PONE-D-21-28384Suillin: a strong cholinesterase inhibitor from Suillus luteus, an edible basidiomycete used by Saraguro indigenous, south of EcuadorPLOS ONE

Dear Dr. Cartuche,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This manuscript describes the isolation, structural analysis and action on acetylcholinesterase of suillin, a terpenoid (tetraprenylphenol) from the basidiomycete Suillus luteus. Suillin is a reversible non-competitive inhibitor of acetylcholinesterase. This compound is interesting from a mechanistic point of view, but likely not for a possible interest as anti-AD drug. However, the study is weak and the enzymologic part needs to be carefully reworked.

Major points

Title has to be changed: suillin is not a strong cholinesterase inhibitor (cf line 231, Table I, Ki = 22 microM)

Introduction: Line 70, AChE is not only mainly found at neuromuscular junction but also in brain where it plays a key role.

Line 71-72 how high level of AChE at neuromuscular junctions can cause neurological disorders ?

Formula of suillin, Line 140: suillin is an acetylester, like aspirin and acetylcholine. Why the authors did not check whether this compound could be also substrate of acetylcholinesterase ? There are many examples of compounds acting both as a substrate and inhibitor of cholinesterases (ex, aprophen).

Lines 163 and 235 and Fig 4: AChE kinetics at concentrations varying between 2.5 and 25 mM cannot be described by the Michaelis-Menten model. Fig 4 shows the rate of AChE as a function of ATC ranging from 0.25 to 2.5 mM. The enzyme should be inhibited by excess substrate beyond 1 mM. How to explain that the enzyme is not inhibited by excess substrate ?

Line 170: this model equation suppose that the inhibitor only affects Vmax. How to rule out that inhibitor does not affect Km or both Km and Vmax?

Lines 221, 23: IC50 and Ki do not match as they should for pure non competitive inhibitor (as it is shown in Fig 4 insert, alpha =0): Ki = IC50.

Minor points

Line 146 : Was MeOH the solvent of suillin for inhibition measurements ? (What was the final MeOH concentration in assay ?)

Typos

Line 21 : acetylcholinesterase

Line 146 : mM

Reviewer #2: In this manuscript, the authors described their findings on suillin, the active ingredient extracted from Suillus luteus, can function as a cholinesterase inhibitor. The methods adopted in this study included thin-layer chromatography (TLC) to determine compounds from extracts of Suillus luteus, Ellman assay to characterize the extracts’ and suillin’s roles in inhibiting AChE and to conduct the kinetic analysis of suillin. As a conclusion, they claimed that suillin can be a candidate for treating neurodegenerative diseases.

Major comments

1. Table 1 & Figure 2: Positive control for Ellman assay is missing. A known drug in inhibiting AChE activity should be included.

2. As several compounds were identified in the extracts. Why suillin was the major component in suppressing AChE? For other compounds identified, same experiments (i.e. Ellman assay and kinetic analysis) should also be conducted in order to compare and confirm suillin as the main functioning chemical.

3. Many fungi have been found to have anti-cholinesterase function in previous studies, so the perspective of this study is not new. Also, if the authors would like to propose it as a candidate for neurodegenerative treatment, the scale of study in this manuscript is not adequate. More studies (e.g. in vitro) should be conducted to make the findings more reliable and persuasive.

Minor comments

1. Some typos need to be rectified. (e.g. ‘acethylcholinesterase’)

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-28384R1Suillin: a mixed-type acetylcholinesterase inhibitor from <suillus luteus=""> which used by Saraguros indigenous, southern Ecuador</suillus>PLOS ONE

Dear Dr. Cartuche,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

In your revised manuscript please address the very minor ​concerns raised by the two reviewers.

Please submit your revised manuscript by May 28 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Israel Silman

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript was greatly improved. However, line 216 page 10, the formula used for determination of Ki is wrong. Is it a typo or a real mistake? Anyway, with such a "mistake" there is still doubt about the values of alpha and Ki.

Reviewer #2: In this manuscript, the authors described their findings on suillin, the active ingredient extracted from the herb Suillus luteus can function as a mixed-type acetylcholinesterase inhibitor. The methods adopted in this study included thin-layer chromatography (TLC) to determine compounds from extracts of Suillus luteus, Ellman assay to characterize the extracts’ and suillin’s roles in inhibiting AChE and to conduct the kinetic analysis of suillin. As a conclusion, they proposed that suillin can be a candidate for treating neurodegenerative diseases. Although the most of problems have been addressed, there are still some questions:

Major comments

- Table 1: IC50 of other chemicals isolated (i.e. linoleic acid, ergosterol etc.) should be included for comparison. What is the extraction efficiency of suillin from the herbal extract?

- In the discussion, how mixed inhibitors to AChE can be discussed. Propose the mechanism of how suillin works on AChE, e.g. docking analysis.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Suillin: a mixed-type acetylcholinesterase inhibitor from <suillus luteus=""> which used by Saraguros indigenous, southern Ecuador

PONE-D-21-28384R2</suillus>

Dear Dr. Cartuche,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Israel Silman

Academic Editor

PLOS ONE

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