Inhibition of dengue virus through suppression of host pyrimidine biosynthesis

Abstract

Viral replication relies on the host to supply nucleosides. Host enzymes involved in nucleoside biosynthesis are potential targets for antiviral development. Ribavirin (a known antiviral drug) is such an inhibitor that suppresses guanine biosynthesis; depletion of the intracellular GTP pool was shown to be the major mechanism to inhibit flavivirus. Along similar lines, inhibitors of the pyrimidine biosynthesis pathway could be targeted for potential antiviral development. Here we report on a novel antiviral compound (NITD-982) that inhibits host dihydroorotate dehydrogenase (DHODH), an enzyme required for pyrimidine biosynthesis. The inhibitor was identified through screening 1.8 million compounds using a dengue virus (DENV) infection assay. The compound contains an isoxazole-pyrazole core structure, and it inhibited DENV with a 50% effective concentration (EC(50)) of 2.4 nM and a 50% cytotoxic concentration (CC(50)) of >5 μM. NITD-982 has a broad antiviral spectrum, inhibiting both flaviviruses and nonflaviviruses with nanomolar EC(90)s. We also show that (i) the compound inhibited the enzymatic activity of recombinant DHODH, (ii) an NITD-982 analogue directly bound to the DHODH protein, (iii) supplementing the culture medium with uridine reversed the compound-mediated antiviral activity, and (iv) DENV type 2 (DENV-2) variants resistant to brequinar (a known DHODH inhibitor) were cross resistant to NITD-982. Collectively, the results demonstrate that the compound inhibits DENV through depleting the intracellular pyrimidine pool. In contrast to the in vitro potency, the compound did not show any efficacy in the DENV-AG129 mouse model. The lack of in vivo efficacy is likely due to the exogenous uptake of pyrimidine from the diet or to a high plasma protein-binding activity of the current compound.

Fig. 1.

(A) Structure of NITD-982. (B) Effect of NITD-982 on expression of viral envelope protein. A549 cells were infected with DENV-2 (MOI of 0.3) in the presence of 2-fold serial dilutions of NITD-982. After incubation at 37°C for 48 h, the expression of viral envelope protein was quantified by CFI assay. (C) Effect of NITD-982 on the growth of DENV-2. Vero cells were infected with DENV-2 (MOI of 0.1). After incubation at 37°C for 48 h, cell culture supernatants were harvested for plaque assay in BHK-21 cells. The limit of quantification (L.O.Q.) (indicated by a dotted line) for the plaque assay is around 100 PFU/ml. (D) Effect of NITD-982 on DENV-2 replicon. A549 cells containing a luciferase replicon of DENV-2 were treated with NITD-982 at the indicated concentrations for 48 h. The inhibition of viral replication by the compound was measured by the luciferase activity. (E) Cytotoxicity of NITD-982 in Vero and A549 cells. Cytotoxicity was examined by incubation of Vero and A549 cells for 2 days with the indicated concentrations of NITD-982. Cell viability was measured by an MTS assay. Average results and standard deviations (n = 3) are presented.

Fig. 2.

Time-of-addition analysis. DENV-2 (strain New Guinea C) containing a Renilla luciferase reporter was used to infect Vero cells in a 96-well plate. The luciferase gene was engineered at the capsid gene of the DENV-2, as previously reported by Shustov and colleagues (44). Approximately 2 × 10⁴ Vero cells were seeded per well. After an overnight incubation, the cells were infected with the reporter virus at an MOI of 1. NITD-982 was then added to the infected cells at a final concentration of 25 nM at the indicated time points. At 24 h p.i., the cells were assayed for luciferase activities. As negative controls, an equivalent concentration of DMSO (0.45%) was included at various time points. Average results and standard deviations (n = 4) are presented.

Fig. 3.

Antiviral spectrum of NITD-982. (A) Vero cells were infected with indicated viruses at an MOI of 0.1; the infected cells were immediately treated with NITD-982. Virus production was quantified using plaque assays. The detection limit of the plaque assay is about 100 PFU/ml for all tested viruses. (B) Huh-7 cells containing a luciferase replicon of HCV were treated with NITD-982 at the indicated concentrations for 48 h. The inhibition of viral replication was measured by luciferase activity (left panel). Replicon cell viability (right panel) was determined by CellTiter-Glo luminescent-cell viability assay (Promega). Average results and standard deviations (n = 3) are presented.

Fig. 4.

DHODH is the target of NITD-982. (A) Recombinant human DHODH protein made in E. coli. Human DHODH containing an N-terminal His tag was overexpressed in an E. coli expression system. The cell lysates were purified through an affinity nickel column. Samples from total cell lysate, soluble cell lysate (soluble fraction), column flowthrough, washings, and elution were analyzed by SDS-PAGE with Coomassie blue staining. (B) NITD-982 and its analogue NITD-102 inhibit DHODH activity in vitro. DHODH activity was measured with a chromogen reduction assay as described in Materials and Methods. Brequinar was included as a positive control. Average results and standard deviations (n = 3) are presented. (C) Calorimetric titration of DHODH with NITD-102 and brequinar. The concentration of DHODH was 23 μM, and the syringe contained NITD-102 (233 μM) or brequinar (200 μM). The structures of NITD-102 and brequinar are shown in the bottom panels. The ITC titration curves and the calculated K_d values are shown in the top panels.

Fig. 5.

Uridine reverses the antiviral effect of NITD-982. A549 cells were infected with DENV-2 and treated with different doses of NITD-982 and uridine. At 48 h p.i., viral envelope protein production was quantified by the CFI assay (see experimental details in Materials and Methods). Average results and standard deviations (n = 3) are presented.

Fig. 6.

(A) Brequinar-resistant viruses are cross resistant to NITD-982. Vero cells were infected with wild-type or recombinant DENV-2 (strain TSV01; MOI of 0.1) in the presence or absence of NITD-982. At 48 h p.i., viral titers were quantified by plaque assays. A dotted line indicates the limit of quantification (L.O.Q.) of the plaque assay. (B) Resistance analyses of WT DENV-1 (strain Western Pacific) and DENV-2 (strain TSV01). Vero cells were infected with WT DENV-1 and DENV-2 at an MOI of 0.1 in the presence of NITD-982. Viral titers were quantified by plaque assays at 48 h postinfection. (C) Inhibition of de novo RNA synthesis by NITD-982. The RdRp assay was performed as described in Materials and Methods. Average results and standard deviations (n = 3) are presented.

Fig. 7.

In vivo testing of NITD-982. (A) Plasma concentrations of NITD-982 after oral and subcutaneous (s.c.) administration in cotton rats at a dosage of 10 mg/kg of body weight (n = 3 per time point). A dotted line indicates the limit of quantification (L.O.Q.) by LC–MS-MS. (B) In vivo efficacy of NITD-982. AG129 mice were intraperitoneally inoculated with 2 × 10⁶ PFU of DENV-2 (strain TSV01) on day 0. The mice (6 animals per group) were subcutaneously dosed with NITD-982 twice a day. The peak viremia on day 3 p.i. was quantified by plaque assay. A dotted line indicates the L.O.Q. of the plaque assay. (C) Plasma uridine level in NITD-982-treated mice. Uridine levels in plasma samples were quantitatively analyzed by LC–MS-MS. The asterisk denotes a Student t test P value of < 0.05. A dotted line indicates the L.O.Q.