6-Thioguanine blocks SARS-CoV-2 replication by inhibition of PLpro protease activities.

A recently emerged betacoronavirus, SARS-CoV-2, has led to a global health crisis that calls for the identification of effective therapeutics for COVID-19 disease. Coronavirus papain-like protease (PLpro) is an attractive drug target as it is essential for viral polyprotein cleavage and for deconjugation of ISG15, an antiviral ubiquitin-like protein. We show here that 6-Thioguanine (6-TG) inhibits SARS-CoV-2 PLpro-catalyzed viral polyprotein cleavage and ISG15 deconjugation in cells and inhibits replication of SARS-CoV-2 in Vero-E6 cells and Calu3 cells at submicromolar levels. As a well-characterized FDA-approved orally delivered drug, 6-TG represents a promising therapeutic for COVID-19 and other emerging coronaviruses. ONE SENTENCE
SUMMARY: A repurposed drug that targets an essential enzymatic activity of SARS-CoV-2 represents a promising COVID-19 therapeutic.

Coronavirus Disease-2019 (COVID-19) is caused by SARS-CoV-2, a betacoronavirus highly similar to SARS (now SARS-CoV-1) (1, 2). An urgent need exists for treatment strategies, and repurposed FDA-approved drugs with existing supply chains and well-characterized pharmacologic properties represent a rapid and efficient approach toward COVID-19 therapeutics (3).

Enzymes encoded by SARS-CoV-2 are attractive drug targets, including PLpro, an essential cysteine protease activity within the nsp3 protein (4). PLpro cleaves the pp1a polyprotein at three sites, generating mature nsp1, 2 and 3 proteins. The PLpro recognition site (LXGG) is also found at the C-terminus of ubiquitin and ISG15, an antiviral ubiquitin-like modifier, although SARS-CoV-1 and -2 PLpro preferentially catalyze de-ISGylation over de-ubiquitylation (5, 6). Therapeutic inhibition of PLpro would therefore be predicted to have two antiviral effects: restoration of the antiviral effect of ISGylation and inhibition of viral replication by blocking polyprotein cleavage. Further, de-ISGylation by PLpro, through generation of free (unconjugated) ISG15, enhances the secretion and extracellular signaling function of ISG15, which in turn promotes pro-inflammatory cytokine production from cells of the immune system (7). Therefore, a potential third effect of inhibiting PLpro would be a decrease in pro-inflammatory “cytokine storms” associated with COVID-19 (8).

6-Thioguanine (6-TG) is an FDA-approved drug that has been used in the clinic since the 1950s, originally for the treatment of childhood leukemias and subsequently for treatment of inflammatory bowel and Crohn’s disease (9). 6-TG was previously reported to inhibit the SARS-CoV-1 and MERS PLpro catalytic domain in vitro (10, 11), with an IC50 of 5 µM and 24.4 µM, respectively, however there was no further follow up of its ability to inhibit de-ISGylation or viral polyprotein cleavage in cells or its ability to inhibit viral replication. Fig. 1A shows that co-expression of PLpro (residues 746-1061 of nsp3) with ISG15 and its conjugation enzymes (Uba7, Ube2L6, and Herc5) in HEK293T cells resulted in nearly complete loss of ISG15 conjugates, while expression of the active-site C-to-A variant of PLpro (C856 of nsp3; PLproCA) was inactive. Addition of 6-TG resulted in a dose-dependent inhibition of PLpro de-ISGylation activity, with half-maximal inhibition at approximately 0.1 µM (Fig. 1A; quantitation shown in Supplementary Fig. 1A).

Figure 1.

6-TG inhibits PLpro mediated de-ISGylation and viral polyprotein cleavage.

A. The structure of 6-Thioguanine is shown. HEK293T cells were transfected with plasmids expressing FLAG-ISG15 and the ISG15 E1/E2/E3 enzymes and 0.2 μg of PLpro plasmid (WT or CA) where indicated. 6-TG was added at the time of transfection at the indicated final concentrations (μM). Total cell lysates were prepared 48 hours post-transfection and analyzed by immunoblotting to detect FLAG-ISG15 and FLAG-ISG15 conjugates. B. (Top) Schematic representation of the TAP-nsp123 fusion protein, with molecular masses of subdomains indicated. Arrows indicate sites of PLpro-catalyzed cleavage. (Bottom) HEK293T cells were transfected with plasmids expressing either TAP-nsp1, TAP-nsp123WT or TAP-nsp123CA. 6-TG was added at the time of transfection at the indicated final concentrations (μM) and total cell lysates were prepared 48 hours post-transfection and analyzed by immunoblotting with anti-TAP antibody. Bands corresponding to TAP-nsp1 and the full-length fusion proteins are indicated. Bracketed bands (*) represent breakdown products of the full-length WT or CA fusion proteins.

The PLpro domain, located within nsp3, generates mature nsp-1, 2 and 3 proteins from the pp1a polyprotein through self-catalyzed cleavage reactions (Fig. 1B, schematic). We determined whether 6-TG inhibited polyprotein processing, as this is an essential function of Plpro and the natural context in which PLpro is expressed. An N-terminally TAP-tagged fusion of nsp1, 2 and 3 (TAP-nsp123WT) was expressed in HEK293T cells (Fig. 1B, schematic), as well as the active-site C-to-A variant (TAP-nsp123CA). The fusion protein was detected with anti-TAP antibody, and TAP-nsp1 was expressed separately as a size marker for the fully processed fusion protein. As shown in Fig. 1B, expression of TAP-nsp123WT resulted primarily in a product the size of TAP-nsp1 (42 kD), as expected if the fusion protein was fully processed, while expression of TAP-nsp123CA resulted in the expected full-length protein (~336 kD) along with several breakdown products of the full-length protein. Increasing concentrations of 6-TG inhibited PLpro-mediated processing of the TAP-nsp123WT polyprotein, with loss of the TAP-nsp1 product and accumulation of the full-length fusion protein, with the same pattern of breakdown products seen with the TAP-nsp123CA protein. Half-maximal inhibition of polyprotein cleavage occurred between 0.1 and 0.5 µM (quantitation shown in Supplementary Fig. 1B). This result indicates that 6-TG inhibits an essential function of PLpro activity and that it inhibits PLpro when it is expressed in the context of the full-length nsp3 protein.

In addition to its function as a ubiquitin-like modifier, free (unconjugated) ISG15 exits cells and signals to LFA-1-expressing cells of the immune system (e.g., NK cells, T cells) to release specific cytokines, including IFN-γ and IL-10 (7, 12), while ISG15 conjugates are retained in cells. Therefore, a secondary effect of PLpro-mediated de-ISGylation is the enhanced secretion of ISG15 (7). We determined whether 6-TG would block this effect. Fig. 2A shows that expression of PLpro in HEK293T cells led to decreased intracellular ISG15 conjugates (upper blot, total cell lysates) and increased free ISG15 in the extracellular space (lower blot, IP-western of ISG15 from cell culture supernatants), and that 6-TG not only restored intracellular conjugates but also blocked the production of free extracellular ISG15.

Figure 2.

6-TG inhibits PLpro-enhanced extracellular ISG15 signaling and production of IL-6 from PBMCs.

A. HEK293T cells were transfected and treated with 6-TG, as indicated. Total cell lysates were prepared and cell culture supernatants were collected 48 hours post-transfection. The upper panel shows a FLAG-ISG15 immunoblot analysis of cell lysates and the lower panel shows a FLAG-ISG15 immunoblot of an anti-FLAG immunoprecipitation of the cell culture supernatants. B. HEK293T cells were plated in the lower chamber of a transwell plate and transfected with plasmids expressing FLAG-ISG15, the ISG15 E1/E2/E3 enzymes, and PLpro, as indicated. 6-TG (0.5 µM) was added to the cells at the time of transfection. PMBCs were added to the upper chamber of the transwell plate; supernatants were collected from the upper chamber 24 hours post-transfection and assayed for IL-6 by ELISA. Significance was assessed by ordinary one-way ANOVA comparison to treatment with 6-TG alone. Asterisks indicate p-values: ****<0.0001, and non-significant changes are indicated by ns.

IFN-γ, IL-10 and IL-6 are associated with uncontrolled cytokine release syndrome and have been shown to be elevated in COVID-19 patients (13, 14). The complete range of cytokines affected by extracellular ISG15 signaling is unknown, but the relationship of ISG15 to IFN-γ and IL-10 (12) led us to test whether ISG15 also induced the secretion of IL-6 from human PBMCs. In a trans-well culture system, HEK293T cells (lower chamber) were transfected with ISG15, with or without the ISG15 conjugation enzymes (E1/E2/E3) and/or PLpro. PBMCs were placed in the upper chamber, separated from the lower chamber by a 0.4 micron membrane. In this system, ISG15 released from cells in the lower chamber signals to LFA-1-expressing cells in the upper chamber, and cytokine release in the upper chamber is measured by ELISA (7). Fig. 2B shows that expression of ISG15, alone, in the HEK293T cells led to secretion of IL-6 from PBMCs, and this effect was diminished when the conjugation enzymes were co-expressed with ISG15 (compare lanes 1, 3, and 5). De-ISGylation catalyzed by PLpro resulted in enhanced IL-6 secretion (compare lanes 5 and 7), and the addition of 6-TG reversed the effect of PLpro, leading to diminished IL-6 production (compare lanes 7 and 8). These results identify IL-6 as an additional cytokine that is responsive to extracellular ISG15 signaling and suggest that PLpro may be at least partially responsible for enhanced cytokine responses in COVID-19 patients. Therapeutically, 6-TG inhibition of PLpro might therefore have the additional benefit of limiting excessive cytokine responses associated with poor patient outcomes.

6-TG was next analyzed for its ability to inhibit SARS-CoV-2 replication (strain 2019 n-CoV/USA_WA1/2020; 18) in Vero-E6 African green monkey kidney cells and in the Calu3 human lung epithelial cell line. As shown in Fig. 3, 6-TG inhibited viral replication in Vero-E6 cells with a half-maximal effective concentration (EC50) of 0.647 ± 0.374 μM. By comparison, Remdesivir inhibited SARS-CoV-2 replication in Vero-E6 cells similarly, with an EC50 of 0.77 µM (15). 6-TG inhibited virus replication in Calu3 cells at a lower EC50, 0.061 ± 0.049 μM. While the basis of the difference between the two cell lines is not known, cell lines can vary significantly with respect to thiopurine uptake and metabolism (16). 6-TG did not elicit significant cellular toxicity in either Vero-E6 or Calu3 cells (CC50 >50 μM), yielding selectivity indices (SI) of >77 in Vero-E6 cells and >819 Calu3 cells. Together, the results presented here indicate that 6-Thioguanine is a direct-acting SARS-CoV-2 antiviral, inhibiting PLpro de-ISGylation, polyprotein cleavage, and viral replication at submicromolar concentrations.

Figure 3.

6-TG inhibits SARS-CoV-2 viral replication.

Vero-E6 (A, C) and Calu3 (B, D) cells were treated with various concentrations of 6-TG and assessed for viability or were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.001. A and B: Cellular cytotoxicity of 6-TG was determined using a real-time SYTOX assay 48 hours post-treatment (closed squares) and the median cytotoxic concentration (CC50) was >50 mM in both cell lines. Inhibition of viral replication was assayed by SARS-CoV-2 N1 RT-qPCR of cell supernatants 48 hours post-infection (closed circles) and the effective median concentration for inhibition (EC50) was determined. The left and right y-axis of each graph represent mean percent inhibition of virus yield/titer and mean percent cell viability at the indicated concentration of 6-TG. C. and D. Viral genome equivalents in the cell supernatants were quantified by SARS-CoV-2 N1 RT-qPCR. Data are means ± SED; n = 3 – 5 biological replicates.

To our knowledge, 6-TG is the first identified FDA-approved inhibitor of PLpro demonstrated to inhibit SARS-CoV-2 replication. 6-TG is a widely available orally delivered generic drug on the World Health Organization list of essential medicines (17). Dosing regimens vary significantly depending on its use, from 10 mg per day for long-term treatment of inflammatory diseases, to up to 3 mg/kg/day in acute lymphocytic leukemia treatments (18). While toxicity can be significant at higher dosages, it is anticipated that its use as an antiviral would be over a relatively short time period and that toxicity issues would likely be minimal. We propose that the results presented here warrant the initiation of human clinical trials of 6-TG as a SARS-CoV-2 therapeutic. As PLpro is a conserved and essential enzymatic activity of the beta coronaviruses, 6-TG may prove useful in the current and future coronavirus pandemics and as a complement to other antivirals in development.