Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography

  1. Omur Guven
  2. Mehmet Gul
  3. Esra Ayan
  4. J Austin Johnson
  5. Baris Cakilkaya
  6. Gozde Usta
  7. Fatma Betul Ertem
  8. Nurettin Tokay
  9. Busra Yuksel
  10. Oktay Gocenler
  11. Cengizhan Buyukdag
  12. Sabine Botha
  13. Gihan Ketawala
  14. Zhen Su
  15. Brandon Hayes
  16. Frederic Poitevin
  17. Alexander Batyuk
  18. Chun Hong Yoon
  19. Christopher Kupitz
  20. Serdar Durdagi
  21. Raymond G. Sierra
  22. Hasan DeMirci

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Abstract

Since early 2020, COVID-19 has grown to affect the lives of billions globally. A worldwide investigation has been ongoing for characterizing the virus and also for finding an effective drug and developing vaccines. As time has been of the essence, a crucial part of this research has been drug repurposing; therefore, confirmation of in silico drug screening studies have been carried out for this purpose. Here we demonstrated the possibility of screening a variety of drugs efficiently by leveraging a high data collection rate of 120 images/second with the new low-noise, high dynamic range ePix10k2M Pixel Array Detector installed at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS). The X-ray Free-Electron Laser (XFEL) is used for remote high-throughput data collection for drug repurposing of the main protease (Mpro) of SARS-CoV-2 at ambient temperature with mitigated X-ray radiation damage. We obtained multiple structures soaked with nine drug candidate molecules in two crystal forms. Although our drug binding attempts failed, we successfully established a high-throughput Serial Femtosecond X-ray crystallographic (SFX) data collection protocol.

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  1. Version published to 10.3390/cryst11121579
  2. ScreenIT

    SciScore for 10.1101/2021.11.28.468932: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Experimental Models: Organisms/Strains
    SentencesResources
    Bacterial transformations were performed on a chemically competent Escherichia coli BL21 Rosetta-2 strain for both constructs.
    Rosetta-2
    suggested: None
    Software and Algorithms
    SentencesResources
    During the data collection the MESH injector system had no blockages.
    MESH
    suggested: (MeSH, RRID:SCR_004750)
    We performed our initial rigid-body refinements through the PHENIX [40] software package.
    PHENIX
    suggested: (Phenix, RRID:SCR_014224)
    The water molecules and strong difference density positions of the structures were checked using the COOT [41] software.
    COOT
    suggested: (Coot, RRID:SCR_014222)
    Structural alignments and all figures were generated using the PyMOL [42] software.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    To overcome this limitation, we performed our soaking experiments with two different crystal forms to increase the likelihood of drug binding. Despite our attempts, we obtained structural results with only subtle differences in the active site during the HTS process. Co-crystallization, used as an alternative method, may provide more accurate structural data to demonstrate the interaction between Mpro and, not only montelukast and ebselen, but also the other drug candidates. In this study, 9 drug candidates were screened for binding in two different crystal forms. However, none of the samples displayed noticeable experimental electron densities, suggesting at 100 μg/ml concentration soaking experiments failed. Interestingly, a high amount of precipitation was observed in two drug-protein samples containing montelukast and ebselen. Montelukast is a selective leukotriene receptor antagonist and an FDA-approved drug used in the treatment of chronic and prophylactic asthma [17]. Previously, montelukast was observed to have immunomodulatory and antiviral activities against dengue and ZIKA viruses. Therefore, it was hypothesized that montelukast may also have antiviral activity against SARS-CoV-2 [27]. Studies related to SARS-CoV-2-montelukast interaction are mostly in silico- [44,45,46] and in vitro- [17,42] oriented. Accordingly, we suggest that although montelukast interacts with Mpro, it may interfere through an allosteric effect, disrupting its conformation and perturbing the ...

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

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  3. Version published to 10.1101/2021.11.28.468932v1 on bioRxiv