From the journal RSC Chemical Biology Peer review history

06-Dec-2021

Dear Dr Kunz:

Manuscript ID: CB-ART-11-2021-000220
TITLE: Phosphorylated resveratrol as a protein aggregation suppressor in-vitro and in-vivo

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Prof Seung Bum Park

Associate Editor, RSC Chemical Biology
Professor, Chemistry Department, Seoul National University, Korea

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Reviewer 1

Authors report anti-amyloidogenic performance of phosphorylated resveratrol under in vitro and in vivo fly model. They have shown that phosphorylated resveratrol has advantage of good water solubility and induce amyloid fibril inhibition. This work is thorough and results are well explained. I suggest following moditications:
1) Discuss and compare other polyphenols and if phosphorylation can be important for inhibiting protein aggregation.

Reviewer 2

This manuscript describes the anti-aggregation properties of a phosphorylated derivative of trans-resveratrol through in vitro and in vivo experiments. The trans-resveratrol reportedly has a variety of effects for radical scavenging, interacting with gene expression, and preventing the fibrillation of pathogenic proteins; however, its low solubility blocked off the extended biological applications. Here, the authors introduced the phosphorylate group into the molecular structure via esterification of the hydroxyl groups to overcome the intrinsic low bioavailability of trans-resveratrol. The phosphorylated resveratrol (PR) exhibited highly water-soluble features and specific ion effects, as well as antioxidant and anti-aggregation effects towards free radicals and disordered proteins, e.g., amyloid- (A) and human insulin (HI), respectively. These studies perform multiple studies for analysis and provide a potent small molecule for the research and pharma; however, several experimental data are not convincing and some results were discussed with a lack of analysis. Below are some concerns about the utility of this manuscript.

1. The authors used the mixture of PR derivatives in this study and the structural composition was analyzed by 1H- and 31P-NMR spectroscopy. We would like to ask why the authors use the mixture instead of each PR derivatives depicted in Figure 1. Please include the rationale about this part for helping the readers’ understanding.

2. The authors conducted the Trolox equivalent antioxidant capacity (TEAC) assay to assess the radical scavenging ability of PR. If authors perform the same experiment using a higher concentration of PR and Trolox to find the saturation point, we think it will help the accuracy of the experiment a little more.

3. We would like to ask how the authors describe the reductive effects of PR towards the formation of fiber species. Why do the authors believe that the resveratrol derivatives with a low degree of phosphorylation are predominantly responsible for this effect without any experimental evidence for other type resveratrol derivatives? Please provide the additional experiments or discussion with previous literature for components other than the monophosphated form constituting the mixture.

4. The thioflavin T (ThT) assay was used to kinetically trace the formation of β-sheet rich fibril species in the presence of PR with various concentrations. Due to the intervening property of polyphenolic substances, the authors conducted the control experiments as shown in Figure 3b; however, there are still questions about the suitability of the ThT assay to measure the activity of PR with consideration of the stability. We would like to suggest additional control experiments with incubated PR structures under the same experimental conditions.

5. For fitting the sigmoidal curve in the ThT assay, more dense points will make it easy to understand and distinguish the impact of PR with various concentrations on the aggregation profiles.

Reviewer 1
Q: Discuss and compare other polyphenols and if phosphorylation can be important for inhibiting protein aggregation.
A: This is an interesting idea. We will address this in a short comparison with other polyphenols and the role of phosphorylation in a revised version of the manuscript.
Reviewer 2
Q1: The authors used the mixture of PR derivatives in this study and the structural composition was analyzed by 1H- and 31P-NMR spectroscopy. We would like to ask why the authors use the mixture instead of each PR derivatives depicted in Figure 1. Please include the rationale about this part for helping the readers’ understanding.
A1: The PR was used as a mixture because it is/was commercially available in this form. It is advertised as a triphosphate, suggesting a pure product. However, limitations in the production procedure and subsequent isolation of the pure RTP product make this compound a mixture with lesser phosphorylated compounds in reality. This is almost impossible to resolve post-hoc due to high similarities between the derivatives, as mentioned in the manuscript. We also mentioned that enzymatic degradation in-vivo will most likely reduce the compounds to versions with lower degree of phosphorylation anyway. In the revised manuscript, we will try to make this section more accommodative for the reader.
Q2: The authors conducted the Trolox equivalent antioxidant capacity (TEAC) assay to assess the radical scavenging ability of PR. If authors perform the same experiment using a higher concentration of PR and Trolox to find the saturation point, we think it will help the accuracy of the experiment a little more.
A2: The Trolox experiment was conducted to measure for antioxidant behaviour of the PR mixture as used in the study. To yield a Trolox value, the measurement needs to be conducted in the linear range (cf. Sochor et al., Molecules, 2010). This is achieved in the current setup and gives a Trolox value that fits the experimental data reasonably well (cf. r-square = 0.997) (cf. also Lucas-Abellan, Food Chem. Tox, 2011). Already now the range extends far the concentrations used in the rest of this study (≈ 180 mM) and what is genuinely believed to be physiologically feasible. Therefore, we reckon that using even higher concentrations contributes little to the overall information about antioxidant activity of PR. The key take-home message for this section is the robust anti-oxidant activity of PR.
Q3: We would like to ask how the authors describe the reductive effects of PR towards the formation of fiber species. Why do the authors believe that the resveratrol derivatives with a low degree of phosphorylation are predominantly responsible for this effect without any experimental evidence for other type resveratrol derivatives? Please provide the additional experiments or discussion with previous literature for components other than the monophosphated form constituting the mixture.
A3: This is a very interesting point. As we state in the manuscript, we believe it to be likely that differently phosphorylated compounds exhibit different anti-fibrillation effects. From previous work by Sciatta et al. we know that monophosphates (RMP) appear more effective than free t-resveratrol. Our experiments showed that the PR mixture is somewhat less effective than free t-resveratrol at the respective concentrations. From this we assume that some resveratrol-compounds in the PR mixture must then be less effective than free t-resveratrol, which leaves only RTP or RDP. Therefore, we suggested a better effectiveness for lower phosphorylated compounds. This is, of course, only an assumption based on previous literature and our experiments. This is in no way supposed to be seen as a scientific proof. We will rephrase this section to make it clearer to the reader.
Resolving the influence of individual PR derivatives is, however, outside the scope of this work, as we focus on the commercially available mixture (cf. A1). It is mentioned in the conclusion that an in-depth analysis on this issue would be a worthy follow up investigation.
Q4: The thioflavin T (ThT) assay was used to kinetically trace the formation of β-sheet rich fibril species in the presence of PR with various concentrations. Due to the intervening property of polyphenolic substances, the authors conducted the control experiments as shown in Figure 3b; however, there are still questions about the suitability of the ThT assay to measure the activity of PR with consideration of the stability. We would like to suggest additional control experiments with incubated PR structures under the same experimental conditions.
A4: Indeed, ThioT assays (as most experimental procedures) come with certain caveats. To approach this, we conducted the control experiments to evaluate the interaction of PR with the fluorescent dye binding itself (cf. Jameson et al., ACS Chem. Neurosci., 2012). The experiments with amyloid beta were done not kinetically, but the endpoint was assessed. In the setup we used, this is achieved after 1 hour. (After this, no change in signal can be detected.) The control experiments were also incubated for one hour, after which the PR was added with subsequent measurement to mirror the conditions for the main experiment. We were careful not to make any claims on the stability other than that there is no immediate disbandment of fibers. Incubation of pure PR with Thio T added afterwards was used as background correction in all measurements, hence we feel that additional controls would yield little further information.
Q5: For fitting the sigmoidal curve in the ThT assay, more dense points will make it easy to understand and distinguish the impact of PR with various concentrations on the aggregation profiles.
A5: While we agree that more data is always better, the number of data points for this type of experiment should normally suffice to make out a trend (e.g. Patel et al., Science, 2017). We will, however, use a different fitting function to improve the sigmoidal fit to our existing data for the revised manuscript. This way, the results should be more accessible.

26-Dec-2021

Dear Dr Kunz:

Manuscript ID: CB-ART-11-2021-000220.R1
TITLE: Phosphorylated resveratrol as a protein aggregation suppressor in-vitro and in-vivo

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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Prof Seung Bum Park

Associate Editor, RSC Chemical Biology
Professor, Chemistry Department, Seoul National University, Korea

Reviewer 2

The authors have tried to address the comments and questions relatively well.