Promising new antivirals in opposition to arboviruses and SARS-CoV-2: nodosome inhibitors

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Even though the 2019 coronavirus disease (COVID-19) pandemic continues to threaten a second wave of SARS-CoV-2 infection, no vaccine has yet been approved in phase III studies. Therefore, the search for effective antivirals is still ongoing.

A new study describes two promising small molecules that inhibit a host cell factor called nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) that inhibit the replication and spread of many arboviruses. This could lead to the development of broad spectrum antivirals. The results of the study were published on the bioRxiv * preprint server.

Many highly effective direct-acting antivirals have been identified for many viruses such as HIV, herpes viruses, and hepatitis C viruses. However, these tend to work specifically against a virus or closely related virus families. The current study aims to find broad spectrum antivirals that can block a number of different viruses.

The researchers examined a number of small molecules that specifically inhibit the pattern recognition receptor NOD2, as well as a molecule called RIPK2 (receptor-interacting serine / threonine protein kinase 2), which is essential for NOD2 signaling. NOD2 receptors have bacterial cell wall ligands called muramyl dipeptide (MDP) which are a peptidoglycan, but they also bind to viral RNA.

Two faces of NOD2

After infection with the Zika virus, NOD2 is expressed in higher amounts in human fetal brain cells and stimulates this virus replication. The viral RNA-NOD2 complex causes the formation of a nodosome and promotes innate immunity.

NOD2 is a double-edged weapon that induces primary innate immunity to multiple viruses, but also causes myocarditis as a result of infection with the Coxsackievirus B3.

Targeting NOD2 signaling

The researchers tested the effects of two drugs, GSK717, which inhibits NOD2, and GSK583, which inhibits the downstream protein RIPK2. These were originally developed to treat inflammatory diseases.

They studied their effects on several human primary cell lines and tissue explants and concluded that these molecules prevent the replication of SARS-CoV-2, flaviviruses, and alphaviruses. This ability to inhibit a range of viruses is due in part to their ability to improve innate immunity.

Zika-induced nodosomes stimulate virus replication

Previous studies have shown that Zika virus replicates in human fetal brain cells and persists in the human fetal brain. It was later found that this caused increased expression of many inflammasome genes, including NOD2, which was increased by more than a hundred fold. Other inflammasome stimulators include viral double-stranded RNA and the human recombinant interferon-alpha.

NOD2 upregulation was key to replicating the Zika virus. It was found that this works via a NOD2-induced downregulation of several interferon-stimulated genes (ISGs) and inflammasome-related genes, which indicates an inhibition of the innate immune response.

The anti-NOD2 drug GSK717 blocks the spread of ZIKV infection. (A) Representative confocal imaging (20X) showing the antiviral effects of GSK717 at 20 µM and 40 µM. A549 cells were infected with ZIKV (MOI = 1) followed by treatment with DMSO or GSK717 at 20 or 40 µM for 48 hours before being processed for indirect immunofluorescence. ZIKV infected cells were identified using mouse monoclonal antibody (4G2) to coat protein and Alexa Fluor 488 donkey anti-mouse to detect the primary antibody. The nuclei were stained with DAPI. The images were recorded using a spinning disc confocal microscope equipped with the Volocity 6.2.1 software.

The NOD2 inhibition prevents Zika replication

When the NOD2 expression was silenced by the tested inhibitors GSK717 and GSK583, no cytotoxic effect was observed, but the multiplication and spread of the virus were inhibited. This was observed not only in fetal brain tissue, but also in human primary embryonic lung fibroblasts.

The researchers found that this anti-Zika effect was present in several cell lines, including those derived from lung and hepatoma cells, and that GSK717 was dose-dependent, whether at high or low infectious doses of the virus.

The NOD2 inhibition prevents Dengue / MAYV replication

They also tested its ability to suppress replication of dengue virus, “the most important arbovirus in terms of morbidity and mortality and the causative agent of 129 dengue hemorrhagic fever / dengue shock syndrome”. It was found that the GSK717 treated cells, compared to untreated cells treated after infection, decreased virus titers by over 90% and became a sharp decrease in the number of cells expressing the dengue virus antigen also observed after 48 min incubation with the drug. This persisted with a 60% reduction in Zika and dengue virus genomic RNA levels in GSK717-treated co-infected cells.

GSK717 also reduced the titer of mosquito-borne alphavirus (MAYV), both alone and in cells co-infected with this virus and dengue virus – with a greater impact on the previous virus. SARS-CoV-2 titers were also reduced to a similar extent by NOD2 inhibition.

RIPK2 inhibition suppresses arbovirus replication

The NOD2-RIPK2 interaction triggered by the binding of MDP to NOD2 causes the transcription of several inflammatory factors and antibacterial proteins. However, Zika infected cells did not show higher levels of RIPK2 transcription.

The researchers therefore asked whether inhibiting RIPK2-NOD2 binding would affect the replication of arboviruses such as Dengue, Zika and MAYV. This was the case both 12 and 24 hours after infection in cells treated with the RIPK2 inhibitor GSK583.

Successful SARS-CoV-2 inhibition in vitro

This virus suppressive effect was also observed with SARS-CoV-2, regardless of whether the drug was used before or after the occurrence of a viral infection. The observed reduction in SARS-CoV-2 titer at the highest drug concentration in two separate human lung and liver tumor-derived cell lines were 60% and 90%, respectively, but no evidence of cytotoxicity was found.

Implications

The arboviruses tested in this study circulate in the same regions of the world and often cause similar clinical signs and symptoms. The serological cross-reactivity further complicates the diagnosis. A drug that is able to fight all of these viruses would be a blessing indeed.

At the same time, the task of developing an effective drug to combat the COVID-19 pandemic is becoming more urgent every day. Therefore, the robust antiviral activity shown by both GSK717 and GSK583 against flaviviruses and against SARS-CoV-2 shows promise for further development.

Gefitinib is a monoclonal antibody that targets both the EGFR growth factor receptor and RIPK2. Its inhibitory effect against dengue virus replication and inflammatory cytokine release has been documented. The authors suggest that it could be useful in dengue patients, and similarly, NOD2 and RIPK2 inhibitors could prove to be effective anti-COVID-19 (as well as anti-flaviviral) drugs.

Both drugs were actually designed to fight inflammatory immunological diseases. Therefore, an additional benefit of these drugs is their anti-inflammatory effects. Since the viral diseases caused by Alpha-, Flavi- and SARS-CoV-2 owe their severity mainly to the associated hyperinflammatory phenomena, these drugs could alleviate these clinical syndromes.

Finally, several vaccine adjuvants enhance NOD2 to improve the immune response. In the light of this study, however, this effect could be undesirable, since higher NOD2 expression increases the replication of several RNA-pathogenic viruses. Indeed, this may be one of the virus evasion mechanisms by which the virus successfully prevents host immune responses.

The researchers conclude: “The current study shows how the identification of a drug target through transcriptomic analysis of virus-infected cells can lead to novel, broad-acting, host-directed antiviral strategies with a high resistance barrier.”

Using this research method, they were able to find broad spectrum antivirals among nodosome inhibitors that may attract further attention as potentially effective and safe anti-COVID-19 drugs.

* Important NOTE

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

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