Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces various health complications all over the body. New research published in the journal Nature found the presence of SARS-CoV-2 in the lungs resulted in abnormal pneumocytes and spike protein-mediated cell fusion. Their findings also showed that TMEM16F protein activation induces cell fusion. Therefore, drugs inhibiting the TMEM16F/Anoctamin6 calcium-activated ion channel, such as niclosamide – a medication used to treat tapeworm infestations – could serve as potential treatments for reducing the severity of COVID-19 infection.
The researchers write:
“Niclosamide has already been reported to be active against various enveloped and non enveloped viruses, including SARS-CoV-2. Although this drug has relatively low solubility, men that take premarin there is evidence of considerable absorption, with serum levels that can reach 1-20 µM. Together, our findings provide a mechanism and a rationale for repurposing of niclosamide to treat patients with COVID-19.”
Cell fusion activity in lung cells of COVID patients
The research team evaluated organs from 41 Italian patients deceased due to COVID-19 from March to May 2020. About 90% of patients showed abnormal cell morphology showing syncytia with a variable number of nuclei. The syncytial cells were positive for SARS-CoV-2 RNA.
Based on observations, the researchers hypothesized that the fused cells were likely due to the SARS-CoV-2 spike protein. This was confirmed when an in vitro study introduced codon-optimized spike protein cDNA in cells and later found syncytia.
Niclosamide and salinomycin are most effective in preventing antiviral activity
The next step for researchers was to screen for drugs that could prevent spike-induced syncytia. A total of 83 potential drug candidates were identified, and 43 were tested in cells infected with SARS-CoV-2.
After 5 days, results showed the anti-histaminic deptropine, the antidepressant sertraline, and the antileprotic antibiotic clofazimine was selected for their ability to protect cells from viral cytopathic effects. Niclosamide and salinomycin were also selected based on screening results.
The five drugs were tested on their effectiveness in preventing SARS-CoV-2-induced cell death in a dose-dependent manner. Niclosamide, clofazimine, salinomycin were able to provide cell protection and moved on to the next round of testing.
Niclosamide and salinomycin prevented viral production at low and high doses, while clofazimine was 10-fold less potent than the other two drugs. All three drugs inhibited viral replication.
Anti-syncytial drugs inhibit calcium ion channels
A common feature among the selected drugs is their ability to block calcium release. The researchers proposed regulation of calcium levels could be a potential mechanism of action for anti-syncytial drugs.
When pitting cells expressing the calcium indicator GCaMP6s, co-cultured with U2OS cells expressing Spike and mCherry fluorescent protein, the team found several calcium level changes, which coincided with the fusion of cells expressing the spike protein.
“The presence of Spike increased the amplitude of Ca2+ transients in individual cells, without a significant difference in frequency, suggesting that Spike expression amplifies spontaneous Ca2+ transients,” wrote the researchers.
Administering niclosamide and clofazimine inhibited the amplitude and frequency of calcium oscillations in cells infected with SARS-CoV-2. In contrast, salinomycin was successful in blocking cell fusion but did not alter calcium levels.
The calcium release occurs in the endoplasmic reticulum of the cell. This was confirmed when researchers added two non-competitive inhibitors of the sarco/endoplasmic reticulum Ca2+ ATPase, showing calcium oscillations to cease once the calcium stores in the endoplasmic reticulum were drained.
Because niclosamide inhibits the calcium-activated TMEM16/Anoctamin family of chloride channels and scramblases, they looked into TMEM16 as a potential cause for syncytia in lung epithelial cells. They found TMEM16F was expressed in all cells and TMEM16 protein activation by the SARS-CoV-2 spike protein increased the amplitude of calcium signaling.
Blocking TMEM16F currents was possible through niclosamide but not through clofazimine and salinomycin.
Downregulation of TMEM16F inhibited phosphatidylserine externalization, which would generally signal for cell apoptosis in cells with calcium ionophore ionomycin. Results suggest TMEM16F is necessary for responding to calcium level changes.
A one-hour treatment of niclosamide or clofazimine reduced suppression of phosphatidylserine externalization.
Overexpression of TMEM16F significantly produced SARS-CoV-2 spike protein-induced syncytia. This effect was not seen in TMEM16A.
The researchers suggest drug therapies targeting the TMEM16 family could alleviate COVID-19 pathogenesis.
“It could participate in inflammation (TMEM16A promotes NK-κB activation and IL-6 secretion), thrombosis (TMEM16F is essential for lipid scrambling in platelets during blood coagulation), dysfunction of endothelial cells, and alveolar edema and diarrhea through increased chloride secretion,” concluded the researchers.
- Braga L. et al. Drugs that inhibit TMEM16 proteins block SARS-CoV-2 Spike-induced syncytia. Nature, 2021. doi: https://doi.org/10.1038/s41586-021-03491- 6, https://www.nature.com/articles/s41586-021-03491-6
Posted in: Drug Trial News | Medical Science News | Medical Research News | Disease/Infection News
Tags: Antibiotic, Antidepressant, Apoptosis, Blood, Calcium, Cell, Cell Death, Codon, Coronavirus, Coronavirus Disease COVID-19, Diarrhea, Drugs, Edema, Fluorescent Protein, Frequency, in vitro, Inflammation, Ion, Ion Channel, Lungs, Microscope, Morphology, Platelets, Protein, Research, Respiratory, RNA, Salinomycin, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spike Protein, Syndrome, Tapeworm, Thrombosis
Jocelyn Solis-Moreira graduated with a Bachelor's in Integrative Neuroscience, where she then pursued graduate research looking at the long-term effects of adolescent binge drinking on the brain's neurochemistry in adulthood.
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