Cellular Activities of SARS-CoV-2 Main Protease Inhibitors Reveal Their Unique Characteristics

  1. Wenyue Cao
  2. Chia-Chuan Dean Cho
  3. Zhi Zachary Geng
  4. Xinyu R. Ma
  5. Robert Allen
  6. Namir Shaabani
  7. Erol C. Vatansever
  8. Yugendar R. Alugubelli
  9. Yuying Ma
  10. William H. Ellenburg
  11. Kai S. Yang
  12. Yuchen Qiao
  13. Henry Ji
  14. Shiqing Xu
  15. Wenshe Ray Liu

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

As an essential enzyme of SARS-CoV-2, the pathogen of COVID-19, main protease (M Pro ) triggers acute toxicity to its human cell host, an effect that can be alleviated by an M Pro inhibitor with cellular potency. By coupling this toxicity alleviation with the expression of an M Pro -eGFP fusion protein in a human cell host for straightforward characterization with fluorescent flow cytometry, we developed an effective method that allows bulk analysis of cellular potency of M Pro inhibitors. In comparison to an antiviral assay in which M Pro inhibitors may target host proteases or other processes in the SARS-CoV-2 life cycle to convene strong antiviral effects, this novel assay is more advantageous in providing precise cellular M Pro inhibition information for assessment and optimization of M Pro inhibitors. We used this assay to analyze 30 literature reported M Pro inhibitors including MPI1-9 that were newly developed aldehyde-based reversible covalent inhibitors of M Pro , GC376 and 11a that are two investigational drugs undergoing clinical trials for the treatment of COVID-19 patients in United States, boceprevir, calpain inhibitor II, calpain inhibitor XII, ebselen, bepridil that is an antianginal drug with potent anti-SARS-CoV-2 activity, and chloroquine and hydroxychloroquine that were previously shown to inhibit M Pro . Our results showed that most inhibitors displayed cellular potency much weaker than their potency in direct inhibition of the enzyme. Many inhibitors exhibited weak or undetectable cellular potency up to 10 μM. On contrary to their strong antiviral effects, 11a, calpain inhibitor II, calpain XII, ebselen, and bepridil showed relatively weak to undetectable cellular M Pro inhibition potency implicating their roles in interfering with key steps other than just the M Pro catalysis in the SARS-CoV-2 life cycle to convene potent antiviral effects. characterization of these molecules on their antiviral mechanisms will likely reveal novel drug targets for COVID-19. Chloroquine and hydroxychloroquine showed close to undetectable cellular potency to inhibit M Pro . Kinetic recharacterization of these two compounds rules out their possibility as M Pro inhibitors. Our results also revealed that MPI5, 6, 7, and 8 have high cellular and antiviral potency with both IC 50 and EC 50 values respectively below 1 μM. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular M Pro inhibition IC 50 value of 31 nM that matches closely to its strong antiviral effect with an EC 50 value of 30 nM. Given its strong cellular and antiviral potency, we cautiously suggest that MPI8 is ready for preclinical and clinical investigations for the treatment of COVID-19.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2021.06.08.447613: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Transfection and inhibition tests using pLVX-MPro-eGFP-1 and pLVX-MPro-eGFP-2: We grew 293T cells to 60% confluency and transfected them with pLVX-MPro-eGFP-1 or pLVX-using Lipofectamine 3000.
    293T
    suggested: None
    We set up five groups of experiments including 1) HEK 293T/17, 2) HEK 293T/17 + MPI8 (1 μM), 3) HEK 293T/17 cells stably expressing MPro-eGFP, 4) HEK 293T/17 cells stably expressing MPro-eGFP + MPI8 (1 μM), and 5) HEK 293T/17 or HEK 293T/17 cells stably expressing MPro-eGFP + antimycin A (1 μM).
    HEK 293T/17
    suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
    Plaque reduction neutralization tests of SARS-CoV-2 by MPI5-8: We seeded 18 × 103 Vero cells per well in flat bottom 96 well plates in a total volume of 200 uL of a culturing medium (DMEM + 10% FBS + glutamine) and incubated cells overnight at 37 °C and under 5 % CO2.
    Vero
    suggested: None
    Recombinant DNA
    SentencesResources
    16 Plasmid construction: We amplified MPro with an N-terminal KTSAVLQ sequence using two primers FRET-Mpro-for and FRET-Mpro-rev primers (Table S1) and cloned it into the pECFP-18aa-EYFP plasmid (Addgene, #109330) between XhoI and HindIII restriction sites to afford pECFP-MPro-EYFP.
    pECFP-18aa-EYFP
    suggested: None
    To facilitate the ligation of three fragments, we used a ratio of MPro, eGFP and pLVX-EF1α-IRES-Puro digested products as 3:3:1.
    pLVX-EF1α-IRES-Puro
    suggested: None
    Transfection and MPI8 inhibition tests using pECFP-MPro-EYFP: We grew 293T cells to 60% confluency and then transfected them with pECFP-MPro-EYFP using Lipofectamine 3000.
    pECFP-MPro-EYFP
    suggested: None
    Transfection and inhibition tests using pLVX-MPro-eGFP-1 and pLVX-MPro-eGFP-2: We grew 293T cells to 60% confluency and transfected them with pLVX-MPro-eGFP-1 or pLVX-using Lipofectamine 3000.
    pLVX-MPro-eGFP-1
    suggested: None
    pLVX-using
    suggested: None
    Briefly, we transfected 293T cells at 90% confluency with three plasmids including pLVX-MPro-eGFP-2, pMD2.G and psPAX2 using 30 μg/mL polyethyleneimine.
    pMD2.G
    suggested: RRID:Addgene_12259)
    psPAX2
    suggested: RRID:Addgene_12260)
    Cellular MPro inhibition analysis for 29 selected compounds: We grew HEK 293T/17 cells in high-glucose DMEM with GlutaMAX Supplement and 10% fetal bovine serum in 10 cm culture plates under 37 □ and 5% CO2 to 80%~90% and then transfected cells with the pLVX-MPro-eGFP-2 plasmid.
    pLVX-MPro-eGFP-2
    suggested: None
    Software and Algorithms
    SentencesResources
    We then analyzed these files using a self-written MATLAB program for massive data processing.
    MATLAB
    suggested: (MATLAB, RRID:SCR_001622)
    It was then plotted and fitted non-linearly with an agonist curve (three parameters) against drug concentrations in the program Prism 9 (from GraphPAD for IC50 determination.
    GraphPAD
    suggested: (GraphPad, RRID:SCR_000306)
    Initial product formation slopes at the first 5 minutes were calculated by simple linear regression and data were plotted in GraphPad Prism 9.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: 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: Please consider improving the rainbow (“jet”) colormap(s) used on page 31. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

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

    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.

  2. Version published to 10.1101/2021.06.08.447613v1 on bioRxiv