Abstract
COVID-19 is a disease with unique characteristics including lung thrombosis1, frequent diarrhoea2, abnormal activation of the inflammatory response3 and rapid deterioration of lung function consistent with alveolar oedema4. The pathological substrate for these findings remains elusive. Here we show that the lungs of patients with COVID-19 contain infected pneumocytes with abnormal morphology and frequent multinucleation. Generation of these syncytia results from activation of the SARS-CoV-2 Spike protein at the cell plasma membrane level. Based on these observations, we performed two high-content microscopy-based screenings with over 3000 approved drugs to search for inhibitors of Spike-driven syncytia. We converged on the identification of 83 drugs that inhibited Spike-mediated cell fusion, several of which belonged to defined pharmacological classes. We focussed our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was Niclosamide, which markedly blunted calcium oscillations and membrane conductances in Spike-expressing cells by suppressing the activity of TMEM16F/Anoctamin6, a calcium-activated ion channel and scramblase responsible for phosphatidylserine exposure on the cell surface. These findings suggest a potential mechanism for COVID-19 disease pathogenesis and support the repurposing of Niclosamide for therapy.
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Video 1
: Heterologous cell fusion Twelve-hr time lapse imaging of co-cultures of human U2OS cells transfected with a control plasmid (left) or with pEC117-Spike-V5, expressing SARS-CoV-2 S protein (right), together with Vero cells expressing EGFP. At the end of the imaging period, cells were fixed and nuclei were counterstained with DAPI.
Video 2
: Formation of syncytia Twelve-hr time lapse imaging of Vero cells transfected with GCaMP6s (green), showing progressive cell fusion and formation of syncytia.
Video 3
: Formation of syncytia Twelve-hr time lapse imaging of Vero cells transfected with GCaMP6s (green), showing progressive cell fusion and formation of syncytia.
Video 4
: Calcium oscillations in cells undergoing fusion Time-lapse imaging of 12-hr co-cultures of Vero cells expressing the GCaMP6s Ca2+ sensor with U2OS cells expressing Spike and mCherry. Spike-mediated fusion events correlated with numerous oscillations in intracellular Ca2+ levels. Individual frames from this movie are in Fig. 2b.
Video 5
: Effect of syncytia-inhibiting drugs on Ca2+ oscillations Time-lapse imaging of Vero cells expressing the GCaMP6s Ca2+ sensor in the presence of the indicated drugs (1 µM Niclosamide and 5 µM Clofazimine and Salinomycin) or of DMSO Control. A reduction in Ca2+ transients is evident with Niclosamide and Clofazimine. Quantification of this Ca2+ activity is in Fig. 2d. Individual frames from the movie with DMSO (Control) are in Fig. 2c.
Video 6
: Effect of Calcium depletion and SERCA inhibition on Ca2+ oscillations Time-lapse imaging of Vero cells co-expressing the GCaMP6s Ca2+ sensor and either an empty vector (Control) or Spike. Cells were cultured in normal medium (upper panels), medium without Ca2+, or normal medium supplemented with the SERCA inhibitors thapsigargin (TG) or cyclopiazonic acid (CPA). A reduction in Ca2+ transients is evident in all the three treatments. Quantification of this Ca2+ activity is in Extended Data Figs. 6c and 6d.
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Braga, L., Ali, H., Secco, I. et al. Drugs that inhibit TMEM16 proteins block SARS-CoV-2 Spike-induced syncytia. Nature (2021). https://doi.org/10.1038/s41586-021-03491-6
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