Varenicline blocks SARS-CoV-2 infection in cells and animal models

As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced coronavirus disease 2019 (COVID-19) pandemic continues to spread in the face of vaccination roll-outs, albeit at a reduced rate, the realization that global vaccination will not be achieved for another year or two has led to continued attempts to find a suitable antiviral therapy.

A new paper released on the bioRxiv* preprint server suggests that the nicotinic acetylcholine receptor (nACHR) agonist, varenicline tartrate, has antiviral activity against the virus.

Background

The need for protective immunity against the virus is heightened not only by the inevitable delay in universal immunization against COVID-19, but by the rapid emergence of newer variants of concern (VOCs) that are resistant to neutralization by therapeutic antibodies and convalescent serum.

This includes the alpha variant, which rapidly rose to a dominant position, before being itself overtaken by the delta variant. These are both much more transmissible than the original Wuhan variant. The alpha variant also has a 63% higher mortality within 28 days.

The SARS-CoV-2 pathogen enters host cells via the angiotensin-converting enzyme-2 (ACE2), while the host transmembrane serine protease 2 (TMPRSS2) enzyme also plays a major role in priming the virus spike protein. Since the nasal mucosa expresses both these proteins on the epithelium, it could be the most vulnerable to infection within the airway.

Involvement of nicotinic cholinergic system

Since early studies have shown that a low prevalence of smoking is associated with reduced hospitalization rates for COVID-19, some scientists have postulated that the nicotinic cholinergic system is implicated in the spread of this virus.

The virus spike protein is predicted to be able to bind to nAChR, according to binding simulations, and one study has shown a putative sequence within the receptor binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 that acts similarly to nAChR-binding snake venom.

Such interactions could account for immunologic pathology in COVID-19, including the disease flares or acute myasthenia-like findings in patients with autoimmune disease on contracting SARS-CoV-2 infection or after receiving mRNA vaccines against this virus.

Varenicline as antiviral

In this scenario, the researchers explored the potential role of the selective small-molecule nAChR agonist varenicline tartrate. This compound mimics the activity of nACh at the α7 receptor and is a partial agonist at the α3β4, α3α5β4, α4β2, and α6β2 receptors.

Its clinical approval at present is as an oral tablet meant to help quit smoking, though it is being studied for its use in treating dry eye syndromes. The current study is based on computational modeling, showing that it may bind at high affinity to the spike protein of SARS-CoV-2 at the hinge site.

In vitro efficacy

The study showed that varenicline indeed reduced viral titers in two different cell cultures to half the expected level at concentrations of 0.3 μM to 0.5 μM – the half-maximal inhibitory concentration (IC50). When tested against the alpha variant, the IC50 was even lower, at 0.13 μM, while for the beta VOC, it was 4μM.

In neither of these conditions did the cells show any toxicity effects.

In vivo efficacy

A second phase of the study explored the efficacy of the drug in rhesus macaques exposed to SARS-CoV-2. This animal model has shown consistent mild to moderate disease following infection with this virus, with high viral loads in the respiratory tract and several patches of lung inflammation.

Varenicline inhibited SARS-CoV-2 in monkeys, with no changes in the rapidity of breathing, respiratory trouble, or fecal consistency. There were only small changes in body weight and temperature.

When treated with varenicline OC-01 nasal spray, viral loads were dramatically reduced as judged by reverse transcriptase-polymerase chain reaction (RT PCR) measuring genomic ribonucleic acid (RNA), as well as subgenomic (sg) RNA. These are expected to be at high levels when the virus is actively replicating.

Both genomic and subgenomic RNA levels went down approximately 100-fold and 200-fold, compared to control cells. At 4 days from exposure, sgRNA levels had fallen below detectable levels.

What are the implications?

The researchers have presented the results of a pioneering study showing the antiviral activity of a nAChR agonist against SARS-CoV-2. The growth of the virus in cell cultures, following treatment with varenicline over a range of dosages and using wild-type, alpha and beta variants, was severely suppressed, while leaving the cell viability intact.

In vivo studies using a rhesus monkey model showed that varenicline applied as a nasal spray, at an estimated dose of 1 mM, successfully prevented infection with the virus, and its replication in the nasal cavity, within 24 hours of administration.

The results suggest a sound rationale for the use of OC-01 (varenicline) nasal spray as a therapeutic for pre-exposure/post-exposure prophylaxis to prevent infection, to decrease viral load, and/or to lessen severity and transmission of SARS-CoV-2.”

Since varenicline can be used topically as an aqueous nasal spray, it can achieve high local concentrations in the nasal mucosa. Moreover, this averts potential side effects due to systemic administration. In addition, it tackles the virus at the site of the first entry. And finally, it retains efficacy against alpha and beta variants as with the wild-type virus.

This confirms earlier in silico studies that indicate the ability of varenicline to bind directly to the SARS-CoV-2 RBD, at the hinge site, showing high affinity. This is thought to block the change of the spike protein into the ‘up’ conformation, which is necessary for binding to the ACE2 receptor or the nAChR and subsequent infection of the host cell.

The spike also binds to the nAChR directly, though not via the RBD, but at the Y674-R685 region. In fact, this part of the protein may actually take on distinct conformations after binding to the α4β2 and α7 nAChR subtypes, which are observed to have high binding affinity for this agent.

This second action suggests that varenicline will continue to show efficacy against new VOCs since its affinity for the Y674-R685 region of the spike or for nAChRs does not depend on the RBD conformation.

Finally, the nicotinic cholinergic system is thought to prevent severe COVID-19, which is due in part to the induction of a hyperactive immune-inflammatory response, triggering a cytokine storm, which results in multi-organ dysfunction.

What is the conclusion?

Given the in vitro and in vivo effectiveness seen in the studies, varenicline nasal spray warrants further investigation as an antiviral agent for pre-exposure/post-exposure prophylaxis, and/or prevention of transmission of SARS-CoV-2 wild-type and variants,” write the researchers.

*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.

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