Hydroxychloroquine: mechanism of action inhibiting SARS-CoV2 entry
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
Hydroxychloroquine (HCQ) has been proposed in the treatment of SARS-coronavirus 2 (SARS-CoV-2) infection, albeit with much controversy. In vitro , HCQ effectively inhibits viral entry, but its use in the clinic has been hampered by conflicting results. A better understanding of HCQ’s mechanism of actions in vitro is needed to resolve these conflicts. Recently, anesthetics were shown to disrupt ordered monosialotetrahexosylganglioside1 (GM1) lipid rafts. These same lipid rafts recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to an endocytic entry point, away from phosphatidylinositol 4,5 bisphosphate (PIP 2 ) domains. Here we employed super resolution imaging of cultured mammalian cells to show HCQ directly perturbs GM1 lipid rafts and inhibits the ability of ACE2 receptor to associate with the endocytic pathway. HCQ also disrupts PIP 2 domains and their ability to cluster and sequester ACE2. Similarly, the antibiotic erythromycin inhibits viral entry and both HCQ and erythromycin decrease the antimicrobial host defense peptide amyloid beta in cultured cells. We conclude HCQ is an anesthetic-like compound that disrupts GM1 lipid rafts similar to anesthetics. The disruption likely decreases viral clustering at both endocytic and putative PIP 2 entry points.
KEY POINTS
Question: What is the molecular basis for antiviral activity of hydroxychloroquine?
Findings: Hydroxychloroquine disrupt lipid rafts similar to general anesthetics.
Meaning: Since lipids cluster ACE2 and facilitate viral entry, hydroxychloroquine appears to inhibit viral entry by disrupting the lipid clustering of the SARS-CoV2 receptor.
Article activity feed
-
-
SciScore for 10.1101/2020.08.13.250217: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources For labelling, cells were incubated with primary antibody (anti-ACE2 antibody (Abcam, #ab189168), anti-PLD2 antibody, or anti-PIP2 antibody) for 60 min in antibody buffer (PBS with 5% BSA and 0.05% TritonX-100) at room temperature followed by 5 washes with wash buffer (PBS with 1% BSA and 0.05% TritonX-100) for 15 min each. anti-ACE2suggested: Noneanti-PLD2suggested: Noneanti-PIP2suggested: NoneSecondary antibodies (donkey anti-rabbit Cy3B and Alexa 647 … SciScore for 10.1101/2020.08.13.250217: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources For labelling, cells were incubated with primary antibody (anti-ACE2 antibody (Abcam, #ab189168), anti-PLD2 antibody, or anti-PIP2 antibody) for 60 min in antibody buffer (PBS with 5% BSA and 0.05% TritonX-100) at room temperature followed by 5 washes with wash buffer (PBS with 1% BSA and 0.05% TritonX-100) for 15 min each. anti-ACE2suggested: Noneanti-PLD2suggested: Noneanti-PIP2suggested: NoneSecondary antibodies (donkey anti-rabbit Cy3B and Alexa 647 conjugated CTxB) were added with antibody buffer for 30 min at room temperature followed by 5 washes as stated above. anti-rabbitsuggested: NoneAfter a 3 h incubation, the plate was washed with 200 μL PBST for 4 times and 100 μL HRP-linked goat anti-rabbit IgG secondary antibody (Invitrogen™ #31460) at 0.4 μg/ml concentration in PBST buffer was added for 1 h incubation in the dark. anti-rabbit IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources In vivo and in vitro PLD Assay: In vivo PLD2 activity was measured in cultured HEK 293T cells by an enzyme-coupled product release assay using amplex red reagent as described previously14. HEK 293Tsuggested: NoneSoftware and Algorithms Sentences Resources Images were acquired by Andor iXon 897 EMCCD camera and Zen 10D software with an exposure time of 18 ms per acquisition. Zensuggested: NoneStatistical Analyses: Data calculations and graphs were performed in Prism 6 (GraphPad software) or Microsoft Excel. GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Microsoft Excelsuggested: (Microsoft Excel, RRID:SCR_016137)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.
- No funding statement was detected.
- No protocol registration statement was detected.
-