Mauro Giacca - May 12, 2021

Mauro Giacca, MD PhD, Professor of Cardiovascular Sciences, King’s College London, School of Cardiovascular Medicine & Sciences, "From COVID-19 pathology to therapy: drugs that inhibit SARS-CoV-2 Spike-induced cellular syncytia"

The histopathological investigation has shown lung thrombosis and abnormal pneumocytes in COVID-19 patients.

Especially, syncytia formation was observed in cytological altered pneumocytes.SARS-CoV-2 spike (S) protein-mediated cell fusion was investigated. SARS-CoV-2 S gene was cloned and transfected to ACE2 expressed AGM Vero cells and observed powerful fusogenic activity induced by SARS-CoV-2 S protein (under a scope and live images). SARS-CoV-2 S protein-dependent cell fusion assay was applied to confirm SARS-Cov-2 S protein-mediated cell fusion. U2OS cell which does not express ACE2 was used to transfect SARS-Cov-2 S protein probed with Green Q-dot and ACE2 expressed Vero cells probed with Red-Q-dot. These two U2OS and Vero cells were co-cultured and SARS-CoV-2 S protein and ACE2/TMPRSS2 mediated cell fusion was occurred and observed both green and red Q-dot in their cytoplasm as well as syncytia formation while Control U2OS which did not transfect with SARS-CoV-2 protein failed to induce cell fusion with Vero cells indicated by single color Q-dots in the cells.

High throughput screening was performed with FDA/EMA-approved drug libraries to find molecules that block the syncytia formation. About ~3800 FDA/EMA-approved drugs were screened. Niclosamide, Clofazimine and Salinomycyin showed protection effect against SARS-CoV-2 induced cell death and inhibited SARS-CoV-2 replication. The selected drugs are classified as H1R1 antagonists, Antipsychotics and Antidepressants. They found the common function of these molecules is blocking signal through G protein-coupled receptor (gPCR) which coupled with G alpha Q protein(GaQP) and this ends up stimulate Ca2+ channel through the PLC and IP3 pathways. Therefore, activated gPCR can cause Ca2+ release from ER. SARS-CoV-2 S protein induces intracellular Calcium oscillation and cell fusion and this can make syncytia. They showed Niclosamide and Clofazimine inhibit calcium oscillation. Previous study shown Niclosamide has known as an inhibitor of TMEM 16A/F. TMEM16 proteins are Ca2+ activated chloride channels and phosphatidylserine scramblases.

They investigated the cells that they were using (Vero, HEK293, Huh-7, Calu-3 U2OS and primary human bronchial epithelial cells) and found a high expression level of TMEM 16F. THEM 16F also showed as the main phosphatidylserine scramblase in Vero cells. Niclosamide and Clofazimine can block phosphatidylserine externalization. Their model suggested Calcium induced by the S spike protein activates the chloride channel in the membrane and activates TEME16.