NITD-982

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 (EC50) of 2.4 nM and a 50% cytotoxic concentration (CC50) of >5 μM. NITD-982 has a broad antiviral spectrum, inhibiting both flaviviruses and nonflaviviruses with nanomolar EC90s. 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 thein vitropotency, the compound did not show any efficacy in the DENV-AG129 mouse model. The lack ofin vivoefficacy is likely due to the exogenous uptake of pyrimidine from the diet or to a high plasma protein-binding activity of the current compound.

The replication of virus is dependent on the host metabolism; nucleotide precursors for the synthesis of viral genome or viral mRNAs are supplied from the host nucleotides pool. As viral replication requires a massive synthesis of viral mRNA or viral genome, the inhibition of cellular nucleotide synthesis is considered a strategy for broad-spectrum antivirals. Indeed, the inhibition of GTP synthesis showed antiviral effects against many viruses. Mycophenolic acid (MPA) has broad-spectrum antiviral activity and the mechanism is to decrease cellular GTP concentration by inhibiting inosine-5’-monophosphate dehydrogenase (IMPDH), which catalyzes the rate-limiting reaction of de novo GTP biosynthesis [1]. Many IMPDH inhibitors, however, show noticeable cytotoxicity at the effective concentrations, making them less attractive as an antiviral therapeutic [2–5]. On the other hand, pyrimidine synthesis inhibitors are getting more attention as a novel antiviral strategy. Several pyrimidine synthesis inhibitors have been discovered as active hit compounds from high-throughput screenings for antivirals [6–8]. Unlike MPA, these pyrimidine synthesis inhibitors did not show toxicity to the cells at the effective concentrations. Recently, a novel antiviral mechanism was discovered that could interpret the robust antiviral activity of pyrimidine synthesis inhibitors. Marianne et al. has shown that brequinar or DD264, a dihydroorotate dehydrogenase (DHODH) inhibitor, has broad-spectrum antiviral activity and the treatment of the cells with the compounds induced the expression of IFN-stimulated genes (ISGs) that are associated with the antiviral effects [9]. The compound decreased cellular pyrimidine concentration; however, the decrease of pyrimidine concentration was not the main antiviral mechanism. More importantly, the antiviral effect was dependent on the synthesis of new proteins under the control of interferon regulatory transcription factor 1 (IRF1). This finding clearly illustrates how pyrimidine synthesis inhibitors could exert potent broad-spectrum antiviral activity without cytotoxicity unlike MPA. This finding could lead to the development of broad-spectrum antivirals from pyrimidine synthesis inhibitors. In support of the mechanism, leflunomide, an immunosuppressant drug that inhibits DHODH, the fourth enzyme of the pyrimidine biosynthesis (Figure 1), has been reported to have an antiviral effect against several viruses in a clinical study [10]. Figure 1 Brequinar and DHODH, Structure of brequinar (A), and the pyrimidine de novo biosynthesis pathway (B).DHODH is the rate limiting step and inhibited by brequinar. Despite this prominent antiviral effect in vitro, none of the pyrimidine synthesis inhibitors have shown antiviral effect in vivo models using mice [6–8]. For this reason, pyrimidine synthesis inhibition has not been accepted as a viable antiviral strategy. It has been speculated that the concentration of exogenous pyrimidines in the serum is too high to inhibit viral replication. This argument, however, can’t explain the lack of antiviral effect in mice completely. Wang et al. showed about a 50% decrease in uridine levels in mice treated with their compound, NITD-982 [8]. With the decrease in the pyrimidine concentration, the induction of ISGs was expected after the treatment of the mice, which could lead to an antiviral activity. As mentioned earlier, no antiviral effect was observed in various in vivo models, which is contradicting to the clinical finding with leflunomide. In this study, we sought to understand better why pyrimidine synthesis inhibitors are not successful in inhibiting virus replication in mouse models. During the study of a novel pyrimidine inhibitor as a broad-spectrum antiviral, we observed results that are similar to Marianne et al. in that cells treated with pyrimidine synthesis inhibitors reduced virus replication significantly. More interestingly, we found that such antiviral effect was cell line-specific: i.e., human cell lines established an antiviral state by the treatment of pyrimidine synthesis inhibitors, but mouse cell lines did not. This observation could explain the lack of antiviral effect of pyrimidine synthesis inhibitors in mouse models. This finding may imply the fundamental difference in the mechanism of innate immune system in response to the inhibition of pyrimidine biosynthesis between human and mouse.