Novel carbon skeletons activate human NicotinAMide Phosphoribosyl Transferase (NAMPT) enzyme in biochemical assay

Karen H. Almeida; Lisbeth Avalos-Irving; Steven Berardinelli; Kristen Chauvin; Silvia Yanez

doi:10.1371/journal.pone.0283428

Peer Review History

Original SubmissionNovember 4, 2022
20 Dec 2022 Decision Letter - Qiu Sun, Editor PONE-D-22-30462Novel carbon skeletons activate human NicotinAMide PhosphoribosylTransferase (NAMPT) enzyme in biochemical assayPLOS ONE Dear Dr. Almeida, 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. Please submit your revised manuscript by Feb 03 2023 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, Qiu Sun Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating in your Funding Statement: "Research reported in this publication was supported in part by grant subawards to KHA from the Rhode Island Institutional Development Award (IDeA) Network of Biomedical Research Excellence (https://web.uri.edu/riinbre/) from the National Institute of General Medical Sciences of the National Institutes of Health (https://www.nigms.nih.gov/) under grant number P20GM103430 and from the National Center for Research Resources of the National Institutes of Health under grant number 5P20RR016457-11. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIGMS or the NIH. Furthermore, this research is based in part upon work conducted using the Rhode Island Genomics and Sequencing Center that is supported in part by the National Science Foundation (https://www.nsf.gov/) under EPSCoR Grants Nos. 0554548 & EPS-1004057. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf. 3. Thank you for stating the following in your Competing Interests section: "No competing interest exists." Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now This information should be included in your cover letter; we will change the online submission form on your behalf. [Note: HTML markup is below. Please do not edit.] 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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes ****** 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 ****** 4. 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: Yes ****** 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: The manuscript titled “Novel carbon skeletons activate human NicotinAMide PhosphoribosylTransferase (NAMPT) enzyme in biochemical assay” by Almeida et al describes the results of a docking-based virtual screening coupled with biochemical assay aimed at finding novel compounds activating NAMPT, which is the rate-limiting enzyme in the NAD+ salvage pathway, regulates the NAD+ biosynthetic capacity of cells. The concerns regarding the manuscript are uploaded as an attachment. Reviewer #2: The paper presented the novel compounds which stimulates human NAMPT activities. The approach and methods are sound and writing is appropriate. Minor point: His-tag protein (NAMPT) binding step was not described:Column size, kind of resin etc. ****** 6. 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. Attachments Attachment Submitted filename: PONE-D-22-30462_review.docx https://doi.org/10.1371/journal.pone.0283428.r001
Revision 1
31 Jan 2023 Author Response Response to Reviewers PONE-D-22-30462_review Novel carbon skeletons activate human NicotinAMide PhosphoribosylTransferase (NAMPT) enzyme in biochemical assay Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf I have completed this to the best of my ability. 2. Thank you for stating in your Funding Statement: "Research reported in this publication was supported in part by grant subawards to KHA from the Rhode Island Institutional Development Award (IDeA) Network of Biomedical Research Excellence (https://web.uri.edu/riinbre/) from the National Institute of General Medical Sciences of the National Institutes of Health (https://www.nigms.nih.gov/) under grant number P20GM103430 and from the National Center for Research Resources of the National Institutes of Health under grant number 5P20RR016457-11. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIGMS or the NIH. Furthermore, this research is based in part upon work conducted using the Rhode Island Genomics and Sequencing Center that is supported in part by the National Science Foundation (https://www.nsf.gov/) under EPSCoR Grants Nos. 0554548 & EPS-1004057. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf. I have made the requested changes and included them in the Cover Letter. 3. Thank you for stating the following in your Competing Interests section: "No competing interest exists." Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now This information should be included in your cover letter; we will change the online submission form on your behalf. I have included the amended Competing Interests Statement in the Cover Letter. Review Comments to the Author Reviewer #1: The manuscript titled “Novel carbon skeletons activate human NicotinAMide PhosphoribosylTransferase (NAMPT) enzyme in biochemical assay” by Almeida et al describes the results of a docking-based virtual screening coupled with biochemical assay aimed at finding novel compounds activating NAMPT, which is the rate-limiting enzyme in the NAD+ salvage pathway, regulates the NAD+ biosynthetic capacity of cells. The concerns regarding the manuscript are as follows (in bold): It is not clear which the NCI Diversity set was used for screening? III or IV? Is the screening library derived from only natural products? The authors should discuss that the both of virtual screening hits are polyphenolic compound? The authors thank the reviewers for the opportunity to clarify our work. The NCI Diversity Set III was used as the docking library for the virtual screen. All reference to the Diversity Set IV has been removed from the manuscript. The Diversity Set III screening library is a diverse set of potential pharmacophores and is not exculsively polyphenolic in nature. To help clarify, the authors have included the following on page 12 lines 273-275: …The library contains both synthetically generated compounds and naturally derived compounds that display a diverse array of functional groups with the potential to interact in a biological setting. This library is not a collection of solely polyphenolic compounds. Additionally, we have included a statement highlighting the result that out of >1500 compounds the two activators are both polyphenolic compounds. Page 14 lines 348-349: …Interestingly, both of these candidates are polyphenolic in nature although they exhibit a dramatically different carbon skeleton. Is there any information about the fluorescence intensity of the compounds (2 and 20) themselves? If not, should be checked. The fluorescence of each individual modulator candidate was incorporated into the activity assay and described in the data analysis portion of the material and methods section, page 11 lines 256-258. It reads: …Percent activity data was calculated with equation: Activity % = (FRXN – FRXN 0) / (F100% - F0 ) where FRXN contains the modulating substance and NAM substrate, FRXN 0 contains the modulating substance and water, F100% contains DMSO and NAM, and F0 contains DMSO and water. Additionally, the following has been added to the results and discussion section, page 14, lines 335-341: …To ensure that the recorded fluorescence was a function of NMN product formation and not intrinsic fluorescence from our lead compounds, the fluorescence of each lead compound was incorporated into the percent activity calculation. Each reported percent activity includes the baseline fluorescence of the buffer without either the candidate compound or the substrate NAM (F0), the fluorescence of the candidate compound without substrate NAM (FRXN 0), and the fluorescence of the NMN product without exposure to the candidate compound (F100). Although the studies on NAT revealed the critical role of K189 in boosting NAMPT activity, the binding modes of other activators with diverse scaffolds should also be considered and docking studies should be performed focusing on additional important residues (https://doi.org/10.1016/j.apsb.2022.07.016). The authors thank the reviewers for directing us to an additional reference investigating the structural relationship of small molecule activation of NAMPT. We agree that the mechanism of action is still under investigation and performing additional docking studies focusing on the interaction between compound 2 and 20 at the proposed site of interaction will help to clarify the activation mechanism. This is the subject of a future investigation. We have included a reference to this study on page 6, lines 135-138 that includes …However, structure-activity studies between NAMPT and a family of small molecule modulators suggests that increased product formation may be achieved through a critical water mediated interaction within the NAMPT active site but independent of K189. (20) Clearly the mechanism of NAMPT activation is still an open investigation. Since the importance of water-mediated h-bonds in the interaction for both inhibitors and activators is known, water molecules in active site should be kept during docking simulations. The Tang and Butterworth study (20) hypothesized that NAMPT activation by small molecules interaction is mediated by water molecule bridges within the active site. We agree that water should not be excluded however, this study was not available when our docking experiments were ongoing and thus any future work will include water molecules as potential interacting partners. The NAMPT activators reported to date should be added to the screening library. The authors agree that inclusion of the known activators of NAMPT would add to the study however, at the time of the screening, there were no known activators of NAMPT. Additionally, the undergraduate student completing the study has since graduated and is currently compleing a PhD program in a non-computational field. We have included the following explanation into the manuscript text. Page 12 lines 280-283 …FK866, a potent inhibitor of NAMPT, was incorporated into the Diversity Set III library as a binding control within the active site. However, at the time of the enzymatic docking procedure, there were no known activators of NAMPT and thus no activating molecules were included in the virtual library. The proposed binding modes of compounds 2 and 20 did not present the binding site properly. The figures need to be improved. Figure 10 showing the proposed binding site has been improved to highlight the binding surface and represent the NAMPT:20 and NAMPT:2 interactions in a more standard format. The text and figure caption has been updated to reflect the new figüre, page 19-20, lines 532-554 The MD simulations should be performed for 2:NAMPT and 20:NAMPT complexes which would be support the findings. The authors agree that MD simulations between 2:NAMPT and 20:NAMPT would support our findings. However, we do not feel that the absence of MD calculations precludes publication. We are currently collaborating with another undergraduate research lab to complete this analysis. Please check the words throughout the manuscrip, fig or Fig? All reference to fig has been changed to Fig per PLOS One Formatting requirements. Reviewer #2: The paper presented the novel compounds which stimulates human NAMPT activities. The approach and methods are sound and writing is appropriate. Minor point: His-tag protein (NAMPT) binding step was not described:Column size, kind of resin etc. We have included additional information on the His tagged purification of the NAMPT protein, page 9 lines 211-213: …Protein lysate was applied to 5ml of Ni-NTA resin (Quiagen) in a gravity drip format, washed with lysis buffer to remove weakly bound protein, and eluted in lysis buffer supplemented with 200mM and 350mM imidazole. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0283428.r002
8 Mar 2023 Decision Letter - Qiu Sun, Editor Novel carbon skeletons activate human NicotinAMide PhosphoribosylTransferase (NAMPT) enzyme in biochemical assay PONE-D-22-30462R1 Dear Dr. Almeida, 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. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Qiu Sun Academic Editor PLOS ONE Additional Editor Comments (optional): 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 ******** 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: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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 seems to be corrected and modified according to reviewer suggestions. In my opinion, it might be now accepted for publication in PLOS ONE. ****** 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 ******** https://doi.org/10.1371/journal.pone.0283428.r003
Formally Accepted
20 Mar 2023 Acceptance Letter - Qiu Sun, Editor PONE-D-22-30462R1 Novel carbon skeletons activate human NicotinAMide Phosphoribosyl Transferase (NAMPT) enzyme in biochemical assay Dear Dr. Almeida: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Qiu Sun Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0283428.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .