Phenotypic screening of 1,953 FDA-approved drugs reveals 26 hits with potential for repurposing for Peyronie’s disease

Marcus M. Ilg; Alice R. Lapthorn; David J. Ralph; Selim Cellek

doi:10.1371/journal.pone.0277646

Peer Review History

Original SubmissionFebruary 19, 2022
14 Mar 2022 Decision Letter - Donald Gullberg, Editor PONE-D-22-05095Phenotypic screening of 1,953 FDA-approved drugs reveals 26 hits with potential for repurposing for Peyronie’s diseasePLOS ONE Dear Dr. Cellek, 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 Apr 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. 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Additional Editor Comments: An expert in the field has taken part of your work together with an editor. They both find your work potentially interesting, and the reviewer has a number of suggestions for additional experiments that need to be performed. We ask your to study the reviewer comments carefully and return a point by point response letter where your have addressed each of the issues raised in the review report. Please let me know if you need additional time to do this. I look forward to receiving your revised version. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know ****** 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: No ****** 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 ****** 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 investigates whether phenotypic screening can be used to repurpose drugs for the treatment of Peyronie’s disease (PD). Authors have used primary fibroblasts from PD patients in a phenotypic screening assay that measures TGF-β1 induced αSMA protein synthesis. Using this screening assay, authors have screened 1,953 FDA-approved drugs and have identified 26 drugs that inhibits TGF-β1 induced αSMA protein synthesis. Moreover, the authors have also identified 10 drugs that increases TGF-β1 induced αSMA protein synthesis. The authors suggest phenotypic screening of FDA-approved drugs for PD is a viable method to predict drugs for repurposing to treat early PD. Although, the approach is interesting and supported with some experimental data, many issues remain to be addressed. Major comments: 1. Secondary experiments like western blotting or qPCR or αSMA stress fibers by immunofluorescence for those 36 drugs that affected αSMA protein synthesis would be helpful to validate the phenotypic screening assay. 2. Apart from αSMA, other assays such as collagen gel contraction assay or TGF-β reporter assay using tMLC system would help to strengthen the data on myofibroblast transformation 3. Cell proliferation assay for those selected 36 drugs would be useful to evaluate the effects of these drugs Minor comments: 1. The phenotypic screening assay measures the ability of the drugs to inhibit TGF-β1 induced αSMA protein synthesis but does not measures the ability of the drugs to inhibit TGF-β1 activation 2. In the results section, the isolated fibroblasts were mentioned as vimentin negative and needs to be clarified. In methods, section it is written as vimentin positive. 3. Low serum or serum-free conditions would have helped to reduce the effects from growth factors or other protein in the serum ****** 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 [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. 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Revision 1
22 Jun 2022 Author Response Reviewer #1: The manuscript investigates whether phenotypic screening can be used to repurpose drugs for the treatment of Peyronie’s disease (PD). Authors have used primary fibroblasts from PD patients in a phenotypic screening assay that measures TGF-β1 induced αSMA protein synthesis. Using this screening assay, authors have screened 1,953 FDA-approved drugs and have identified 26 drugs that inhibits TGF-β1 induced αSMA protein synthesis. Moreover, the authors have also identified 10 drugs that increases TGF-β1 induced αSMA protein synthesis. The authors suggest phenotypic screening of FDA-approved drugs for PD is a viable method to predict drugs for repurposing to treat early PD. Although, the approach is interesting and supported with some experimental data, many issues remain to be addressed. Major comments: 1. Secondary experiments like western blotting or qPCR or αSMA stress fibers by immunofluorescence for those 36 drugs that affected αSMA protein synthesis would be helpful to validate the phenotypic screening assay. We thank the reviewer for the suggestion of these secondary assays. We expanded the chapter ‘Confirmation of hits’ and have now included a figure that uses immunocytochemistry that demonstrates the effect of the selected three hit drugs on αSMA stress fibre formation, as suggested (Fig 3). We further tested these three drugs on ECM production and cell viability (please see the point below). We used these assays to confirm the three randomly selected hit drugs that we followed up via concentration-response-curves previously. Whilst it would be interesting to follow up all 26 hits that decreased myofibroblast transformation, it is beyond the scope of this manuscript which aims to report the findings from 1,953 drugs We have previously validated the assay after it revealed drugs in a smaller screening campaign and these hits (PDE5 inhibitors and selective estrogen receptor modulators) were validated using both, in vitro and in vivo experiments, highlighting the ability of the assay to identify relevant drugs (Ilg et al, 2019). Furthermore, the combination of these two drug classes have been shown to be effective in patients with early PD in the clinic (Megson et al 2022) which further validates the applicability of our screening assay. 2. Apart from αSMA, other assays such as collagen gel contraction assay or TGF-β reporter assay using tMLC system would help to strengthen the data on myofibroblast transformation We thank the reviewer for the suggestion and have included the outcome of an ECM production assay to further confirm the anti-myofibroblast activity of the three randomly selected hit drugs (Fig 4). As can be seen, ECM production is significantly reduced upon drug treatment, further confirming anti-myofibroblast activity of the drugs. 3. Cell proliferation assay for those selected 36 drugs would be useful to evaluate the effects of these drugs Whilst we agree with the reviewer that it would be of interest to include data on cell proliferation, we only included the data for the three drugs that we previously confirmed via concentration response curve. We have now included a separate figure (Fig 5) to showcase that cell proliferation increases in cells treated with TGF- β1, which is reduced after treatment with the drugs. The graph depicts cells number (proliferation by proxy) derived from the nuclear stain DRAQ5 which emits at 700nm. Minor comments: 1. The phenotypic screening assay measures the ability of the drugs to inhibit TGF-β1 induced αSMA protein synthesis but does not measures the ability of the drugs to inhibit TGF-β1 activation The reviewer is right to point this out. Whilst we cannot claim that the drugs prevent TGF- β1 activation, we can say that the drugs inhibit TGF- β1-induced myofibroblast transformation, a downstream consequence of TGF- β1 activation. Given the agnostic nature of a phenotypic assay, it is not possible to suggest a potential mechanism of action, without target deconvolution. We have now reworded the text where this was unclear. 2. In the results section, the isolated fibroblasts were mentioned as vimentin negative and needs to be clarified. In methods, section it is written as vimentin positive. We apologize for this mistake. The cells were desmin negative and vimentin positive. This has now been corrected in the results section. We thank the reviewer for pointing out this mistake. 3. Low serum or serum-free conditions would have helped to reduce the effects from growth factors or other protein in the serum We agree with the reviewer on this, as this was our initial thought as well. When we developed the assay, we determined whether the use of serum free media would improve the TGF- β1-induced fold-change and/or well/well consistency. We tested serum concentrations of 10%, 1%, and 0.1% and assessed the influence on TGF- β1-induced fold-change of αSMA protein expression. No significant difference could be noted in fold-change, however, serum deprivation caused cell loss, leading to well/well inconsistency resulting in a less robust screening assay. Therefore, it was concluded that serum free conditions were not beneficial in our settings. We have now added a section in the discussion chapter to explain all optimisation steps we have taken while developing this assay. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0277646.r002
20 Jul 2022 Decision Letter - Donald Gullberg, Editor PONE-D-22-05095R1Phenotypic screening of 1,953 FDA-approved drugs reveals 26 hits with potential for repurposing for Peyronie’s diseasePLOS ONE Dear Dr. Cellek, 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 Sep 03 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, Donald Gullberg, PhD Academic Editor PLOS ONE Additional Editor Comments (if provided): Thank you for the revised version which one editor and one reviewer have read. Both think that data is interesting but reviewer has raised some serious concerns ( please see reviewer comments) and has proposed additional experiments and also ask to see raw data to clarify concerns. Please study reviewer comments carefully. I look forward to a revised version where you address the concerns raised point by point and also provide the raw data asked for. I would be happy to give you extra time should this be needed. [Note: HTML markup is below. Please do not edit.] 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: (No Response) ******** 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 ****** 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 authors have made considerable efforts to address some of the issues mentioned in the last review. However, there are still some major concerns that needs to be addressed. Major comments: 1. The resolution of the images in figure 3 could have been better and quantification was not presented. Quantification of αSMA normalized to F-actin (Phalloidin) would make the data solid. Alternatively, the authors should consider either western blotting or qPCR for the three hit drugs 2. The ECM production assay to validate the myofibroblast function is appreciated but it would have been much better if the authors have performed either western blotting or qPCR for Fibronectin or Collagen I/III as these methods are not dependent on cell numbers Minor comments: 1. According to the description in the figure legend, the images were captured at 200x magnification but scale bar was not presented. The nucleus size appear different across the images. Raw images of these data would help to evaluate the data better. 2. The amount of ECM measured using this assay can be influenced by cell proliferation and washing steps with ammonium hydroxide. 3. It is very hard to find a difference between the data presented in Figure 2 and figure 5 with reference to cell proliferation and cell viability as it uses the same method. The authors should have used an alternative method to assess cell proliferation. ****** 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 ******** [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. https://doi.org/10.1371/journal.pone.0277646.r003
Revision 2
19 Oct 2022 Author Response 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 authors have made considerable efforts to address some of the issues mentioned in the last review. However, there are still some major concerns that needs to be addressed. Major comments: 1. The resolution of the images in figure 3 could have been better and quantification was not presented. Quantification of αSMA normalized to F-actin (Phalloidin) would make the data solid. Alternatively, the authors should consider either western blotting or qPCR for the three hit drugs >>>We thank the reviewer for the suggestion and have now included both RT-qPCR data for ACTA2 as well as Western blot images for ASMA to support the findings (new Fig 4). As can be seen in the figure, they concur with our previously reported observations. 2. The ECM production assay to validate the myofibroblast function is appreciated but it would have been much better if the authors have performed either western blotting or qPCR for Fibronectin or Collagen I/III as these methods are not dependent on cell numbers >>>We have now included both RT-qPCR data for FN2 and Western blot images for fibronectin to support our findings (new Fig 6). The initial ECM production assay data is still included to show the effect of the drugs on total ECM formation, without differentiating the type of matrix protein. We have included a paragraph in the discussion to explain the effects of solifenacin succinate which we believe keeps the cells in the protomyofibroblast state instead of completely preventing myofibroblast transformation. Minor comments: 1. According to the description in the figure legend, the images were captured at 200x magnification but scale bar was not presented. The nucleus size appear different across the images. Raw images of these data would help to evaluate the data better. >>>We thank the reviewer for the comment. We have now included scale bars to make comparison easier. It should be noted that it has been reported that cellular size is affected in myofibroblast transformation. Myofibroblasts have been described as larger cells with more prominent Golgi apparatus and rough endoplasmic reticulum with elongated, serrated nuclei and extensive stress fibre network (PMID: 21297493, PMID: 32673537). This has now been added to the discussion. 2. The amount of ECM measured using this assay can be influenced by cell proliferation and washing steps with ammonium hydroxide. >>>We agree with the reviewer and have now included Western blot images for fibronectin as suggested (new Fig 6). It might not have been evident from the manuscript. but data was normalised to cell count before lysis. We have revised the methods to reflect this better (Line 217). 3. It is very hard to find a difference between the data presented in Figure 2 and figure 5 with reference to cell proliferation and cell viability as it uses the same method. The authors should have used an alternative method to assess cell proliferation. >>>We do agree with the reviewer that cell proliferation assay would have been nice to show one of the characteristics of transformation of fibroblasts to myofibroblasts. However, several methods of assessing cell viability only capture a fraction of it (mitochondrial activity, metabolic activity, protease activity, membrane integrity) often overlapping with cell proliferation assays (DNA synthesis, metabolic activity). In order to measure cell proliferation adequately, several different assays have to deployed. We believe this would be outside the scope of our manuscript. We have now highlighted this as a point for future research in the limitations section. Attachments Attachment Submitted filename: Response to reviewer.docx https://doi.org/10.1371/journal.pone.0277646.r004
2 Nov 2022 Decision Letter - Donald Gullberg, Editor Phenotypic screening of 1,953 FDA-approved drugs reveals 26 hits with potential for repurposing for Peyronie’s disease PONE-D-22-05095R2 Dear Dr. Cellek, 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, Donald Gullberg, PhD 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 authors have addressed the questions raised in the last review with new experiments. Minor comment: In figure 6A, fibronectin gene is denoted as FN2 instead of FN1 ****** 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.0277646.r005
Formally Accepted
1 Dec 2022 Acceptance Letter - Donald Gullberg, Editor PONE-D-22-05095R2 Phenotypic screening of 1,953 FDA-approved drugs reveals 26 hits with potential for repurposing for Peyronie’s disease Dear Dr. Cellek: 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 Professor Donald Gullberg Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0277646.r006

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