Zinc supplementation bolsters repurposed antivirals against SARS-CoV-2

Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen, serious efforts have been made to identify new or repurposable drugs that effectively inhibit or counteract the infection. A new preprint on the bioRxiv* server reports the efficacy of two repurposed drugs that could be useful in this role, especially when supplemented with zinc.

The angiotensin-converting enzyme 2 (ACE2) receptor is widely expressed in the human body, and plays a role in the functioning of the renin-angiotensin system (RAS). SARS-CoV-2 binds to the receptor by its spike protein, which has two subunits, S1 and S2.

The S1 subunit mediates the initial attachment of the virus, leading to the cleavage of the spike at the S1/S2 interface. Following the spike-ACE2 binding, S2 is cleaved by the host protease TMPRSS2, an essential step for endocytosis and internalization of SARS-CoV-2 into the host cell.

The fundamental importance of the ACE2 receptor has led to the exploration of many compounds which may prevent the virus-ACE2 interactions.

This includes soluble ACE2 molecules, used as a decoy for the virus, ACE2 inhibitors, and TMPRSS2 inhibitors. The hope is that these will entrap the virus and inhibit spike-ACE2 binding. However, no effective and safe drugs have yet been identified.

The viral spike or the double-stranded genetic material of the virus may activate the NF-κB pathway in the cells of the alveolar epithelium or the vascular endothelium. The triggering of this inflammatory pathway may finally result in a cytokine storm, characteristic of severe COVID-19.

The inhibition of this pathway is, therefore, a therapeutic target to prevent disease progression and adverse outcomes in COVID-19. The glucocorticoid dexamethasone has been found to reduce mortality among patients with severe COVID-19 who are on invasive mechanical ventilation or supplemental oxygen, but not on those with less severe disease.

This has suggested to many researchers that this beneficial activity of dexamethasone is in part at least mediated by its suppression of the pro-inflammatory NF-κB activation in patients with severe disease.

NF-κB inhibitor, PDTC, decreases ACE2 expression

The researchers found that the NF-κB inhibitor, ammonium pyrrolidine dithiocarbamate (PDTC), as well as the drugs triclabendazole and emetine, were capable of suppressing ACE2 expression. Both the mRNA encoding ACE2 and ACE2 protein expression were found to be increased in cells treated with PDTC, in a time-dependent manner.

This indicates that the expression of ACE2 is regulated by the NF-κB pathway, such that inhibitors of NF-κB could potentially be helpful in reducing the severity of COVID-19.

N-acetyl-cysteine blocks PDTC-derived ACE2 suppression

The researchers also found that inhibition of the p50 subunit of NF-κB by PDTC was not responsible for inhibiting ACE2 expression. Instead, PDTC is a chelator with both pro-oxidant and antioxidant effects.

The potential role of reactive oxygen species (ROS) in inducing ACE2 suppression in response to cell exposure to PDTC was explored by pretreating the cells with N-acetyl-cysteine (NAC). This thiol compound is known to inhibit ROS production induced by PDTC.

The researchers observed an increase in ACE2 mRNA and protein levels in cells treated with NAC alone. When NAC was used for pretreatment, followed by PDTC, the expected PDTC-induced suppression of ACE2 was not observed, at transcriptional and translational levels.

The conclusion was that PDTC is an oxidant. It suppresses ACE2 expression by inducing oxidant activity, similar to the effects produced by treatment with hydrogen peroxide on cells lacking the p50 subunit of NF-κB. This effect of hydrogen peroxide was not observed in control cells.

Thus, both NF-κB inhibition and oxidant activity induced by PDTC are required for the latter to suppress ACE2 expression.

These results together suggest that PDTC suppresses ACE2 expression through ROS induction and NF-κB inhibition.”

Triclabendazole or emetine suppress ACE2 expression

Drug repurposing is a method to rapidly identify drugs capable of treating a disease condition without the time and expense involved in conventional drug discovery protocols.

The drugs emetine and triclabendazole, which have anti-protozoal and anthelmintic activity, respectively, have been found to suppress this signaling pathway. The current study seeks to explore their effects on ACE2 expression.

The researchers used non-toxic dosages of both triclabendazole and emetine on cultured cells, and observed a dose- and time-dependent suppression of ACE2 expression.

These findings suggest that triclabendazole and emetine exhibit anti-SARS-CoV-2 activity via ACE2 suppression."

Supplemental zinc enhances the effects of these drugs

The study also explored the potential for combination therapy, using either of these repurposed drugs with zinc. This could increase the efficacy of treatment as well as reduce the required dose and thus the adverse effects.

Zinc is a trace mineral that enhances the clearance of virus particles through the mucociliary apparatus, augments epithelial barrier function, reduces viral replication, modulates inflammatory processes, and promotes anti-oxidant activity. Hospitalized adult COVID-19 patients have shown correlations between lower basal zinc levels and increased mortality, complication rate, and length of hospital stay.

The researchers found that zinc was able to produce a small decrease in ACE2 mRNA and protein levels. When combined with either triclabendazole or emetine, vs. the use of each of these drugs alone, the researchers found that monotherapy with any of these three compounds failed to produce a significant drop in ACE2 levels.

However, when zinc was combined with either of the other two drugs, ACE2 levels were suppressed, with no signs of cell toxicity.

What are the implications?

The exact changes in ACE2 expression following viral binding are unclear, but some studies suggest an increase on airway epithelium by about threefold. If so, viral amplification could be promoted by the increase in ACE2 levels following infection.

Triclabendazole or emetine plus zinc supplementation may be more effective in reducing virus amplification through decreasing ACE2 protein expression than triclabendazole or emetine monotherapy.”

These findings indicate the suitability of this combination for further studies, to investigate its efficacy in alleviating the severity of COVID-19 as well as possibly prevent further infection. The researchers suggest a future focus on the development of these drugs in an inhalable form, to optimize the efficacy on airway epithelium.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.