COVID-19 Breakthrough: Canadian Study On Unfolded Protein Response (UPR) Uncovers Thiopurines To Stop Viral Replication In Human Coronaviruses

COVID-19 Breakthrough: Canadian researchers have made a major breakthrough during a study on UPR or unfolded protein response during coronaviruses viral replication leading them to a discovery of thiopurines to stop the viral replication of human coronaviruses including SARS-CoV-2. This breakthrough with further research could lead to a new and effective broad-spectrum therapeutic antiviral that could effectively inhibit not only SARS-CoV-2 coronavirus but also all other human coronaviruses and future coronaviruses.
 

The study team from the Department of Microbiology & Immunology, Dalhousie University, University of Calgary, and Department of Biochemistry and Molecular Biology, University of British Columbia initiated a detailed study to fully comprehend the unfolded protein response (UPR) in human coronaviruses in various cell cultures in order to look for prospective drug candidates that might prevent accumulation and replication of the SARS-CoV-2 coronavirus.
 
Compared to current direct-acting antiviral drugs, effective host-targeting antivirals may provide a higher barrier to the emergence of antiviral drug-resistant viruses. However, it remains challenging to identify cellular pathways that can be targeted to disrupt viral replication without causing adverse effects on bystander uninfected cells. In this study, the Canadian researchers report that two chemically similar FDA-approved thiopurine analogues, 6-TG and 6-TGo, have broad antiviral effects that result from activation of UPR and disruption of viral glycoprotein synthesis and maturation. Importantly, their data demonstrate for the first time that 6-TG and 6-TGo are effective antivirals against influenza virus and coronavirus and may be effective against other glycoprotein-containing viruses including the SARS-CoV-2 coronavirus.
 
Their study findings are published on a preprint server and are currently being peer-reviewed. https://www.biorxiv.org/content/10.1101/2020.09.30.319863v1
 
The study team  explained that viruses that are enveloped, such as the coronavirus, have genetic material that can code for membrane proteins that can be synthesized and modified in the endoplasmic reticulum (ER) before they can be transported to the areas of assembly of the parts of the virion.
 
However should the ER protein folding capacity be overwhelmed by too many virion particles, there is an overload of unfolded proteins in the ER. This triggers an unfolded protein response (UPR). This activates the transcription factor-6 (ATF6), inositol requiring enzyme-1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK). These can sense that the ER is under stress, and there is thus a synthesis of basic leucine zipper (bZIP) transcription factors.
 
When the UPR gets activated, the protein folding capacity of the ER is increased. This also triggers the ER-associated degradation (ERAD). All proteins that are not folded properly are brought out of the ER and degraded via the 26S proteasome.
 
Upon a coronavirus particle invading a cell, it first  tries to replicate fast, and this burdens the ER. The virus releases bursts of glycoproteins that overwhelm the ER. The virus, however, is capable of bypassing the UPR and promotes efficient repli cation.
 
It was observed that Influenza A viruses (IAVs) can encode three integral membrane proteins: hemagglutinin (HA), neuraminidase (NA), and matrix protein 2 (M2). While the IAV replication causes selective activation of the UPR, specific mechanisms can activate the UPR but then bypass it to promote effective viral replication.
 
The study team explains that the effects of NA and M2 proteins on UPR are not clear, but HA can promote UPR.
 
Similarly several coronaviruses (CoVs) can activate UPR. This includes the “infectious bronchitis virus (IBV), mouse hepatitis virus (MHV), transmissible gastroenteritis virus (TGEV), human coronavirus (HCoV)-OC43, and SARS-CoV-1.
 
However the whole of the genetic sequence does not react similarly to the CoV replication.
 
The study team identified two FDA-approved thiopurine analogs called “6- thioguanine (6-TG) and 6-thioguanosine (6-TGo)”. These were found to block IAV and HCoV-OC43 replication when their dose was increased in a graded manner.
 
Both Pateamine A and Silvestrol had been tested previously. These two thiopurines, however, were found to disrupt the process of accumulation of viral glycoproteins that could activate the UPR.
 
Pateamine A (PatA) and silvestrol are natural products that are eukaryotic translation initiation factor 4A that disrupt eIF4A function and arrest translation. (eIF4A) inhibitor 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744162/#:~:text=Pateamine%20A%20(PatA)%20and%20silvestrol,as%20stress%20granules%20(SGs).


 
Both drugs are made from phytochemicals extracted from the fruits and twigs of Aglaia foveolata plant which is also used in Thailand Medical News Therapeutic Teas. https://www.thailandmedical.news/news/new-therapeutic-teas-
 
It was found that in the cells that had been treated with 6-TG, the viral glycoprotein synthesis could be partially restored by the chemical inhibition of the UPR.
 
Interestingly, CoV Spike (S) proteins that are expressed on the surface of the virus showed UPR activation. The S protein from the novel coronavirus or SARS-CoV-2 S also caused UPR activation.
 
 6-TG inhibited the accumulation of full-length S0 or furin-cleaved S2 fusion proteins, they noted. It did not affect the S1 ectodomain. 6-TG could induce UPR that accelerates ERAD-mediated turnover of membrane-anchored S0 and S2 glycoproteins, the team found.
 
However it should be noted that not all thiopurines have this effect that can lead to viral replication inhibition.
 
The study team experimented and found that a chemically similar compound thiopurine 6-mercaptopurine (6-MP) had little effect on UPR and did not affect the replication of IAV HCoV-OC43.
 
Further studying the mechanism of UPR induction by the thiopurine compounds 6-TG and 6-TGo, the researchers wrote that these effects are not likely to be mediated through DNA or RNA incorporation of 6-TG because of several reasons.
 
The initial reason is that the stress associated with viral replication does not specifically induce UPR. The next reason is that among viral proteins, the accumulation of glycoproteins and their processing was disrupted selectively. The last reason was that the messenger RNA levels of HA and NA in the IAV were not significantly affected.
 
6-MP, on the other hand, can be converted into 6-thioguanosine triphosphate but did not induce UPR and had no effects on IAV glycoproteins or OC43 replication.
 
The study team wrote that their data reveals that “UPR-inducing molecules could be effective host-targeted antivirals against viruses that depend on ER processes to support efficient replication.”
 
Hence induction of UPR by 6-TG and 6-TGo thus could be a novel method by which an antiviral mechanism could be triggered by the host cell itself. This has been a previously unrecognized unique mechanism of action, the team wrote.
 
From the study findings it can be safely concluded that 6-TG and 6-TGo are effective host-targeted antivirals that trigger the UPR and disrupt accumulation of viral glycoproteins.
 
The study team is next planning more vitro studies with different cell lines and also in vivo studies using animal models before proceeding to human trials despite the fact that these two thiopurines’ Pateamine A and Silvestrol are already U.S.FDA approved drugs with human safety records and can be easily repurposed.
 
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