N-acylethanolamine acid amide hydrolase is a novel target for drugs against SARS-CoV-2 and Zika virus

  1. Michele Lai
  2. Veronica La Rocca
  3. Rachele Amato
  4. Elena Iacono
  5. Carolina Filipponi
  6. Elisa Catelli
  7. Lucia Bogani
  8. Rossella Fonnesu
  9. Giulia Lottini
  10. Alessandro De Carli
  11. Alessandro Mengozzi
  12. Stefano Masi
  13. Paola Quaranta
  14. Pietro Giorgio Spezia
  15. Giulia Freer
  16. Paola Lenzi
  17. Francesco Fornai
  18. Daniele Piomelli
  19. Mauro Pistello

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Abstract

Several compounds have been tested against SARS-CoV-2; at present, COVID-19 treatments decrease the deleterious inflammatory response and acute lung injury. However, the best therapeutic response would be expected by combining anti-inflammatory properties, while concomitantly blocking viral replication. These combined effects should drastically reduce both infection rate and severe complications induced by novel SARS-CoV-2 variants. Therefore, we explored the antiviral potency of a class of anti-inflammatory compounds that inhibit the N-Acylethanolamine acid amidase (NAAA). This enzyme catalyzes the hydrolysis of palmitoylethanolamide (PEA), a bioactive lipid that mediates anti-inflammatory and analgesic activity through the activation of peroxisome proliferator receptor-α (PPAR-α). Similarly, this pathway is likely to be a significant target to impede viral replication since PPAR-α activation leads to dismantling of lipid droplets, where viral replication of Flaviviruses and Coronaviruses occurs.

Here, we show that either genetic or pharmacological inhibition of the NAAA enzyme leads to five-fold reduction in the replication of both SARS-CoV-2 and ZIKV in various cell lines. Once NAAA enzyme is blocked, both ZIKV and SARS CoV-2 replication decrease, which parallels a sudden five-fold decrease in virion release. These effects induced by NAAA inhibition occurs concomitantly with stimulation of autophagy during infection. Remarkably, parallel antiviral and anti-inflammatory effects of NAAA antagonism were confirmed in ex-vivo experiments, within SARS-CoV-2 infected human PBMC cells, in which both viral genomes and TNF-α production drop by ~60%. It is known that macrophages contribute to viral spread, excessive inflammation and macrophage activation syndrome that NAAA inhibitors might prevent, reducing the macrophage-induced acute respiratory distress syndrome and subsequent death of COVID-19 patients.

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  1. ScreenIT

    SciScore for 10.1101/2022.02.08.479661: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Human peripheral blood mononuclear isolation and infection: Clinical study was approved by local ethics committee (protocol number 19204) in accordance with institutional guidelines; participants were aware of the nature of the study and signed written informed consent), PBMCs derived from healthy donors (age 30-60, no chronic comorbidities, absence of medications) were isolated by gradient centrifugation using Lympholite-H Cell Separation Media.
    Consent: Human peripheral blood mononuclear isolation and infection: Clinical study was approved by local ethics committee (protocol number 19204) in accordance with institutional guidelines; participants were aware of the nature of the study and signed written informed consent), PBMCs derived from healthy donors (age 30-60, no chronic comorbidities, absence of medications) were isolated by gradient centrifugation using Lympholite-H Cell Separation Media.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: Cells were tested for mycoplasma contamination as previously described(41) CRISPR/Cas9 design and transfection: A549 and Huh-7 cells were transfected with CRISPR/Cas9 RNP (IDT, Coralville, Iowa) by nucleoporation

    Table 2: Resources

    Antibodies
    SentencesResources
    anti-LC3 I-II (1:1000, L7543, Sigma-Aldrich, St. Louis, MO 63103, USA), anti-ATG5 (1:1000, Anti-APG5L/ATG5 antibody, ab228668, Abcam, Cambridge, UK) and anti-β-actin (1:1000, A2066 Sigma-Aldrich, St. Louis, MO 63103, USA).
    anti-LC3 I-II
    suggested: None
    anti-ATG5
    suggested: None
    Anti-APG5L/ATG5
    suggested: None
    anti-β-actin
    suggested: None
    Cells were fixed using 3.7% formaldehyde and stained with the following primary antibodies: anti-NAAA (1:300
    anti-NAAA
    suggested: None
    0, Abnova, Taipei, Taiwan), ZIKA: Anti-Flavivirus NS1 antibody (ab214337), Anti-SARS-CoV-2 spike protein (Sino Biological, Beijing, China, 1:200).
    ZIKA: Anti-Flavivirus NS1
    suggested: None
    Anti-Flavivirus
    suggested: None
    Anti-SARS-CoV-2 spike protein
    suggested: None
    Ultrathin sections were collected on a nickel-coated grid, processed for LC3 detection using a primary antibody (Rabbit anti-LC3, Abcam, ab128025, AB_11143008).
    anti-LC3
    detected: (Abcam Cat# ab128025, RRID:AB_11143008)
    After washing in cold PBS, ultrathin sections were incubated in the gold-conjugated secondary antibodies (20 nm gold particles, EM Goat anti-Rabbit IgG, gold conjugated antibody BBInternational EM.
    anti-Rabbit IgG
    suggested: None
    GAR 20 AB_1769136), diluted 1:20 in blocking buffer (1% goat serum and 0.2% saponin in PBS) for 1 h at 22 °C.
    GAR 20
    suggested: (BBI Solutions Cat# EM GAR20/0.25, RRID:AB_1769136)
    Experimental Models: Cell Lines
    SentencesResources
    A549 and Huh-7 cells and NAAA-/- cells were infected with 1 M.O.
    Huh-7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    NAAA-/-
    suggested: None
    Western blot analysis: A549 and Huh-7 cells were lysed with RIPA lysis buffer (Millipore, Massachusetts, USA).
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)

    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: 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.

  2. Version published to 10.1101/2022.02.08.479661 on bioRxiv