An expedited approach towards the rationale design of non-covalent SARS-CoV-2 main protease inhibitors with in vitro antiviral activity

  1. Naoya Kitamura
  2. Michael Dominic Sacco
  3. Chunlong Ma
  4. Yanmei Hu
  5. Julia Alma Townsend
  6. Xiangzhi Meng
  7. Fushun Zhang
  8. Xiujun Zhang
  9. Adis Kukuljac
  10. Michael Thomas Marty
  11. David Schultz
  12. Sara Cherry
  13. Yan Xiang
  14. Yu Chen
  15. Jun Wang

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Abstract

The main protease (M pro ) of SARS-CoV-2 is a validated antiviral drug target. Several M pro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II and XII, each containing a reactive warhead that covalently modifies the catalytic Cys145. In this study, we report an expedited drug discovery approach by coupling structure-based design and Ugi four-component (Ugi-4CR) reaction methodology to the design of non-covalent M pro inhibitors. The most potent compound 23R had cellular antiviral activity similar to covalent inhibitors such as GC376. Our designs were guided by overlaying the structure of SARS-CoV M pro + ML188 (R), a non-covalent inhibitor derived from Ug-4CR, with the X-ray crystal structures of SARS-CoV-2 M pro + calpain inhibitor XII/GC376/UAWJ247. Binding site analysis suggests a strategy of extending the P2 and P3 substitutions in ML188 (R) to achieve optimal shape complementary with SARS-CoV-2 M pro . Lead optimization led to the discovery of 23R , which inhibits SARS-CoV-2 M pro and SARS-CoV-2 viral replication with an IC 50 of 0.31 μM and EC 50 of 1.27 μM, respectively. The binding and specificity of 23R to SARS-CoV-2 M pro were confirmed in a thermal shift assay and native mass spectrometry assay. The co-crystal structure of SARS-CoV-2 M pro with 23R revealed the P2 biphenyl fits snuggly into the S2 pocket and the benzyl group in the α-methylbenzyl faces towards the core of the enzyme, occupying a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study revealed the most potent non-covalent SARS-CoV-2 M pro inhibitors reported to date and a novel binding pocket that can be explored for M pro inhibitor design.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.19.423537: (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.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-100, blocked with DMEM containing 10% FBS, and stained with a rabbit monoclonal antibody against SARS-CoV-2 NP (GeneTex, GTX635679) and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (ThermoFisher Scientific).
    SARS-CoV-2 NP
    suggested: None
    GTX635679
    suggested: (GeneTex Cat# GTX635679, RRID:AB_2888553)
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2, isolate USA-WA1/2020 (NR-52281), was obtained through BEI Resources and propagated once on VERO E6 cells before it was used for this study.
    VERO E6
    suggested: RRID:CVCL_XD71)
    Antiviral assay in Calu-3 cells: Calu-3 cells (ATCC, HTB-55) grown in Minimal Eagles Medium supplemented with 0.1% non-essential amino acids, 0.1% penicillin/streptomycin, and 10% FBS are plated in 384 well plates.
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    Calu3 cells are pretreated with controls and test drugs (in triplicate) for 2 hours prior to infection.
    Calu3
    suggested: KCLB Cat# 30055, RRID:CVCL_0609)
    Software and Algorithms
    SentencesResources
    The total number of cells, as indicated by the nuclei staining, and the fraction of the infected cells, as indicated by the NP staining, were quantified with the cellular analysis module of the Gen5 software (BioTek)
    Gen5
    suggested: (Gen5, RRID:SCR_017317)
    A non-linear regression curve fit analysis (GraphPad Prism 8) of POC Infection and cell viability versus the log10 transformed concentration values to calculate IC50 values for Infection and CC50 values for cell viability.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    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

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  2. Version published to 10.1101/2020.12.19.423537v1 on bioRxiv