Drug targeting Nsp1-ribosomal complex shows antiviral activity against SARS-CoV-2

  1. Mohammad Afsar
  2. Rohan Narayan
  3. Md Noor Akhtar
  4. Deepakash Das
  5. Huma Rahil
  6. Santhosh Kambaiah Nagaraj
  7. Sandeep M Eswarappa
  8. Shashank Tripathi
  9. Tanweer Hussain

  • Curated by eLife

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    Evaluation Summary:

    This work reports on a drug repurposing effort to target the non-structural protein 1 (Nsp1) of SARS-Cov-2, involved in suppressing host immune function. The authors use a combination of computational, in vitro and cellular assays to this end. They start by virtual screening of an FDA approved drug library, followed by biophysical assays to measure the binding affinity of the top hits as well as molecular dynamics simulations and free energy calculations to confirm binding poses. Finally, in vitro and cellular assays were used to quantify translation inhibition and rescue as well as the production of infectious virus particles. One of the FDA approved drugs, Montelukast, emerges as a promising antiviral drug candidate.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

The SARS-CoV-2 non-structural protein 1 (Nsp1) contains an N-terminal domain and C-terminal helices connected by a short linker region. The C-terminal helices of Nsp1 (Nsp1-C-ter) from SARS-CoV-2 bind in the mRNA entry channel of the 40S ribosomal subunit and blocks mRNA entry, thereby shutting down host protein synthesis. Nsp1 suppresses host immune function and is vital for viral replication. Hence, Nsp1 appears to be an attractive target for therapeutics. In this study, we have in silico screened Food and Drug Administration (FDA)-approved drugs against Nsp1-C-ter. Among the top hits obtained, montelukast sodium hydrate binds to Nsp1 with a binding affinity (K D ) of 10.8 ± 0.2 µM in vitro. It forms a stable complex with Nsp1-C-ter in simulation runs with –95.8 ± 13.3 kJ/mol binding energy . Montelukast sodium hydrate also rescues the inhibitory effect of Nsp1 in host protein synthesis, as demonstrated by the expression of firefly luciferase reporter gene in cells. Importantly, it shows antiviral activity against SARS-CoV-2 with reduced viral replication in HEK cells expressing ACE2 and Vero-E6 cells. We, therefore, propose montelukast sodium hydrate can be used as a lead molecule to design potent inhibitors to help combat SARS-CoV-2 infection.

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  1. eLife

    Evaluation Summary:

    This work reports on a drug repurposing effort to target the non-structural protein 1 (Nsp1) of SARS-Cov-2, involved in suppressing host immune function. The authors use a combination of computational, in vitro and cellular assays to this end. They start by virtual screening of an FDA approved drug library, followed by biophysical assays to measure the binding affinity of the top hits as well as molecular dynamics simulations and free energy calculations to confirm binding poses. Finally, in vitro and cellular assays were used to quantify translation inhibition and rescue as well as the production of infectious virus particles. One of the FDA approved drugs, Montelukast, emerges as a promising antiviral drug candidate.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  2. eLife

    Reviewer #1 (Public Review):

    The Non-structural protein (Nsp)-1 from SARS-CoV2 mimics the binding mode of eukaryotic initiation factor 3 (eIF3j) to the mRNA entry tunnel of the 40S ribosomal subunits and blocks the entry of mRNA, which shuts down host protein synthesis. As a result, the host immune function is suppressed. This makes Nsp-1 an attractive target. The manuscript reports on the repurposing of Montelukast, an FDA-approved drug, which forms a complex with the C-terminal helices of Nsp-1 and prevents it from binding to the mRNA channel on the 40S ribosomal subunit. The drug displays a binding affinity (KD) of 10.8{plus minus}0.2 μM in vitro and reverses the effects of Nsp-1 on host protein synthesis. The authors demonstrate this using the firefly luciferase reporter gene assay and by observing a reduced expression of viral spike protein in HEK-ACE2 and Vero-E6 cells. Short molecular dynamics simulations are also used to study the binding mode of Montelukast to Nsp-1.

  3. eLife

    Reviewer #2 (Public Review):

    In this manuscript Afsar et al. report on a drug repurposing effort to target the non-structural protein 1 (Nsp1) of SARS-Cov-2, a promising target involved in suppressing the host immune function. The authors use a combination of computational, in vitro and in vivo studies. They start with a virtual screening of the FDA approved drug library against the C-terminal domain of Nsp1, then they use biophysical assays (BLI and NanoDSF) to measure the binding affinity of the top hits emerging from the virtual screening as well as molecular dynamics simulations and MM-PBSA based free energy calculations to confirm the binding poses. Finally, luciferase-based and plaque assays were used to quantify the translation inhibition and rescue as well as infectious virus particles. One of the FDA approved drugs, Montelukast, emerges as a promising antiviral drug candidate.

    Overall, the proposed drug seems to have some activity against SARS-Cov-2 (albeit the error bars reported in panel A and D of Figure 4 are quite sizeable), but there are a number of questions raised by different aspects of the pipeline.

  4. eLife

    Reviewer #3 (Public Review):

    The strength of this work lies in the simplicity of the screening procedure using computational techniques resulting in the identification of an inhibitor that has the expected effects in in vitro and cellular assays. Often, this is not the case, but the current study is a good example of a successful computational screening campaign. In my opinion, one weakness of the manuscript is the lack of structural experimental data for the nsp1-montelukast complex, such as NMR or x-ray crystallography, although the authors provide a potential binding mode using computational methods. Most of the claims are justified with two minor exceptions. I think the authors conducted cellular assays rather than in vivo assays as they claim. In vivo assays would include for example mouse models, ferrets or non-human primates. This can be easily corrected and there is often confusion (in the literature) over what are cellular and in vivo assays. The authors also state that montelukast can be used to treat SARS-CoV-2 ("montelukast as a potential antiviral drug against SARS-CoV-2 infection that may help in combatting the COVID-19 pandemic"), but I don't think this statement is correct. Although the authors mention a clinical trial on montelukast during the discussion it is not obvious that the observed effects were related to inhibiting nsp1. I would rather argue that montelukast is a good starting point for the development of more potent drugs.

  5. Version published to 10.7554/elife.74877 on eLife
  6. ScreenIT

    SciScore for 10.1101/2021.11.02.466951: (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

    Antibodies
    SentencesResources
    The blots were then probed with SARS-CoV-2 spike antibody (NR-52947, BEI Resources, NIAID, NIH) in blocking buffer for 12 hr at 4°C, followed by secondary Goat Anti-Rabbit IgG antibody (ab6721, Abcam, RRID:AB_955447) incubation for 2hr.
    Anti-Rabbit IgG
    detected: (Abcam Cat# ab6721, RRID:AB_955447)
    Actin was labelled using antibody against beta-actin [AC-15] (HRP) (ab49900, Abcam, RRID: AB_867494).
    antibody against beta-actin
    detected: (Abcam Cat# ab49900, RRID:AB_867494)
    Experimental Models: Cell Lines
    SentencesResources
    The HEK293 cells were transfected with 100 ng/well of pGL3-Fluc plasmid using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol at around 75-90% confluency in a 96 well plate.
    HEK293
    suggested: None
    Cells and virus: The following cell lines were used in this study, namely HEK 293T cells (CRL-1573, ATCC, RRID: CVCL_0045)
    HEK 293T
    detected: (NIH-ARP Cat# 103-306, RRID:CVCL_0045)
    , HEK 293T cells stably expressing human ACE2 (NR-52511, BEI Resources, NIAID, NIH, RRID:
    HEK 293T
    suggested: None
    , Vero-E6 cells (CRL-1586, ATCC, RRID: CVCL_0574).
    Vero-E6
    detected: (IZSLER Cat# BS CL 87, RRID:CVCL_0574)
    NR-52282, BEI Resources, NIAID, NIH) was propagated and quantified by plaque assay in Vero-E6 cells as described before (Case et al., 2020)
    Vero-E6
    suggested: None
    Cytotoxicity assay: HEK-ACE2 cells were seeded in 0.1 mg/mL poly-L-lysine (P9155-5MG, Sigma-Aldrich) coated 96-well plate to reach 70-80% confluency after 24 Hrs.
    HEK-ACE2
    suggested: None
    Virus infection: HEK ACE2 cells were seeded in poly-L-lysine coated 24-well plate to reach 80% confluency at the time of infection.
    HEK ACE2
    suggested: None
    Recombinant DNA
    SentencesResources
    Nsp1 expression and purification: The gene construct encoding the Nsp1 from SARS-CoV-2 in pCDNA 5-3X-Flag-Nsp1 was amplified and sub-cloned into pET28a with N-terminal His-tag (Schubert et al., 2020; Thoms et al., 2020) using appropriate primers (Supplementary table 1).
    pCDNA 5-3X-Flag-Nsp1
    suggested: None
    pET28a
    suggested: RRID:Addgene_114156)
    the cultures were then transferred at 16°C at 120 rpm, and the expression of pET28a-His-Nsp1 and pET28a-His-Nsp1Δ40 were induced by adding 1 mM of Isopropyl β-d-1-thiogalactopyranoside (IPTG) and allowed to grow for 18 hours.
    pET28a-His-Nsp1
    suggested: None
    pET28a-His-Nsp1Δ40
    suggested: None
    The HEK293 cells were transfected with 100 ng/well of pGL3-Fluc plasmid using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol at around 75-90% confluency in a 96 well plate.
    pGL3-Fluc
    suggested: None
    The plasmid expressing the Nsp1 protein (pcDNA 3.1-Nsp1) was co-transfected at 100 ng/well concentration.
    pcDNA 3.1-Nsp1
    suggested: None
    After 48 hr incubation, cells were fixed with 4% paraformaldehyde, and crystal violet (C6158, Merck) staining was done to visualize the plaques. Plasmids: pLVX-EF1alpha-SARS-CoV-2-nsp1-2xStrep-IRES-Puro expressing SARS CoV-2 NSP1 was a kind gift from Prof. Nevan Krogan (Gordon et al., 2020).
    pLVX-EF1alpha-SARS-CoV-2-nsp1-2xStrep-IRES-Puro
    suggested: RRID:Addgene_141367)
    Other plasmids used in this study include Plasmids pRL-TK (mammalian vector for weak constitutive expression of wild-type Renilla luciferase), pGL4 (mammalian vector expressing firefly luciferase), pIFN-β Luc (IFN beta promoter-driven firefly luciferase reporter).
    pRL-TK
    suggested: RRID:Addgene_11313)
    pGL4
    suggested: RRID:Addgene_48744)
    The plasmid pMTB242 pcDNA5 FRT-TO-3xFLAG-3C-Nsp1_SARS2 was a kind gift from Prof. Ronald Beckmann.
    pMTB242
    suggested: None
    Software and Algorithms
    SentencesResources
    Finally, the library was screened against the 18S rRNA interacting interface of Nsp1-C-ter using the Surflex-dock program, which is available in SYBYL v2.1 (Jain, 2003).
    Surflex-dock
    suggested: (Surflex-Dock, RRID:SCR_000196)
    The data was analysed by using ThermControl software.
    ThermControl
    suggested: None
    The two subsequent 100 ns runs from MD simulations were further subjected to perform the MM-PBSA by using the python script (mmpbsa.py) to calculate the binding energy of the two drugs.
    python
    suggested: (IPython, RRID:SCR_001658)
    Relative intensity of bands was quantified using Fiji/imageJ.
    Fiji/imageJ
    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: 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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

    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.

  7. Version published to 10.1101/2021.11.02.466951v1 on bioRxiv