GSK-3 inhibitors show promise in the treatment of coronavirus infections

Researchers from the United States have proposed a new approach to treating coronavirus infection, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) – the agent that causes coronavirus disease 2019 (COVID-19).

The study showed that inhibition of phosphorylation of a key structural protein expressed on coronaviruses damages SARS-CoV-2 infection in human lung epithelial cells.

The team used inhibitors of the host cell protein glycogen synthase kinase 3 (GSK-3) to block the phosphorylation of the nucleocapsid protein SARS-CoV-2, which is necessary for coronavirus replication.

The researchers also showed that the lithium inhibitor GSK-3 was associated with a significantly reduced risk for COVID-19.

“Targeting GSK-3 may therefore provide antiviral therapy for COVID-19 and for coronavirus infections that may occur in the future,” says Peter Klein of the University of Pennsylvania and colleagues.

A pre-print version of the research paper is available at medRxiv* server, while the article is being reviewed.

An outbreak of coronavirus is likely to occur in the future

Klein and colleagues warn that given the large outbreaks of coronavirus that have occurred in the last two decades, new outbreaks are likely to occur in the future.

“In addition to developing effective vaccines, antiviral strategies that target preserved mechanisms in coronavirus replication and transmission may be needed for COVID-19 and potential future coronavirus epidemics,” they say.

What is already known about the nucleocapsid protein?

The nucleocapsid (N) protein is required for replication, transcription, and assembly of the coronavirus.

However, N protein expressed by the SARS-CoV-1 virus (which caused the outbreak of SARS 2002-2004) requires phosphorylation by the GSK-3 host cell to the arginine-serine (RS) domain.

Furthermore, the GSK-3 lithium inhibitor, which is already a common therapy for bipolar disorder, impairs the replication of various coronaviruses, including SARS-CoV-1, swine epidemic virus, and transmissible gastroenteritis virus.

“Interfering with the conserved dependence of N protein on the host protein GSK-3 may be a sustainable approach to the treatment of COVID-19 and potential future coronavirus epidemics,” writes Klein and team.

Studies have already shown that despite the SARS-CoV-1 N protein sharing only 20–30% of the sequence identity with the N proteins of many other coronaviruses, all viruses have an RS domain located between the N-terminus and the C-terminal of the conserved domain.

The RS domain of the SARS-CoV-1 N protein includes repeat motifs (SXXXS) that are often associated with GSK-3 phosphorylation.

Furthermore, one study recently found that the SARS-CoV-2 N protein is highly phosphorylated within the RS domain. However, no study to date has examined whether GSK-3 phosphorylates the SARS-CoV-2 N protein or whether lithium exerts any antiviral effect against SARS-CoV-2.

What did the researchers do?

Klein and colleagues have now shown that the RS domain of the SARS-CoV-2 N protein is 90% similar to that of the SARS-CoV-1 N protein and that each contains two sets of three SXXXS motifs.

Although the N protein sequences of the other coronaviruses diverge, the team also showed that they still retain SXXXS motifs.

“As we find GSK-3 consensus sites in the N proteins of various coronaviruses, GSK-3 inhibitors may also be effective antiviral therapy in other coronavirus infections, including those that may occur in the future,” the researchers say.

Klein and colleagues then tested the effects of various GSK-3 inhibitors on the SARS-CoV-2 N protein expressed in 293T human embryonic kidney cells.

They found that lithium chloride and several other small molecule inhibitors of GSK-3, including CHIR99021, AR-A014418, and Enzastaurin, all inhibited N-phosphorylation, strongly supporting that GSK-3 is key to N-protein phosphorylation.

Furthermore, the team found that the GSK-3 inhibitor CHIR99021 also disrupted the replication of SARS-CoV-2 in the Calu-3 cell line derived from human lung epithelium..

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The GSK-3 inhibitor blocks replication in SARS-CoV2-infected cells: A. Dose response analysis of Calu-3 cells treated with GSK-3 inhibitors CHIR99021 or Enzastaurin (UPenn).  Cells were treated with the drug at the indicated concentrations and then inoculated with SARS-CoV-2.  Cells were fixed at 48 hpi and the total number of cells (green) and the percentage of viral infection (blue) detected by immunofluorescence for dsRNA were estimated.  B. Calu-3 cells were treated with vehicle or indicated concentrations of CHIR99021, inoculated with SARS-CoV-2, fixed at 48 hpi, and Spike protein was detected by immunofluorescence (UCLA).  Enzastaurin had no effect on viral infection in Calu-3 cells.

The GSK-3 inhibitor blocks replication in SARS-CoV2-infected cells: A. Dose response analysis of Calu-3 cells treated with GSK-3 inhibitors CHIR99021 or Enzastaurin (UPenn). Cells were treated with the drug at the indicated concentrations and then inoculated with SARS-CoV-2. Cells were fixed at 48 hpi and the total number of cells (green) and the percentage of viral infection (blue) detected by immunofluorescence for dsRNA were estimated. B. Calu-3 cells were treated with vehicle or indicated concentrations of CHIR99021, inoculated with SARS-CoV-2, fixed at 48 hpi, and Spike protein was detected by immunofluorescence (UCLA). Enzastaurin had no effect on viral infection in Calu-3 cells.

Examination to see if lithium reduces the risk of COVID-19

Because lithium is already widely used to treat bipolar disorder, the researchers investigated whether patients taking lithium have a reduced risk of developing COVID-19, compared to the general population.

They conducted a retrospective analysis of data from the three major health systems in the United States.

The researchers included data for 121,589 people from the University of Pennsylvania Health System (UPHS), 115,073 from Mount Sinai Medical Center (MSMC) and 102,420 from hospitals and clinics at the University of Iowa (UIHC). All patients have been tested for SARS-CoV-2 polymerase chain reaction (PCR) since February 2021.

Among these individuals, 8,856 (7.2%) patients with UPHS, 10,597 (9.2%) patients with MSMC, and 16,170 (15.8%) patients with UIHC were confirmed to be positive for SARS-CoV-2.

In all three health systems, 7% of patients taking lithium developed COVID-19, compared with 15% among the general population.

Furthermore, a meta-analysis of data using a random effects model showed that patients taking lithium were 49% less likely to develop COVID-19 than patients not taking lithium.

What did the authors conclude?

The team suggests that inhibition of N protein protein phosphorylation underlies the antiviral activity of lithium and other GSK-3 inhibitors against SARS-CoV-2.

“The development of GSK-3 inhibitors that safely and effectively inhibit N phosphorylation is a promising potential approach to controlling SARS-CoV-2 and other coronavirus infections that may occur in the future,” say Klein and colleagues.

The researchers also point out that the approach is based on a clear mechanism and uses clinically tested drugs that are well tolerated and can be rapidly repurposed for COVID-19.

* Important notice

medRxiv publishes preliminary scientific reports that have not been reviewed and, therefore, should not be considered definitive, guide clinical practice / behavior related to health, or be treated as established information.

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