Inhibiting pyrimidine biosynthesis impairs Ebola virus replication through depletion of nucleoside pools and activation of innate immune responses

Abstract

Specific host pathways that may be targeted therapeutically to inhibit the replication of Ebola virus (EBOV) and other emerging viruses remain incompletely defined. A screen of 200,000 compounds for inhibition of an EBOV minigenome (MG) assay that measures the function of the viral polymerase complex identified as hits several compounds with an amino-tetrahydrocarbazole scaffold. This scaffold was structurally similar to GSK983, a compound previously described as having broad-spectrum antiviral activity due to its impairing de novo pyrimidine biosynthesis through inhibition of dihydroorotate dehydrogenase (DHODH). We generated compound SW835, the racemic version of GSK983 and demonstrated that SW835 and brequinar, another DHODH inhibitor, potently inhibit the MG assay and the replication of EBOV, vesicular stomatitis virus (VSV) and Zika (ZIKV) in vitro. Nucleoside and deoxynucleoside supplementation studies demonstrated that depletion of pyrimidine pools contributes to antiviral activity of these compounds. As reported for other DHODH inhibitors, SW835 and brequinar also induced expression of interferon stimulated genes (ISGs). ISG induction was demonstrated to occur without production of IFNα/β and independently of the IFNα receptor and was not blocked by EBOV-encoded suppressors of IFN signaling pathways. Furthermore, we demonstrated that transcription factor IRF1 is required for this ISG induction, and that IRF1 induction requires the DNA damage response kinase ATM. Therefore, de novo pyrimidine biosynthesis is critical for the replication of EBOV and other RNA viruses and inhibition of this pathway activates an ATM and IRF1-dependent innate immune response that subverts EBOV immune evasion functions.

Figure 1.. GSK983 and SW835 are potent inhibitors of Ebola minigenome activity.

The Ebola MG assay and cell toxicity in the presence of varying concentrations of (A) SW407, (B) SW835, (C) GSK983, (D) SW032 and (E) SW308. F. The effect of SW835 on the counter screen assay for which the readout is firefly and Renilla luciferase expression from a T7 promoter and an RNA polymerase II promoter, respectively. G. Ebola MG assay activity and cell toxicity of brequinar.

Figure 2.. GSK983 and SW835 are potent inhibitors of EBOV replication.

A. Anti-EBOV effects of GSK983 and its enantiomers and SW835. A549 cells were pretreated with compound at the indicated concentrations, infected with EBOV-GFP at an MOI of 2 and two days post-infection culture supernatants were collected, and virus titers were determined by plaque assay. The graphs represent the mean and standard deviation of triplicates. B. Viability of A549 cells treated with indicated compounds 72h post treatment. C. Antiviral activity of brequinar against EBOV-GFP in A549 cells assessed as in panel A. D. Cell viability of brequinar in A549 cells assessed as in panel B. E. The anti-EBOV activity of the compounds in HeLa cells as determined by using EBOV-GFP virus. Percent infectivity was determined by quantification of fluorescence intensities by measuring GFP. The DMSO treated sample to 100%. F. Cell viability was determined in the presence of the indicated compounds in HeLa cells at 24h post-treatment. The cells were fixed, and nuclei were stained with Hoechst stain and were quantified using Cell Profiler software. DMSO treated cells were set at 100%.

Figure 3.. SW835 and brequinar are potent inhibitors of VSV and ZIKV replication.

A. The antiviral activity of SW835 was measured in Vero cells against VSV-GFP. The cells were pretreated with 3-fold serial dilutions starting at 50 μM for 1h, and then infected with VSV-GFP at an MOI of 0.002. Infectivity was determined by quantifying GFP expression in 96 well plates at 21h post-infection. The y-axis represents percent mean fluorescence activity as determined by normalizing to the mock-treated, VSV-GFP infected samples. B. Antiviral activity of SW385 against ZIKV. Vero cells were treated with compound at four-fold serial dilutions with concentration starting from 1 μM. The cells were infected with ZIKV at MOI=1. ZIKV protein production was quantified using antibody staining (pan-flavivirus antibody, 4G2) via a colorimetric assay at 48h post-infection by measuring absorbance at 650nm. The DMSO treated infected sample is set at 100% activity. C. Antiviral activity of brequinar against VSV-GFP virus. D. Antiviral activity of brequinar against ZIKV. Data represent the mean and standard deviation of triplicates for A-D.

Figure 4.. Deoxycytidine reverses growth inhibition of SW835 without affecting its antiviral activity.

A and B. HEK293T cells were transfected in bulk in a T75 flask with the MG complex plasmids. The next day, cells (1*10⁵) were plated in 96 well plates and treated with DMSO or 1 μM SW835 in the presence of the indicated concentrations of (A) uridine and (B) deoxcytidine. MG activity was measured at 24h post drug treatment. In parallel, cell viability was determined. C. Effect of SW835 (3 μM) on replication of EBOV-GFP (MOI 2) in A549 cells in the presence of 1 mM uridine or 1 mM deoxycytidine at 48 post infection. D. Effect of SW835 on VSV-GFP replication with pyrimidine supplementation. The A549 cells were pretreated with SW835 (10 μM) for 8h with or without exogenously added nucleosides. The cells were infected with VSV-GFP at an MOI of 0.1 and further incubated with drugs for another 16h. The cell supernatants were collected, and viral titers were determined by plaque assay. E. Effect of brequinar (3 μM) on the replication of EBOV-GFP. G. Effect of brequinar (10 μM) on the replication of VSV-GFP. F. Effect of uridine and deoxycytidine on SW835 or brequinar-induced growth inhibition in A549 cells. Cell viability was determined following 72h treatment with no exogenous pyrimidines (control), 1 mM uridine or 1mM deoxycytidine at the indicated concentrations of SW835. Error bars represent the standard deviation for triplicates in A-F.

Figure 5.. SW835 induced ISG expression depends on pyrimidine depletion.

Stable HEK293T-IFNβ-FF (A) or ISRE-FF (B) cell lines were plated in 384 well plates. In each case, the cells were allowed to rest for 2h and then treated with DMSO or the indicated amounts of SW835 or brequinar. SeV infection and uIFN (100 units/ml) served as controls. Error bars represent the standard deviation for four replicates. The p-value was determined by one-way ANOVA with Tukey’s test for multiple comparison, *** p< 0.0001. C. The ISG54, ISG56 and MX1 mRNA expression in A549 cells 24h after DMSO, uIFN (100 Units) or SW835 (10μM) addition. D. Stable HEK293T ISRE-FF cells were plated into 96 well plates, treated with compound (10 μM) in the presence or absence of the indicated pyrimidines at 1mM. Luciferase activity was measured at 24h post treatment. E. The endogenous expression of mRNAs for (E) ISG54 and (F) ISG56 in A549 cells after 24h treatment with the DMSO, SW835 or brequinar at 3 μM in the presence or absence of uridine or deoxycytidine at 24h post treatment. Error bars represent the standard deviation for triplicates in A and B-F.

Figure 6.. SW835 ISG induction contributes to antiviral activity.

A. A549 were treated with compounds in presence or absence of nucleosides and infected with EBOV-GFP as described in Figure 4C. EBOV NP (eNP) (A) and ISG54 (B) mRNA levels were measured by quantitative real-time RT-PCR (qRT-PCR) 48h post infection. Alternatively, cells were treated with compound and infected with VSV-GFP as described in Figure 4D. The VSV-N (C) and ISG54 (D) mRNA levels were determined at 16h post-infection. Finally, A549 cells were infected with ZIKV (MOI 1) with similar treatment of compounds and pyrimidines as described in Figure 4D. ZIKV RNA or ISG54 mRNA levels were measured at 40h post-infection. Primers specific to the 5’UTR of the viral genome was used to assess viral RNA levels (E) and an ISG54 specific primer was used to assess ISG induction (F). Error bars represent the standard deviation for triplicates in A-F.

Figure 7.. SW835 induces ISGs independently of virus infection or IFN production.

A. ISRE activation by SW835 is not suppressed by filovirus interferon antagonists. The stable HEK293T-ISRE-FF cells were transfected with plasmids expressing HA-tagged EBOV VP35 (eVP35), EBOV VP24 (eVP24) or MARV VP40 (mVP40) (50 or 500ng of expression plasmid was transfected, as indicated). The next day cells were treated with DMSO, SW835, brequinar or uIFN. 20h post-treatment luciferase activity was determined. The ‘ns’ indicate the values were not significantly different from vector transfected conditions. The p-value was determined by one-way ANOVA with Tukey’s test for multiple comparison, * p< 0.02. Western blotting was performed to detect the expression of eVP35, eVP24 and mVP40 (at 50 and 500ng) using anti-HA antibody. B. The MEF-wt or IFNAR knockout cells were transfected with ISRE and Renilla reporter plasmids. The next day cells were treated with DMSO, SW835, brequinar or uIFN. 20h later, the luciferase activity was determined. Error bars represent the standard deviation for triplicates in A-B. P-value was determined by one-way ANOVA with Tukey’s test for multiple comparison. ** p< 0.001 and “ns” is not significant.

Figure 8.. IRF1 contributes to the antiviral activity of SW835.

A. Stable HEK293T-ISRE-FF cells were transfected with an siRNA targeting IRF1 or a scrambled siRNA. 48h later the cells were treated with SW835 or brequinar (10 μM). Luciferase activity was determined at 20h post treatment. Western blot shows the knockdown of IRF1 expression using IRF1 specific antibody. B. The MEF wt, IFNAR knockout or STAT2 knockout cells were transfected with siRNA targeting IRF1 or scrambled siRNA. 48h post transfection cells were additionally transfected with ISRE-firefly and Renilla reporter plasmids. The next day, cells were treated with compounds at 10 μM. 20h post-treatment luciferase activity was measured. The IRF1 knockdown significantly decreased ISRE luciferase activity upon compound treatment. The p-value was determined by one-way ANOVA with Tukey’s test for multiple comparison; *** p< 0.0001. C. Antiviral activity of SW835 against VSV-GFP infected at MOI of 0.01 in IRF1 knockdown A549 cells. The virus titers were determined by plaque assay. IRF1 expression was detected by using anti-IRF1 antibody and anti-GAPDH served as loading control. D. Endogenous expression of ISG54 was determined by qRT-PCR from RNA isolated from cells infected with VSV-GFP described in Panel C.

Figure 9.. ATM regulates IRF1 levels upon nucleoside depletion by DHODH inhibitors.

A. Stable ISRE-FF cells were treated with ATM Kinase inhibitor (10 μM) 1h prior to addition of DMSO, SW835 (10 μM), brequinar (10 μM) of uIFN. 20h post treatment, luciferase activity was determined. B. Stable ISRE-FF cells were transfected with control or ATM shRNA plasmids and the following day treated with DMSO, SW835 (10 μM), brequinar (10 μM) or universal IFN alpha (uIFN, 100 units/ml). 20h post treatment, luciferase activity was determined. The knockdown of ATM was evaluated by Western blotting using anti-ATM antibody. C. Expression level of IRF1 was determined by Western blotting of HEK293T cells treated with ATM Kinase inhibitor and either SW835, brequinar or uIFN. The ATM Kinase treatment and ATM knockdown significantly decreased ISRE luciferase activity upon compound treatment. The p-value was determined by one-way ANOVA with Tukey’s test for multiple comparison; *** p< 0.0001.

Figure 10.. A proposed model of the antiviral mechanisms of DHODH inhibitors against RNA viruses.

DHODH is a critical enzyme for de novo pyrimidine biosynthesis mediating oxidation of dihydroorotate to orotate. SW835 or other DHODH inhibitors impair the enzymatic activity of DHODH, blocking de novo pyrimidine biosynthesis. This results in the depletion of deoxynucleosides and ribonucleosides, affecting both cell proliferation and RNA virus replication. Depletion of pyrimidine pools also triggers a cellular antiviral response likely through its effect on cellular DNA. This leads to the activation of ATM which induces the expression of IRF1. IRF1 induces ISG expression contributing to inhibition of virus replication.