Drugs that inhibit TMEM16 proteins block SARS-CoV-2 spike-induced syncytia

Abstract

COVID-19 is a disease with unique characteristics that include lung thrombosis 1 , frequent diarrhoea 2 , abnormal activation of the inflammatory response 3 and rapid deterioration of lung function consistent with alveolar oedema 4 . The pathological substrate for these findings remains unknown. Here we show that the lungs of patients with COVID-19 contain infected pneumocytes with abnormal morphology and frequent multinucleation. The generation of these syncytia results from activation of the SARS-CoV-2 spike protein at the cell plasma membrane level. On the basis of these observations, we performed two high-content microscopy-based screenings with more than 3,000 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 focused our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was the antihelminthic drug niclosamide, which markedly blunted calcium oscillations and membrane conductance in spike-expressing cells by suppressing the activity of TMEM16F (also known as anoctamin 6), a calcium-activated ion channel and scramblase that is responsible for exposure of phosphatidylserine on the cell surface. These findings suggest a potential mechanism for COVID-19 disease pathogenesis and support the repurposing of niclosamide for therapy.One of the defining features of coronavirus biology is the coordinated process by which the virus binds and enters the host cell, which involves both docking to receptors at the cell surface (ACE2 for SARS-CoV2 5 ), and proteolytic activation of the spike protein by host encoded proteases at two distinct sites 6 . One activation step is spike cleavage at the S1-S2 boundary, which can occur either before or after receptor binding. A second proteolytic activation exposes the S2 portion, and primes S2 for fusion of virus and cellular membranes. The protease priming event at this S2′ site and subsequent fusion can occur after endocytosis, in which cleavage is carried out by endosomal low pH-activated proteases such as cathepsin B and cathepsin L 7 , or at the plasma membrane, where cleavage can be mediated by TMPRSS2 [8][9][10] . The spike proteins of MERS-CoV and SARS-CoV-2 possess a multibasic amino acid sequence at the S1-S2 interface, which is not present in SARS-CoV 11 , that also allows cleavage by the ubiquitously expressed serine protease furin [12][13][14] . As a consequence, cells that express MERS-CoV and SARS-CoV-2 spike protein at the plasma membrane can fuse with other cells that express the respective receptors and form syncytia.

SARS-CoV-2 transmission via apical syncytia release from primary bronchial epithelia and infectivity restriction in children epithelia

“…Furthermore, we also observed the loss of cilia in many of the syncytia, which could be responsible for a poor mucociliary clearance that impedes the evacuation of viral particles and pathogens. Taken together, our findings are in accordance with previous findings but highlight syncytium formation as an important mechanism to explain the spreading of SARS-CoV-2 and the physiopathology of bronchial epithelium infection (14, 43, 45, 46). Since the beginning of the COVID-19 pandemic, SARS-CoV-2 infection is more virulent in adults compared to children.…”

SARS-CoV-2 B.1.617.2 Delta variant replication, sensitivity to neutralising antibodies and vaccine breakthrough

“…Virus infectivity and fusogenicity mediated by the PBCS is a key determinant of pathogenicity and transmissibility 22,28 and there are indications that giant cells/syncitia formation are associated with fatal disease 29 . We find that P681R is associated with enhanced capacity to induce cell-cell fusion and syncitia formation, and that P681R alone confers this ability on the B.1.617.1 spike with RBD mutations L452R and E484Q.…”

Sequences in the cytoplasmic tail of SARS-CoV-2 Spike facilitate expression at the cell surface and syncytia formation

“…It has recently been reported that syncytia formation by SARS-CoV-2 S can induce pyroptosis, and it has been proposed for other viral-mediated fusion events that cell death following syncytia formation can affect immune responses 60,61 . All this has led to an interest in the possibility that syncytia formation by SARS-CoV-2 is a potential target for therapeutic strategies 18,62 . Indeed, the process appears entirely dependent on the cell surface protease TMPRSS2 to make the activating S2' cleavage, whereas viral entry can be facilitated by either TMPRSS2 or lysosomal cathepsins 11,17 .…”