Computational drug repurposing strategy predicted peptide-based drugs that can potentially inhibit the interaction of SARS-CoV-2 spike protein with its target (humanACE2)

Samuel Egieyeh; Elizabeth Egieyeh; Sarel Malan; Alan Christofells; Burtram Fielding

doi:10.1371/journal.pone.0245258

Peer Review History

Original SubmissionAugust 12, 2020
28 Sep 2020 Decision Letter - Israel Silman, Editor PONE-D-20-25309 Computational drug repurposing strategy identified peptide based drugs that can potentially inhibit the interaction of SARS-CoV-2 Spike proteins with its target (humanACE2) PLOS ONE Dear Dr. Egieyeh, 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 constructive comments of the two reviewers as fully as possible. Please submit your revised manuscript by Nov 12 2020 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. 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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: No Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: N/A ****** 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: No 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 aim of the study presented in this manuscript is to identify drugs from the Drug Bank that can prevent the binding of the SARS-CoV-2 spike protein with the human angiotensin-converting enzyme 2 (hACE2) receptor. However, it is difficult to understand that the authors used the whole S(RBD)-ACE2 complex, instead of the binding site of the S(RBD) protein or ACE2 alone, for molecular docking. In general, the screened compound or peptide is supposed to competitively binding to the S protein or ACE2. It is also quite curious how the binding of candidate peptides with the S(RBD)-ACE2 complex can destroy the stability of the complex. Does it mean that huge conformational changes were induced by the binding of the candidate or there was great conflict inside the molecular docked conformation? The simulation duration (100 ps or 4 ns) is too short to generate reliable information or conclusion. The definition of RMSF is inappropriate: “The RMSF per residue is the distance between a residue in the first frame (structure) of the protein under simulation and the same residue in the subsequent frames (structures) of the same protein.” Most importantly, there is no any in vitro experiment to validate the modeling result. Reviewer #2: abstract - please change language to indicate all results are predictions, nothing was tested experimentally. for example: "The study identified a number of peptide-based drugs, including BV2, Gonadorelin,28 Icatibant, and Thymopentin that bind sufficiently" should be changed to "could potentially bind" or "is predicted to bind" Please clarify how many drugs were screened. The methods section said 7000, line 80 said 1101 and line 83 said 1070. Line 165 - "These observations suggest potential conformational changes and moderate flexibility of the S protein and the hACE2 receptor complex in vivo" - I would suggest removing the in vivo from the sentence. Figure 1 - please indicate which color is the structure from PDB and which is the average of the simulation. Line 178 "3.2 Docking studies identified drugs with good binding affinities to the target site" - since binding affinities were not measured, this should be changed to something like "3.2 Docking studies identified drugs with potential to bind to the target site" Lines 184-186: “The more negative the S score, the stronger the binding affinity, the better the ligand interaction with the protein and the more stable the complex of the drug hits with the protein complex.” Again, language should indicate this is all predicted and not experimentally tested. Figure 4 – please indicate what the red and blue lines are in the figure itself for better readability (it appears in the text). For the text and legend of figure 4, please indicate the drug name (it is only listed as “the top hit”). Line 253 – “Top-ranked drug hits identified in the study could inhibit protein-protein interaction between the SARS-CoV-2 spike protein and hACE2.” This sentence is misleading, as it suggests experimental evidence of inhibiting the binding. Please delete it. Lines 261-263: “Similar hinderance to protein–protein interaction necessary for the establishment of the infection by coronavirus was observed for all of the top six ranked drug hits.” – please provide the data, either in the text or a supplementary file. Figure 5 – please indicate what the red and blue lines are in the figure itself for better readability (it appears in the text). Line 280 – “from our previous study”. Please provide a reference Lines 289-291: “The molecular docking studies identified drugs that bind strongly to the interacting surface of SARS-CoV-2 spike protein hACE2 complex.”. Please change to indicate these MAY bind strongly, as it is only predicted. Lines 322-326: “Although a number of known drugs including hydroxychloroquine [5], Azithromycin [5], remdesivir [6], and idasanutlin [7] have been proposed and reported to be effective in the treatment of COVID-19, however results from our study suggest that hydroxychloroquine and remdesivir may have lower potential (as evidenced by binding free energy lower than -5.0 kcal/mol) to inhibit the interaction between the spike protein and hACE2.” There are several problems here. One – azithromycin and hydroxychloroquine have been widely shown to not be effective in treating COVID-19 patients, and should be removed from this sentence. Two – remdesivir is an RdRp inhibitor and is not expected to interfere with Spike binding to Ace2. Three- Ref #7 is a “thought experiment” is using idasanutlin to treat COVID-19, again in a mechanism unrelated to Spike-Ace2 binding. I suggest you delete these sentances. In the Conclusion section (lines 327-337), please rephrase language to indicate potential inhibition rather than actual inhibition. ****** 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. https://doi.org/10.1371/journal.pone.0245258.r001
Revision 1
12 Dec 2020 Author Response Following the review, which we accept in good faith, we have implemented the following changes according to the reviews (please note that number line of the previous version has changed in this new version): The title has been slightly revised to reflect that the study was a computational prediction. Reviewer #1: The aim of the study presented in this manuscript is to identify drugs from the Drug Bank that can prevent the binding of the SARS-CoV-2 spike protein with the human angiotensin-converting enzyme 2 (hACE2) receptor. However, it is difficult to understand that the authors used the whole S(RBD)-ACE2 complex, instead of the binding site of the S(RBD) protein or ACE2 alone, for molecular docking. In general, the screened compound or peptide is supposed to competitively binding to the S protein or ACE2. It is also quite curious how the binding of candidate peptides with the S(RBD)-ACE2 complex can destroy the stability of the complex. Does it mean that huge conformational changes were induced by the binding of the candidate or there was great conflict inside the molecular docked conformation? The simulation duration (100 ps or 4 ns) is too short to generate reliable information or conclusion. The definition of RMSF is inappropriate: “The RMSF per residue is the distance between a residue in the first frame (structure) of the protein under simulation and the same residue in the subsequent frames (structures) of the same protein.” Most importantly, there is no any in vitro experiment to validate the modeling result. 1. We revised the experiment and conducted the molecular docking on the hACE2 part of the S(RBD)-ACE2 complex. We have clarified that the molecular docking was done on the hACE2 (the spike protein interacting surface on hACE2) (Line 99 – 110) 2. Simulation to get the average structure of the protein was done over a duration of 50 ns. (line 107) 3. The definition of RMSF was revised (Line 156 – 158). Reviewer #2: abstract - please change language to indicate all results are predictions, nothing was tested experimentally. for example: "The study identified a number of peptide-based drugs, including BV2, Gonadorelin,28 Icatibant, and Thymopentin that bind sufficiently" should be changed to "could potentially bind" or "is predicted to bind" The statements were revised accordingly (Line 27 - 29). Please clarify how many drugs were screened. The methods section said 7000, line 80 said 1101 and line 83 said 1070. 5. It has been clarified in the document that the amount compounds docked in this study was 1070. These compounds were the top subset from the drug bank database (over 7000 compounds) virtually screened against the spike protein of Human coronavirus HKU1 (HCoV-HKU1) in an inhouse study we conducted our lab (Line 92 – 94). Line 165 - "These observations suggest potential conformational changes and moderate flexibility of the S protein and the hACE2 receptor complex in vivo" - I would suggest removing the in vivo from the sentence. The word “in vivo” has been removed. Figure 1 - please indicate which color is the structure from PDB and which is the average of the simulation. Correction effected: Inserted is the three-dimensional (3D) superimposition of the average structure of the SARS-CoV-2 S protein and hACE2 complex generated from the molecular dynamics simulation (magenta) and the crystal structure of the SARS-CoV-2 S protein and hACE2 complex downloaded from RSCB protein data bank (cyan blue) (B). (Line 182 – 185) Line 178 "3.2 Docking studies identified drugs with good binding affinities to the target site" - since binding affinities were not measured, this should be changed to something like "3.2 Docking studies identified drugs with potential to bind to the target site" Corrected as suggested and rephrased (Line 188) Lines 184-186: “The more negative the S score, the stronger the binding affinity, the better the ligand interaction with the protein and the more stable the complex of the drug hits with the protein complex.” Again, language should indicate this is all predicted and not experimentally tested. It has been rephrased accordingly (Line 195 – 196) Figure 4 – please indicate what the red and blue lines are in the figure itself for better readability (it appears in the text). For the text and legend of figure 4, please indicate the drug name (it is only listed as “the top hit”). Corrected as directed. Legend inserted for the figure (now Figure 6) and top hit now specified (Line 305 – 314). Line 253 – “Top-ranked drug hits identified in the study could inhibit protein-protein interaction between the SARS-CoV-2 spike protein and hACE2.” This sentence is misleading, as it suggests experimental evidence of inhibiting the binding. Please delete it. Done. Deleted Lines 261-263: “Similar hinderance to protein–protein interaction necessary for the establishment of the infection by coronavirus was observed for all of the top six ranked drug hits.” – please provide the data, either in the text or a supplementary file. Statement has been deleted. Experiment was conducted on one predicted drug hit and presented as such. Figure 5 – please indicate what the red and blue lines are in the figure itself for better readability (it appears in the text). Done as suggested (see Figure 5). Line 280 – “from our previous study”. Please provide a reference Statement rephrased. Lines 289-291: “The molecular docking studies identified drugs that bind strongly to the interacting surface of SARS-CoV-2 spike protein hACE2 complex.”. Please change to indicate these MAY bind strongly, as it is only predicted. Statement rephrased to suggest that it was a prediction (340 – 342). Lines 322-326: “Although a number of known drugs including hydroxychloroquine [5], Azithromycin [5], remdesivir [6], and idasanutlin [7] have been proposed and reported to be effective in the treatment of COVID-19, however results from our study suggest that hydroxychloroquine and remdesivir may have lower potential (as evidenced by binding free energy lower than -5.0 kcal/mol) to inhibit the interaction between the spike protein and hACE2.” There are several problems here. One – azithromycin and hydroxychloroquine have been widely shown to not be effective in treating COVID-19 patients, and should be removed from this sentence. Two – remdesivir is an RdRp inhibitor and is not expected to interfere with Spike binding to Ace2. Three- Ref #7 is a “thought experiment” is using idasanutlin to treat COVID-19, again in a mechanism unrelated to Spike-Ace2 binding. I suggest you delete these sentances. Deleted as suggested. In the Conclusion section (lines 327-337), please rephrase language to indicate potential inhibition rather than actual inhibition. Done! Attachments Attachment Submitted filename: Egieyeh Response to Reviewers Letter.docx https://doi.org/10.1371/journal.pone.0245258.r002
28 Dec 2020 Decision Letter - Israel Silman, Editor Computational drug repurposing strategy predicted peptide based drugs that can potentially inhibit the interaction of SARS-CoV-2 Spike proteins with its target (humanACE2) PONE-D-20-25309R1 Dear Dr. Egieyeh, 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, Israel Silman 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 Reviewer #2: 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 Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: N/A ****** 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: 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 basically addressed the comments. It is thus recommended to accept this manuscript for publication. Reviewer #2: (No Response) ****** 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 https://doi.org/10.1371/journal.pone.0245258.r003
Formally Accepted
30 Dec 2020 Acceptance Letter - Israel Silman, Editor PONE-D-20-25309R1 Computational drug repurposing strategy predicted peptide-based drugs that can potentially inhibit the interaction of SARS-CoV-2 spike protein with its target (humanACE2) Dear Dr. Egieyeh: 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 Prof. Israel Silman Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0245258.r004

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