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 M. 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 compoundmediated 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.

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

“…The antiviral activity of all selected compounds was confirmed, and the overall identification hit rate for this screening pipeline was 1.3‰. This score is very similar to results obtained by other groups with different high-throughput replication-based screening assays for antivirals 27,32 , and falls in the expected range for cell-based phenotypic assays in general (1-5‰ 33 ). However, a hit rate is highly dependent on both the compound library that is screened and the scoring filter thresholds, which can be adjusted depending on the profile of collected data.…”

“…Optional: supplement culture medium with uridine at 20 μg/ml. Note: When screening chemical libraries for viral replication inhibitors, it seems that compounds targeting early steps of pyrimidine biosynthesis pathway are frequently isolated 13,16,27,28 . The addition of uridine to culture medium is used to filter out such antiviral molecules.…”

Broad-spectrum inhibition of common respiratory RNA viruses by a pyrimidine synthesis inhibitor with involvement of the host antiviral response

“…Despite promising in vitro antiviral activity, the use of pyrimidine synthesis inhibitors in animal challenge models has not been successful [27][28][29]. This could be due to the specific uptake and metabolism of the compound, as well as to a reservoir of uridine in living organisms.…”

Activation de la réponse innée antivirale par des inhibiteurs de la biosynthèse des pyrimidines

“…Alors que nous testions sans succès différentes hypothèses, plusieurs groupes ont publié simultanément des travaux montrant que des molécules inhibitrices de la voie de biosynthèse des pyrimidines sont des antiviraux à large spectre [19][20][21][22]. Même si rien ne suggérait un lien fonctionnel entre cette voie métabolique et la réponse innée antivirale, nous avons testé la capacité du composé DD264 à bloquer la biosynthèse de novo des pyrimidines (Figure 3).…”

“…Cependant, plusieurs groupes qui cherchaient à identifier directement des composés antiviraux dans des tests mesurant la réplication d'un virus en culture ont également isolé des inhibiteurs de cette voie métabolique, et qui ciblent notamment la DHODH [19][20][21][22]25]. Les essais réalisés in vivo chez la souris par ces différentes équipes ont donné des résultats mitigés.…”