A novel antiviral mechanism of action for an FDA-approved thiopurine known as 6-thioguanine


In a recent study published in PLoS pathogensIn this study, researchers described a new antiviral mechanism of action by the Food and Drug Administration (FDA) – a thiopurine known as 6-thioguanine (6-TG).

Study: Theopurines prevent the processing of the spike protein of the Corona virus and its incorporation into offspring virions.  Image Credit: Bacsica / Shutterstock
Stady: Theopurines prevent the processing of the spike protein of the Corona virus and its incorporation into the offspring’s virions. Image Credit: Bacsica / Shutterstock

background

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has spurred efforts to repurpose drugs to develop effective and safe antivirals. Host-targeted antivirals (HTAs) indirectly inhibit viral replication by inhibiting host cellular processes and/or stimulating antiviral responses.

The authors of the current study previously demonstrated that thiopurines, 6-thioganosine (6-TGo) and 6-TG inhibit IAV (influenza A virus) replication by activating the unfolded protein response (UPR) and interfering with viral glycoprotein processing and accumulation.

about studying

The current study investigated whether 6-TG and other thiopurines could interfere with coronavirus (CoV) glycoproteins.

Theopurine effectiveness Against severe SARS-CoV-2, inhibition of HCoV-229E replication and disruption of human (HCoV)-OC43 RNA synthesis were evaluated. Cell culture experiments were performed using a 293T cell, HCT-8 (human ileal adenocarcinoma cell line), Huh7.

Furthermore, viral particle release was evaluated by quantitative analysis of reverse transcriptase-polymerase chain reaction (RT-qPCR) of extracellular viral genomes. Immunofluorescence analysis was performed using HCoV-OC43 virus-infected cells for anti-nucleocapsid (N) antibodies and double-stranded RNA (dsRNA). Ribopuromycinylation assays were performed, and 293T cells were transfected with plasmids encoding SARS-CoV-2 structural proteins such as N, spike (S), membrane (M) and envelope (E) proteins to evaluate the effect of 6-TG on SARS. Corona virus 2 structural proteins.

Furthermore, SARS-CoV-2 S was ectopically expressed and evaluated against multiple concentrations of 6-TG, after which immunoblotting was performed with pseudovirions (PV). S-expressing 293T cell lysates were treated with PNGase F (peptide-N-glycosidase) to remove N-linked glycans from the polypeptide chains, and the effects of 6-TG on the secretory pathway were evaluated by Gaussia luciferase assays.

Flow cytometry (FC) analysis and surface staining of 293T-expressing S cells were performed to measure S secretion, and cells were co-transfected with EGFP+ (enhanced green fluorescent protein) plasmids to assess changes in S protein accumulation.

Furthermore, cell co-transfers with structural proteins of SARS-CoV-2 proteins and plasmids were performed to elucidate the defective assembly mechanisms, and the team determined whether any known 6-TG targets cause defects in S-protein maturation. The team also evaluated potential morphological changes. of HCoV virus after 6-TG treatment by electron microscopy (TEM) analysis.

consequences

6-TG inhibits the initial stage of SARS-CoV-2 and HCoV-OC43 replication, limiting full-length viral genomes, structural proteins and genomic RNA accumulation. Analysis of ectopic S expression demonstrated enhanced electrophoretic mobility of S protein from several βCoVs by 6-TG treatment, according to in the laboratory Removal of N-linked enzymatic oligosaccharides from S. SARS-CoV-2 VLPs (virus-like particles) in 6-TG-treated cells lacking S protein.

Similar effects of 6-TG on lentivirus production of SARS-CoV-2 S were observed resulting in S-deficient pseudoviruses that could not infect cells expressing angiotensin converting enzyme 2 (ACE2). The results indicated that 6-TG treatment led to a defect in S-protein processing and trafficking and thus impeded the assembly of the infectious offspring virus. However, conversion of 6-TG to its nucleotide (nt) form by HPRT1 (hypoxanthin phosphoribosyltransferase 1) was essential for antiviral activity, which could be overcome by ML099, the guanosine-5′-triphosphate (GTP) agonist.

There are no GTPase inhibitors [Ras-related C3 botulinum toxin substrate 1 (Rac1), Ras homolog family member A (RhoA), and Cell division control protein 42 homolog (CDC42)] affected S accumulation or processing, indicating that 6-TG inhibits S maturation by inhibiting the unknown cellular GTPase. 6-TG, 6-thioguanosine (6-TGo) and 6-mercapturine (6-MP) reduced four logs in the release of SARS-CoV-2 virion and 6-TGo showed similar inhibition to HCoV-OC43 and HCoV-229E, Whereas 6-MP was ineffective. RT-qPCR analysis showed that 6-TG treatment reduced HCOV-OC43 titer by 20-fold on the first day of infection.

The putative full-length viral genomic RNA was reduced 10-fold at most stages of infection. 6-TG treatment caused similar decreases in viral S- and N-encoded subunit genomic RNAs associated with less protein accumulation. Immune cells infected with HCoV-OC43 with anti-N antibodies showed punctate staining initially and peripheral staining later. After 6-TG treatment, the stained areas were brighter, with significant dots observed 24 h post infection (HPI). Immunostaining for dsRNA showed a significant decrease in dsRNA signaling among 6-TG-treated cells.

6-TG delayed or suppressed downstream UPR transcriptional responses but did not affect translation initiation rates in HCoV-OC43-infected cells. 6-TG inhibition of HCoV-OC43 infection restricted inositol-requiring enzyme 1 (IRE1) activation and accumulation of the X-box protein 1 target gene (XBP1s). The results indicated that although 6-TG interfered with the synthesis of full-length and viral genomic RNA, the host closure was not perturbed.

6-TG downregulation of SARS-CoV-2 structural protein expression (particularly S) and immunoblotting analysis showed a high molecular weight S-band of S0 precursor protein, which was also sensitive to PNGaseF treatment. The results of electrophoresis analysis indicated that 6-TG inhibits S-glycosylation and processing, and Gaussia luciferase experiments showed that 6-TG does not disturb the global secretory pathway. TEM analysis showed fewer viral particles in 6-TG-treated cells.

conclusion

Overall, the study results highlighted small GTPases as potential targets of HTA, and the effects of 6-TG on S in several models, such as ectopic expression, native HCoV infections, and production of PVs and VLPs, suggest an antiviral mechanism bypassing papain. Protease inhibition (PLpro).



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