC correlations are not only H-8' to C-6'. Please be more kind for readers.
(10) In line 177 on page 8, "cotton" should be "Cotton" as this effect was originally reported by Cotton.
(11) In line 190 on page 8, for HMBC correlation, don't you need C-3' to support the elucidation?
(12) In line 272 and 284 on page 10, readers would like to know how the authors obtained mixtures S7 and S15. There is no solid explanation for their origins.
(13) In line 400 on page 13, do you need ORD?

Author Response

Point-by-point responses to the reviewers’ comments

Reviewer 1

1. 1. In Abstract, there is a line about ORD spectra, but discussion about stereochemistry in the manuscript is all based on ECD spectra. Do you need ORD?

Author response: We thank the reviewer for the comment. In lines 192 and 194 on page 9, in the Stereostructural determination of compound 8, ORD spectra were supplemented to further corroborate the stereoconformation.

2. 2. In line 43 on page 2, “withe” should be “white”. The same errors are found in lines 78, 89, and 101.

Author response: Thank you and the typo error has been corrected accordingly.

3. 3. In lines 70 on page 3, the sentence “The latter showed...for1.” is not clear to the reviewer. What the latter and high similarity mean are not obvious. Please improve. The same ambiguities are found in lines 98 and 107.

Author response: Thank you for your suggestion. The corresponding sections in the manuscript have been revised accordingly.

The added content is as follows in lines 84 and 85: The experimental ECD curve of 1 showed a higher similarity to the calculated ECD curve of (2'R, 3'R)-1 (Figure 4).

The added content is as follows in lines 98 and 99: The experimental ECD curve of 2 showed a higher similarity to the calculated ECD curve of (2'S, 3'S)-2.

The added content is as follows in lines 113 and 114: The experimental ECD curve of 3 showed a higher similarity to the calculated ECD curve of (2'S, 3'R)-3 (Figure 4).

The added content is as follows in lines 122 and 123: The experimental ECD curve of 4 showed a higher similarity to the calculated ECD curve of (2'R, 3'S)-4.

4. 4. In line 80 on page 4, “was” should be “be”.

Author response: Thank you and this error has been corrected accordingly.

5. 5. In lines 126-128 on page 5, HMBC correlations are not precisely described. Please be helpful for readers.

Author response: Thank you for your suggestion. The corresponding sections in the manuscript have been revised accordingly. The added content is as follows: “As confirmed by the HMBC correlations from H-7' to C-4', C-3' and C-2', from H-1' to C-6 and C-7, from H-8' to C-6', from H-5' to C-6', from 5-OMe to C-5, from H-8 to C-3a, C-4, and C-5, and from H-3 to C-1, C-3a, C-4 and C-7a.”

6. 6. The format of Table 2 is not complete. ¹³C should be well-shaped. Proton chemical shifts for C1' of compound 6 can be well aligned (not with two-columns). “Recorded in CDCl₃” can put in a better place.

Author response: Thank you for your suggestion. The corresponding sections in the manuscript have been revised accordingly.

7. 7. In line 134 on page 6, “as a yellow oil” should be “as yellow oil”. The same errors are found in lines 196 and 202.

Author response: Thank you for your suggestion. The corresponding sections in the manuscript have been revised accordingly.

8. 8. Figure 5 on page 6 should have plots for 5 and 6 on left and those for 8 on right.

Author response: Thank you for your suggestion. The corresponding sections in the manuscript have been revised accordingly.

9. 9. In line 155 on page 7, important HMBC correlations are not only H-8' to C-6'. Please be more kind for readers.

Author response: Thank you for your suggestion. The corresponding sections in the manuscript have been revised accordingly. The added content is as follows: “Additionally, the key HMBC correlations from H-5' to C-6', from H-4' to C-2', from H-7' to C-2' and C-4', from H-1' to C-3', C-5, C-6 and C-7, from 5-OMe to C-5, from H-8 to C-3a and C-4, and from H-3 to C-1, C-3a, C-4 and C-7a further supported the planar structure of 7 (Figure 1), which was elucidated as 4′S-hydroxy-6′-ethoxy mycophenolic acid and named penicacid U.”

10. 10. In line 177 on page 8, “cotton” should be “Cotton” as this effect was originally reported by Cotton.

Author response: Thank you and this error has been corrected accordingly.

11. 11. In line 190 on page 8, for HMBC correlation, don't you need C-3' to support the elucidation?.

Author response: Thank you for your suggestion. The corresponding sections in the manuscript have been revised accordingly. The added content is as follows: “This shift pattern implies that the methyl acetate group in compound 8 may have been repositioned as confirmed by the HMBC correlations from H-7' to C-9', C-4', C-3' and C-2'. Additionally, the key HMBC correlations from H-10' to C-9', from H-8' to C-6', from H-5' to C-6', from H-4' to C-2', from H-1' to C-3', C-5, C-6 and C-7, from 5-OMe to C-5, from H-8 to C-3a and C-4, and from H-3 to C-1, C-3a, C-4 and C-7a further supported the planar structure of 9.”

12. 12. In line 272 and 284 on page 10, readers would like to know how the authors obtained mixtures S7 and S15. There is no solid explanation for their origins.

Author response: We thank you for your comment. The corresponding sections in the manuscript have been revised accordingly.

The added content is as follows in lines 292-294: “Gradient elution of the de-oiled crude was subjected to silica gel column chromatography, and five fractions (Fr. 1–Fr. 5) were obtained. Fraction Fr. 1 (20.5 g) was subjected to MPLC with MeOH and H2O (2:8–1:0, v/v, per 2.0 L). Subfraction Fr. 1-1 (633.0 mg) was isolated and purified by preparative HPLC with a Titank C18 column (55% ACN in H2O, v/v, 2 mL/min) to give compound 12 (3.1 mg, tR = 31 min) and mixture S7.”

The added content is as follows in lines 301-305: “Subfraction Fr. 3-7 was isolated by preparative HPLC with a Titank C18 column (50% ACN in H2O, v/v, 2 mL/min) to give compound 7 (12.4 mg, t_R = 16 min), compound 8 (10.2 mg, tR = 22 min), compound 13 (11.7 mg, t_R = 14 min), compound 14 (9.9 mg, t_R = 27 min), compound 11 (4.6 mg, t_R = 17 min), compound 10 (3.4 mg, t_R = 24 min) and mixture S15.”

13. 13. In line 400 on page 13, do you need ORD?

Author response: We thank you for your comment. In lines 192 and 193 on page 9, in the Stereostructural determination of compound 8, ORD spectra were supplemented to further corroborate the stereoconformation.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Deng et al. evaluated the inhibitory effects of mycophenolic acids derived from Penicillium senticosum RCDB005 on the proliferation of acute myeloid leukemia cells.

I have a few comments for polishing the manuscript.

1. The authors should emphasize the significance of marine-derived compounds as effective antitumor agents in the Introduction Section, followed by recent evidence regarding various bioactive compounds derived from marine fungi as effective anti-tumor effects.

https://doi.org/10.3390/ijtm4040051 

https://doi.org/10.1007/s12032-024-02594-0

https://doi.org/10.1007/s13659-024-00493-5

2. The biological activity of the compound should be elaborately evaluated. CCK-8 assay alone cannot determine the proper biological activity.  The authors should perform MTT/CyQuant Assay to demonstrate the biological activity further.
3. The authors should investigate whether the compound exhibits any activity related to reactive oxygen species (ROS).
4. The authors should explore the global pathways affected by mycophenolic acid. RNA sequencing (transcriptome analysis) and/or mass spectrometry (proteomics) should be performed to assess the compound’s impact on cellular pathways.
5. The authors should add a graphical abstract for the broader audience.
6. The authors should state the major limitations associated with the study.

Author Response

Point-by-point responses to the reviewers’ comments

Reviewer 2

1. The authors should emphasize the significance of marine-derived compounds as effective antitumor agents in the Introduction Section, followed by recent evidence regarding various bioactive compounds derived from marine fungi as effective anti-tumor effects.

Author response: We thank you for your comments. The corresponding sections in the manuscript have been revised accordingly. The added content is as follows: “With great advantages of unique ecological environments, powerful gene clusters, and high yields of secondary metabolites, marine-derived fungi represent a gigantic and untapped reservoir for the exploration of novel bioactive marine natural products (MNPs). They offer immense potential in cancer treatment due to their diverse mechanisms of action, such as cytotoxic, antiproliferative, and immunomodulatory properties [1]. As a representative example, fucoxanthin (C₄₂H₅₈O₆), found in brown algae such as Undaria pinnatifida, reduces leukemia and breast cancer cell growth [2]. Similarly, highly oxygenated polyketides, aspergilsmins A-G were isolated from Aspergillus giganteus NTU967 obtained from Ulva lactuca. Among them, aspergilsmin C and patulin exhibited sound inhibitory effects on prostate cancer PC-3 cells and human hepatocellular carcinoma SK-Hep-1 cells with IC₅₀ values ranging from 2.7 to 7.3 μM [3]. Importantly, marine-derived compounds have shown a broad spectrum of anticancer activities including some that are resistant to conventional chemotherapy. These compounds offer a unique and largely untapped source of chemical diversity that can be exploited in the search for new and effective cancer treatments. This has led to a growing body of research focused on the discovery and development of marine-derived anticancer drugs, with many compounds undergoing preclinical and clinical evaluation [4].”

2. The biological activity of the compound should be elaborately evaluated. CCK-8 assay alone cannot determine the proper biological activity. The authors should perform MTT/CyQuant Assay to demonstrate the biological activity further.

Author response: Thank you for your suggestion. The CCK-8 assay is a widely used colorimetric method for evaluating the cytotoxic effects of anticancer drugs on tumor cells and is suitable for high-throughput screening of large compound libraries. Its reliability has been fully confirmed. The following are reference papers published in Marine Drugs that used the Cell Counting Kit-8 (CCK8) assay. https://doi.org/10.3390/md13106064

3. The authors should investigate whether the compound exhibits any activity related to reactive oxygen species (ROS).

Author response: Thank you very much for your advice. The current structure data for these tested compounds does not clearly suggest that their antitumor efficacy is related to a ROS-dependent mechanism. We will investigate this potential role further in future studies.

4. The authors should explore the global pathways affected by mycophenolic acid. RNA sequencing (transcriptome analysis) and/or mass spectrometry (proteomics) should be performed to assess the compound’s impact on cellular pathways.

Author response: The reviewer's suggestion about mechanism exploration is highly valuable. However, given the currently modest potency of these compounds, we plan to conduct these analyses when more potent analogs are identified or clearer structure-activity relationships emerge in our future research.

5. The authors should add a graphical abstract for the broader audience.

Author response: Thanks for your suggestion. We have added a graphical abstract, as shown in the attachment.

6. The authors should state the major limitations associated with the study.

Author response: Thank you for your suggestion. The corresponding sections in the manuscript have been revised accordingly. The added content is as follows In lines 433-435 on page 14: “However, given the insufficient sample amount and weak potency of these compounds, mechanistic studies were not performed. Our future work will focus on identifying more potent analogs to enable subsequent in-depth studies.”

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed all the previous comments.  Thus, the manuscript can be accepted in its present form.