A drug repurposing screen identifies hepatitis C antivirals as inhibitors of the SARS-CoV2 main protease

  1. Jeremy D. Baker
  2. Rikki L. Uhrich
  3. Gerald C. Kraemer
  4. Jason E. Love
  5. Brian C. Kraemer

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Abstract

Effective SARS-CoV-2 antiviral drugs are desperately needed. The SARS-CoV-2 main protease (Mpro) appears as an attractive target for drug development. We show that the existing pharmacopeia contains many drugs with potential for therapeutic repurposing as selective and potent inhibitors of SARS-CoV-2 Mpro. We screened a collection of ~6,070 drugs with a previous history of use in humans for compounds that inhibit the activity of Mpro in vitro and found ~50 compounds with activity against Mpro. Subsequent dose validation studies demonstrated 8 dose responsive hits with an IC50 ≤ 50 μM. Hits from our screen are enriched with hepatitis C NS3/4A protease targeting drugs including boceprevir, ciluprevir. narlaprevir, and telaprevir. This work suggests previous large-scale commercial drug development initiatives targeting hepatitis C NS3/4A viral protease should be revisited because some previous lead compounds may be more potent against SARS-CoV-2 Mpro than boceprevir and suitable for rapid repurposing.

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  1. Version published to 10.1371/journal.pone.0245962
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    Reply to the reviewers

    We would like to express our appreciation for both the Editors’ and Reviewers’ efforts as essential contributions to the peer review process. We highly value the Reviewers’ constructive critique of our manuscript#RC-2020_00434R entitled “__A drug repurposing screen identifies hepatitis C antivirals as inhibitors of the SARS-CoV2 main protease.____” __

    We appreciate the Reviewers’ thoughtful consideration of our work and feel their critiques and recommendations have significantly improved our manuscript. Taken together, we believe the additional data, clarification of data presentation, and revised discussion address the heart of the Reviewers’ previous concerns. Thus we feel the work is ready for reconsideration and will be an impactful addition to the literature appropriate for publication. Below we provide a breakdown and a point by point response to previous review critiques.

    Thank you for your attention. We look forward to your response.

    Best Wishes,

    Brian Kraemer, PhD ▪ Associate Director for Research Geriatric Research Education and Clinical Center ▪ Veterans Affairs Puget Sound Health Care System ▪ Research Professor ▪ Departments of Medicine, Psychiatry and Behavioral Sciences, and Pathology ▪ University of Washington ▪ 1660 South Columbian Way ▪ Seattle, WA 98108 ▪ Phone 206-277-1071 ▪ www.kraemerlab.uw.edu

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    In this manuscript, Baker et al. report the screening of a collection of ~6,070 drugs for their inhibitory activity against the enzymatic activity of the SARS-SoV-2 Mpro protein in vitro using two peptide substrates. 50 compounds with activity against Mpro were identified and tested for their dose-dependent effect in the same assay. Several hits were identified, among which are approved drugs that target the HCV protease.

    Indeed, there is an urgent need for effective drugs for SARS-CoV-2 infection, and high throughput screenings can discover novel candidates. However, the novelty of this work is quite limited, as former screens have been published with the same target using the same substrates. Moreover, as discussed below the translational impact of the hits discussed is also quite limited, particularly in the absence of antiviral data. Lastly, there are several overstatements in the write up and it will require major editing.

    **Major comments:**

    1. Were there any positive controls previously shown to potently inhibit the SARS-CoV-2 Mpro included in the screen (e.g. ebselen)? How did these perform in this assay? When first designing our protease assay, we did use ebselen as the initial control. Ebselen showed low potency in all our in our assays and was not considered as a positive control subsequently. It should be noted that Ebselen failed to work against multiple substrates. It is possible that our buffer conditions prevented Ebselen activity. See data plotted below. After identifying boceprevir as a potent inhibitor, it was used in all subsequent assays as a positive control.

    It will be helpful if the authors would provide info re the 50 hits from prior screens conducted with this library of compounds - how promiscuous are they across screens? How toxic in cell based assays?

    We have updated the table to provide additional useful information as well as a footnote explaining statuses. The compounds in the Broad repurposing library are generally non-toxic and information about them can be found here: https://clue.io/repurposing

    The translational potential of the findings appears to be limited. The calculated IC50s for these drugs in the Mpro assay are very high (10-1000 fold higher) relative to their IC50 in an enzymatic assay involving the HCV protease (Boceprevir: IC50 = 0.95 μM vs. 0.084 μM in HCV), Ciluprevir (IC50 = 20.77 μM vs. 0.0087 in HCV), Telaprevir (IC50 = 15.25 μM vs.0.050 μM in HCV) (https://aac.asm.org/content/aac/57/12/6236.full.pdf ). In the absence of antiviral data, the main statement of the manuscript that "the work presented here supports the rapid evaluation of previous HCV NS3/4A inhibitors for repurposing as a COVID-19 therapy." is thus an overstatement. Even is there is some activity, since likely to be limited, as with the HIV protease inhibitors, its chances to elicit a meaningful clinical effect is low. Moreover, when used in monotherapy, some of these protease inhibitors have a very low genetic barrier to resistance.

    We have reworked the discussion to incorporate these concerns and limitations of our results.

    There are additional inaccurate or overstatements - e.g. line 61 "Probably the most successful approved antivirals are protease inhibitors such as atazanavir for HIV-1 and simeprevir for hepatitis C. [reviewed in 10 and 11]."

    We have reworded this statement: (Page 4, Lines 61-62)

    “There is precedence for targeting the protease, as this approach has been successful in treating both HIV-1 and hepatitis C (10,11).”

    The manuscript requires editing - e.g. structure of sentences, commas, spacing (including in the abstract) etc.

    The manuscript has been re-proofed throughout (see tracked changes version of manuscript)

    What is the take home message? The statement "Taken together this work suggests previous large-scale commercial drug development initiatives targeting hepatitis C NS3/4A viral protease should be revisited because some previous lead compounds may be more potent against SARS-CoV-2 Mpro than Boceprevir and suitable for rapid repurposing." is unclear.

    The take home message of the manuscript is that HCV-targeting protease inhibitors have potential in blocking the SARS-Cov2 protease and a more thorough analysis of the space is needed. As the reviewer pointed out, the identified hits boceprevir and narlaprevir are less potent when targeting the SARS-Cov2 protease as compared to the HCV protease. However, we believe this work does show the potential for screening HCV-targeting protease inhibitors that may not have made it to the clinic. For instance, Boceprevir or Narlaprevir analogs may be even more potent against Mrpo. Further, we believe that these compounds would benefit from further optimization through medicinal chemistry.

    We have expanded the discussion to incorporate issues brought up here and in point 3.

    Reviewer #1 (Significance (Required)):

    Limited. As discussed above

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    SARS-CoV-2 pandemic causing serious health crisis globally. There are no specific medicine or vaccines to contain this virus currently. To address this issue, the authors developed one efficient fluorescent Mpro assay system and screened ~6070 previous used drugs in this article. Several compounds with activity against SARS-CoV-2 Mpro in vitro were founded. Most hits are hepatitis C NS3/4A protease inhibitors with fair IC50 value. Besides, the authors found that most identified compounds in in silico screen lack activity against Mpro in kinetic protease assays.

    These research results are well proved and reproducible. But there are two minor questions I present below:

    1. In your Mpro assay optimization process you said substrate MCA-AVLQSGFR-K(Dnp)- K-NH2 had drastically lower rates of Mpro catalyzed hydrolysis and were not considered further in your assay development. And in your Fig.1 I saw extremely low RFU changes. But several nice inhibitors were screened using this substrate that was reported in April. Can you explain this result? The substrates used in our assay appear to be much more efficiently cleaved at least with our buffer conditions and Mpro concentrations tested. Variables including recombinant Mpro purity and activity, differences in assay buffer, reader sensitivity may all play a role, but our best guess is that the substrate identified by Marcin Drag’s group (https://doi.org/10.1101/2020.04.29.068890), is more readily cleaved by Mpro. Although screening with other reported substrates is feasible given previous results, we believe the Ac-Abu-Tle-Leu-Gln-AFC to be superior for use in high throughput screening because of its superior cleavage kinetics yielding an improved signal to background ratio for HTS.

    To exclude inhibitors possibly acting as aggregators, a detergent-based control should do at the same time when you do IC50 value measurement.

    Compound aggregation is a concern, and our assays were all run with detergent in the buffer. Our buffer composition was 20mM Tris pH 7.8, 150mM NaCl, 1mM EDTA, 1mM DTT, 0.05% Triton X-100.

    Reviewer #2 (Significance (Required)):

    Nice work but the significance of this article is losing now. Most screened hits are reported in the last serval months. Some inhibitor complex structures have been published or released on Protein Data Bank. The novelty is missing. I suggest the authors add more results and resubmit it again.

    **Referees Cross-commenting**

    I agree with the other two reviewers' comments. The significance of this work is losing but still has something interest. I think it can be published in the lower-impact journal if they complete our suggestions

    We concur with both reviewers that demonstration of antiviral activity would strengthen the impact of the manuscript. However, this work remains outside of the scope of feasibility at our institution. We believe that our screen and hit identification can stand on their own until further translational work can be completed.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    In this report, Baker et al. show that four inhibitors of hepatitis C virus (HCV) NS3/4 protease (ciluprevir, boceprevir, narlaprevir and telaprevir) are also effective inhibitors of the SARS-CoV-2 main protease (Mpro) in enzymatic assays, with lower IC50 values for narlaprevir and boceprevir (around 1 µM in their assay conditions). HCV NS3/4 inhibitors were identified after screening a library of >6,000 compounds of the Broad Institute, including approved drugs. Screening was done with fluorometric proteolytic assays.

    Experiments have been apparently well-done and results are sound. The manuscript needs editing.

    Reviewer #3 (Significance (Required)):

    Experiments have been apparently well-done and results are sound. However, this is a limited study since there are no data obtained in cell culture and a comparison of IC50 values of the selected drugs against HCV and SARS-CoV-2 proteases is missing. It is difficult to infer whether the drugs would be equally effective against SARS-CoV-2 than against HCV, and otherwise, how much should the doses increase in order to have a therapeutic effect.

    The manuscript needs editing (see below) and the Discussion is poor. The results reported by authors are not new, and a discussion of the effects of HCV inhibitors on SARS-CoV-2 replication, based on previous publications is necessary to provide the appropriate context for the study.

    Here are some references on Covid-19 and HCV inhibitors, that in my opinion should be considered for discussion and proper citation. As correctly pointed out by Baker and co- workers, docking studies should be considered with caution, though.

    We appreciate the feedback and have now reworked and expanded the discussion to incorporate reviewer #1 and #3 comments and suggestions.

    1: Ghahremanpour MM, Tirado-Rives J, Deshmukh M, Ippolito JA, Zhang CH, de Vaca IC, Liosi ME, Anderson KS, Jorgensen WL. Identification of 14 Known Drugs as Inhibitors of the Main Protease of SARS-CoV-2. bioRxiv [Preprint]. 2020 Aug 28:2020.08.28.271957. doi: 10.1101/2020.08.28.271957. PMID: 32869018; PMCID: PMC7457600.

    2: Sacco MD, Ma C, Lagarias P, Gao A, Townsend JA, Meng X, Dube P, Zhang X, Hu Y, Kitamura N, Hurst B, Tarbet B, Marty MT, Kolocouris A, Xiang Y, Chen Y, Wang J. Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L. bioRxiv [Preprint]. 2020 Jul 27:2020.07.27.223727. doi: 10.1101/2020.07.27.223727. PMID: 32766590; PMCID: PMC7402059.

    3: Ma C, Sacco MD, Hurst B, Townsend JA, Hu Y, Szeto T, Zhang X, Tarbet B, Marty MT, Chen Y, Wang J. Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2viral replication by targeting the viral main protease. Cell Res. 2020 Aug;30(8):678-692. doi: 10.1038/s41422-020-0356-z. Epub 2020 Jun 15. PMID: 32541865; PMCID: PMC7294525.

    4: Ke YY, Peng TT, Yeh TK, Huang WZ, Chang SE, Wu SH, Hung HC, Hsu TA, Lee SJ, Song JS, Lin WH, Chiang TJ, Lin JH, Sytwu HK, Chen CT. Artificial intelligence approach fighting COVID-19 with repurposing drugs. Biomed J. 2020 May 15:S2319- 4170(20)30049-4. doi: 10.1016/j.bj.2020.05.001. Epub ahead of print. PMID: 32426387; PMCID: PMC7227517.

    5: Elzupir AO. Inhibition of SARS-CoV-2 main protease 3CLpro by means of α-ketoamide and pyridone-containing pharmaceuticals using in silico molecular docking. J Mol Struct. 2020 Dec 15;1222:128878. doi: 10.1016/j.molstruc.2020.128878. Epub 2020 Jul 10.

    PMID: 32834113; PMCID: PMC7347502.

    Additional computational studies:

    1: Hosseini FS, Amanlou M. Anti-HCV and anti-malaria agent, potential candidates to repurpose for coronavirus infection: Virtual screening, molecular docking, and molecular dynamics simulation study. Life Sci. 2020 Aug 8;258:118205. doi:10.1016/j.lfs.2020.118205. Epub ahead of print. PMID: 32777300; PMCID:PMC7413873.

    2: Hakmi M, Bouricha EM, Kandoussi I, Harti JE, Ibrahimi A. Repurposing of known anti- virals as potential inhibitors for SARS-CoV-2 main protease using molecular docking analysis. Bioinformation. 2020 Apr 30;16(4):301-306. doi:10.6026/97320630016301.

    PMID: 32773989; PMCID: PMC7392094.

    3: Chtita S, Belhassan A, Aouidate A, Belaidi S, Bouachrine M, Lakhlifi T. Discovery of Potent SARS-CoV-2 Inhibitors from Approved Antiviral Drugs via Docking Screening. Comb Chem High Throughput Screen. 2020 Jul 30. doi:10.2174/1386207323999200730205447. Epub ahead of print. PMID: 32748740.

    4: Alamri MA, Tahir Ul Qamar M, Mirza MU, Bhadane R, Alqahtani SM, Muneer I, Froeyen M, Salo-Ahen OMH. Pharmacoinformatics and molecular dynamics simulation studies reveal potential covalent and FDA-approved inhibitors of SARS-CoV-2 main protease 3CLpro. J Biomol Struct Dyn. 2020 Jun 24:1-13. doi:10.1080/07391102.2020.1782768. Epub ahead of print. PMID: 32579061; PMCID:PMC7332866.

    5: Bafna K, Krug RM, Montelione GT. Structural Similarity of SARS-CoV2 Mpro and HCV NS3/4A Proteases Suggests New Approaches for Identifying Existing Drugs Useful as COVID-19 Therapeutics. ChemRxiv [Preprint]. 2020 Apr 21. doi: 10.26434/chemrxiv.12153615. PMID: 32511291; PMCID: PMC7263768.

    6: Eleftheriou P, Amanatidou D, Petrou A, Geronikaki A. In Silico Evaluation of the Effectivity of Approved Protease Inhibitors against the Main Protease of the Novel SARS- CoV-2 Virus. Molecules. 2020 May 29;25(11):2529. doi:10.3390/molecules25112529.

    PMID: 32485894; PMCID: PMC7321236.

    7: Wang J. Fast Identification of Possible Drug Treatment of Coronavirus Disease-19 (COVID-19) through Computational Drug Repurposing Study. J Chem Inf Model. 2020 Jun 22;60(6):3277-3286. doi: 10.1021/acs.jcim.0c00179. Epub 2020 May 4. PMID: 32315171; PMCID: PMC7197972.

    8: Chen YW, Yiu CB, Wong KY. Prediction of the SARS-CoV-2 (2019-nCoV) 3C-like protease (3CL pro) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates. F1000Res. 2020 Feb 21;9:129. doi: 10.12688/f1000research.22457.2. PMID: 32194944; PMCID: PMC7062204.

    Minor comments:

    We appreciate the time that the reviewer has taken to address grammatical changes and have addressed each throughout the manuscript with tracked changes.

    p.2, line 26: > appears as an attractive

    Manuscript edited

    p.2, line 27: > we show that the existing

    Manuscript edited

    p.2, line 33: > separate numbers and units, eg. 1.10 µM (this is a persisting error that should be corrected throughout the whole ms)

    Manuscript edited

    p.4, line 44: SARS virus should be referred as to SARS-CoV-1 throughout the whole manuscript. MERS-CoV is the name of the virus causing MERS

    Manuscript edited

    p.4, lines 61-62: > the selection of the specific compounds seems to be arbitrary... why atazanavir and not darunavir or other? The sentence should be rewritten.

    Rewritten as: “There is precedence for targeting the protease, as this approach has been successful in treating both HIV-1 and hepatitis C.”

    p.6, line 100: Citing Fig. 2B before completing the description of Fig. 1 is distracting. Authors should think of a better way to describe their results.

    This was a mistake and should have cited Fig 1B. Thank you for catching this.

    p.7, line 116: It is not clear what "10m-20,810" means

    This has been clarified to state: “ΔRFU at 10 minutes = 20,810 relative fluorescence units”

    p.7, lines 125-126: These sentences belong to an introduction, not appropriate in results section.

    We have removed these sentences.

    Figure 2. Part A is not necessary in results (ok for introduction). Black and purple dots in part B is not a good choice since they are difficult to distinguish, maybe orange and black is better.

    We have removed panel A, expanded the size of panel B and changed the color.

    Table 1: Status should be explained in a footnote (i.e the distinction between launched, P2/P3, phase 2, preclinical is not clear).

    The one compound indicated in P2/P3 development is now Phase 3 and the table has been updated. We have added a footnote:

    *Launched = compound approved for humans, though may only be approved for veterinary use in some countries

    Discussion. I think that subheadings are not necessary.

    Subheadings have been removed from the discussion.

    **Referees cross-commenting** I agree with reviewer no. 1 on the limited interest of the study. However, it could be published in a specialized lower-impact journal after addressing issues raised by reviewers 2 and 3 (likely to be completed in less than a month)

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    Referee #3

    Evidence, reproducibility and clarity

    In this report, Baker et al. show that four inhibitors of hepatitis C virus (HCV) NS3/4 protease (ciluprevir, boceprevir, narlaprevir and telaprevir) are also effective inhibitors of the SARS-CoV-2 main protease (Mpro) in enzymatic assays, with lower IC50 values for narlaprevir and boceprevir (around 1 µM in their assay conditions). HCV NS3/4 inhibitors were identified after screening a library of >6,000 compounds of the Broad Institute, including approved drugs. Screening was done with fluorometric proteolytic assays.

    Experiments have been apparently well-done and results are sound. The manuscript needs editing.

    Significance

    Experiments have been apparently well-done and results are sound. However, this is a limited study since there are no data obtained in cell culture and a comparison of IC50 values of the selected drugs against HCV and SARS-CoV-2 proteases is missing. It is difficult to infer whether the drugs would be equally effective against SARS-CoV-2 than against HCV, and otherwise, how much should the doses increase in order to have a therapeutic effect. The manuscript needs editing (see below) and the Discussion is poor. The results reported by authors are not new, and a discussion of the effects of HCV inhibitors on SARS-CoV-2 replication, based on previous publications is necessary to provide the appropriate context for the study. Here are some references on Covid-19 and HCV inhibitors, that in my opinion should be considered for discussion and proper citation. As correctly pointed out by Baker and co-workers, docking studies should be considered with caution, though.

    1: Ghahremanpour MM, Tirado-Rives J, Deshmukh M, Ippolito JA, Zhang CH, de Vaca IC, Liosi ME, Anderson KS, Jorgensen WL. Identification of 14 Known Drugs as Inhibitors of the Main Protease of SARS-CoV-2. bioRxiv [Preprint]. 2020 Aug 28:2020.08.28.271957. doi: 10.1101/2020.08.28.271957. PMID: 32869018; PMCID: PMC7457600.

    2: Sacco MD, Ma C, Lagarias P, Gao A, Townsend JA, Meng X, Dube P, Zhang X, Hu Y, Kitamura N, Hurst B, Tarbet B, Marty MT, Kolocouris A, Xiang Y, Chen Y, Wang J. Structure and inhibition of the SARS-CoV-2 main protease reveals strategy for developing dual inhibitors against Mpro and cathepsin L. bioRxiv [Preprint]. 2020 Jul 27:2020.07.27.223727. doi: 10.1101/2020.07.27.223727. PMID: 32766590; PMCID: PMC7402059.

    3: Ma C, Sacco MD, Hurst B, Townsend JA, Hu Y, Szeto T, Zhang X, Tarbet B, Marty MT, Chen Y, Wang J. Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease. Cell Res. 2020 Aug;30(8):678-692. doi: 10.1038/s41422-020-0356-z. Epub 2020 Jun 15. PMID: 32541865; PMCID: PMC7294525.

    4: Ke YY, Peng TT, Yeh TK, Huang WZ, Chang SE, Wu SH, Hung HC, Hsu TA, Lee SJ, Song JS, Lin WH, Chiang TJ, Lin JH, Sytwu HK, Chen CT. Artificial intelligence approach fighting COVID-19 with repurposing drugs. Biomed J. 2020 May 15:S2319-4170(20)30049-4. doi: 10.1016/j.bj.2020.05.001. Epub ahead of print. PMID: 32426387; PMCID: PMC7227517.

    5: Elzupir AO. Inhibition of SARS-CoV-2 main protease 3CLpro by means of α-ketoamide and pyridone-containing pharmaceuticals using in silico molecular docking. J Mol Struct. 2020 Dec 15;1222:128878. doi: 10.1016/j.molstruc.2020.128878. Epub 2020 Jul 10. PMID: 32834113; PMCID: PMC7347502.

    Additional computational studies:

    1: Hosseini FS, Amanlou M. Anti-HCV and anti-malaria agent, potential candidates to repurpose for coronavirus infection: Virtual screening, molecular docking, and molecular dynamics simulation study. Life Sci. 2020 Aug 8;258:118205. doi:10.1016/j.lfs.2020.118205. Epub ahead of print. PMID: 32777300; PMCID:PMC7413873.

    2: Hakmi M, Bouricha EM, Kandoussi I, Harti JE, Ibrahimi A. Repurposing of known anti-virals as potential inhibitors for SARS-CoV-2 main protease using molecular docking analysis. Bioinformation. 2020 Apr 30;16(4):301-306. doi:10.6026/97320630016301. PMID: 32773989; PMCID: PMC7392094.

    3: Chtita S, Belhassan A, Aouidate A, Belaidi S, Bouachrine M, Lakhlifi T. Discovery of Potent SARS-CoV-2 Inhibitors from Approved Antiviral Drugs via Docking Screening. Comb Chem High Throughput Screen. 2020 Jul 30. doi:10.2174/1386207323999200730205447. Epub ahead of print. PMID: 32748740.

    4: Alamri MA, Tahir Ul Qamar M, Mirza MU, Bhadane R, Alqahtani SM, Muneer I, Froeyen M, Salo-Ahen OMH. Pharmacoinformatics and molecular dynamics simulation studies reveal potential covalent and FDA-approved inhibitors of SARS-CoV-2 main protease 3CLpro. J Biomol Struct Dyn. 2020 Jun 24:1-13. doi:10.1080/07391102.2020.1782768. Epub ahead of print. PMID: 32579061; PMCID:PMC7332866.

    5: Bafna K, Krug RM, Montelione GT. Structural Similarity of SARS-CoV2 Mpro and HCV NS3/4A Proteases Suggests New Approaches for Identifying Existing Drugs Useful as COVID-19 Therapeutics. ChemRxiv [Preprint]. 2020 Apr 21. doi: 10.26434/chemrxiv.12153615. PMID: 32511291; PMCID: PMC7263768.

    6: Eleftheriou P, Amanatidou D, Petrou A, Geronikaki A. In Silico Evaluation of the Effectivity of Approved Protease Inhibitors against the Main Protease of the Novel SARS-CoV-2 Virus. Molecules. 2020 May 29;25(11):2529. doi:10.3390/molecules25112529. PMID: 32485894; PMCID: PMC7321236.

    7: Wang J. Fast Identification of Possible Drug Treatment of Coronavirus Disease-19 (COVID-19) through Computational Drug Repurposing Study. J Chem Inf Model. 2020 Jun 22;60(6):3277-3286. doi: 10.1021/acs.jcim.0c00179. Epub 2020 May 4. PMID: 32315171; PMCID: PMC7197972.

    8: Chen YW, Yiu CB, Wong KY. Prediction of the SARS-CoV-2 (2019-nCoV) 3C-like protease (3CL pro) structure: virtual screening reveals velpatasvir, ledipasvir, and other drug repurposing candidates. F1000Res. 2020 Feb 21;9:129. doi: 10.12688/f1000research.22457.2. PMID: 32194944; PMCID: PMC7062204.

    Minor comments:

    p.2, line 26: > appears as an attractive

    p.2, line 27: > we show that the existing

    p.2, line 33: > separate numbers and units, eg. 1.10 µM (this is a persisting error that should be corrected throughout the whole ms)

    p.4, line 44: SARS virus should be referred as to SARS-CoV-1 throughout the whole manuscript. MERS-CoV is the name of the virus causing MERS

    p.4, lines 61-62: > the selection of the specific compounds seems to be arbitrary... why atazanavir and not darunavir or other? The sentence should be rewritten.

    p.6, line 100: Citing Fig. 2B before completing the description of Fig. 1 is distracting. Authors should think of a better way to describe their results.

    p.7, line 116: It is not clear what "10m-20,810" means

    p.7, lines 125-126: These sentences belong to an introduction, not appropriate in results section.

    Figure 2. Part A is not necessary in results (ok for introduction). Black and purple dots in part B is not a good choice since they are difficult to distinguish, maybe orange and black is better.

    Table 1: Status should be explained in a footnote (i.e the distinction between launched, P2/P3, phase 2, preclinical is not clear).

    Discussion. I think that subheadings are not necessary.

    Referees cross-commenting

    I agree with reviewer no. 1 on the limited interest of the study. However, it could be published in a specialized lower-impact journal after addressing issues raised by reviewers 2 and 3 (likely to be completed in less than a month)

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    Referee #2

    Evidence, reproducibility and clarity

    SARS-CoV-2 pandemic causing serious health crisis globally. There are no specific medicine or vaccines to contain this virus currently. To address this issue, the authors developed one efficient fluorescent Mpro assay system and screened ~6070 previous used drugs in this article. Several compounds with activity against SARS-CoV-2 Mpro in vitro were founded. Most hits are hepatitis C NS3/4A protease inhibitors with fair IC50 value. Besides, the authors found that most identified compounds in in silico screen lack activity against Mpro in kinetic protease assays.

    These research results are well proved and reproducible. But there are two minor questions I present below:

    1.In your Mpro assay optimization process you said substrate MCA-AVLQSGFR-K(Dnp)-K-NH2 had drastically lower rates of Mpro catalyzed hydrolysis and were not considered further in your assay development. And in your Fig.1 I saw extremely low RFU changes. But several nice inhibitors were screened using this substrate that was reported in April. Can you explain this result?

    2.To exclude inhibitors possibly acting as aggregators, a detergent-based control should do at the same time when you do IC50 value measurement.

    Significance

    Nice work but the significance of this article is losing now. Most screened hits are reported in the last serval months. Some inhibitor complex structures have been published or released on Protein Data Bank. The novelty is missing. I suggest the authors add more results and resubmit it again.

    Referees Cross-commenting

    I agree with the other two reviewers' comments. The significance of this work is losing but still has something interest. I think it can be published in the lower-impact journal if they complete our suggestions

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Baker et al. report the screening of a collection of ~6,070 drugs for their inhibitory activity against the enzymatic activity of the SARS-SoV-2 Mpro protein in vitro using two peptide substrates. 50 compounds with activity against Mpro were identified and tested for their dose-dependent effect in the same assay. Several hits were identified, among which are approved drugs that target the HCV protease.
    Indeed, there is an urgent need for effective drugs for SARS-CoV-2 infection, and high throughput screenings can discover novel candidates. However, the novelty of this work is quite limited, as former screens have been published with the same target using the same substrates. Moreover, as discussed below the translational impact of the hits discussed is also quite limited, particularly in the absence of antiviral data. Lastly, there are several overstatements in the write up and it will require major editing.

    Major comments:

    1.Were there any positive controls previously shown to potently inhibit the SARS-CoV-2 Mpro included in the screen (e.g. ebselen)? How did these perform in this assay?

    2.It will be helpful if the authors would provide info re the 50 hits from prior screens conducted with this library of compounds - how promiscuous are they across screens? How toxic in cell based assays?

    3.The translational potential of the findings appears to be limited. The calculated IC50s for these drugs in the Mpro assay are very high (10-1000 fold higher) relative to their IC50 in an enzymatic assay involving the HCV proteast (Boceprevir: IC50 = 0.95 μM vs. 0.084 μM in HCV), Ciluprevir (IC50 = 20.77 μM vs. 0.0087 in HCV), Telaprevir (IC50 = 15.25 μM vs. 0.050 μM in HCV) (https://aac.asm.org/content/aac/57/12/6236.full.pdf ). In the absence of antiviral data, the main statement of the manuscript that "the work presented here supports the rapid evaluation of previous HCV NS3/4A inhibitors for repurposing as a COVID-19 therapy." is thus an overstatement. Even is there is some activity, since likely to be limited, as with the HIV protease inhibitors, its chances to elicit a meaningful clinical effect is low. Moreover, when used in monotherapy, some of these protease inhibitors have a very low genetic barrier to resistance.

    4.There are additional inaccurate or overstatements - e.g. line 61 "Probably the most successful approved antivirals are protease inhibitors such as atazanavir for HIV-1 and simeprevir for hepatitis C. [reviewed in 10 and 11]."

    5.The manuscript requires editing - e.g. structure of sentences, commas, spacing (including in the abstract) etc.

    6.What is the take home message? The statement "Taken together this work suggests previous large-scale commercial drug development initiatives targeting hepatitis C NS3/4A viral protease should be revisited because some previous lead compounds may be more potent against SARS-CoV-2 Mpro than Boceprevir and suitable for rapid repurposing." is unclear.

    Significance

    Limited. As discussed above

  6. Version published to 10.1101/2020.07.10.197889v2 on bioRxiv
  7. ScreenIT

    SciScore for 10.1101/2020.07.10.197889: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Data was analyzed using Biotek Gen5 software, Microsoft Excel, and GraphPad Prism 8.
    Gen5
    suggested: (Gen5, RRID:SCR_017317)
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Inhibition was calculated as at each concentration and data fitted to 4-parameter nonlinear regression model using GraphPad Prism 8.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    In Silico Docking of the Broad Repurposing Library with Mpro: We utilized the Schrodinger Maestro software package 29,30 to conduct a computational docking of all compounds in the Broad Repurposing library.
    Schrodinger Maestro
    suggested: None

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    This is also particularly germane given the limitations of animal models of COVID-19 infection and pathogenesis. A diverse variety of initial hits were identified in our high throughput screen of the broad library. Of these, the most potent hits are all known protease inhibitors and there is strong representation from protease inhibitors developed to inhibit HCV protease NS3/4A (Boceprevir, Ciluprevir, Narlaprevir, and Telaprevir). Clearly as approved or well-developed clinical candidates, these drugs exhibit pharmacological and pharmacodynamic properties well suited to repurposing as a COVID-19 antiviral therapy. Boceprevir and Narlaprevir appear the most potent against Mpro and may be suitable for repurposing. Previous clinical evaluation of Boceprevir (also known as Victrelis) showed it to be safe and effective for treating HCV 37. Boceprevir was approved as a first in class HCV NS3/4A serine protease inhibitor for treatment of chronic HCV infection. Boceprevir was FDA approved for use in the USA in 2011 and Boceprevir treatment is given as a combination therapy with interferon α2b and ribavirin. Likewise, clinical evaluation of Narlaprevir (also known as Arlansa or SCH900518) showed it to be both safe and to exhibit antiviral activity when combined with interferon α2b 38. Furthermore, Narlaprevir has been show effective against HCV NS3/4A mutations causing resistance to protease inhibitors 39. Narlaprevir was approved for use against HCV in Russia in 2016. Our findings de...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  8. Version published to 10.1101/2020.07.10.197889v1 on bioRxiv