Characterization of Dengue Virus Resistance to Brequinar in Cell Culture

Abstract

Brequinar is an inhibitor of dihydroorotate dehydrogenase, an enzyme that is required for de novo pyrimidine biosynthesis. Here we report that brequinar has activity against a broad spectrum of viruses. The compound not only inhibits flaviviruses (dengue virus, West Nile virus, yellow fever virus, and Powassan virus) but also suppresses a plus-strand RNA alphavirus (Western equine encephalitis virus) and a negative-strand RNA rhabdovirus (vesicular stomatitis virus). Using dengue virus serotype 2 (DENV-2) as a model, we found that brequinar suppressed the viral infection cycle mainly at the step of RNA synthesis. Supplementing the culture medium with pyrimidines (cytidine or uridine) but not purines (adenine or guanine) could be used to reverse the inhibitory effect of the compound. Continuous culturing of DENV-2 in the presence of brequinar generated viruses that were partially resistant to the inhibitor. Sequencing of the resistant viruses revealed two amino acid mutations: one mutation (M260V) located at a helix in the domain II of the viral envelope protein and another mutation (E802Q) located at the priming loop of the nonstructural protein 5 (NS5) polymerase domain. Functional analysis of the mutations suggests that the NS5 mutation exerts resistance through enhancement of polymerase activity. The envelope protein mutation reduced the efficiency of virion assembly/ release; however, the mutant virus became less sensitive to brequinar inhibition at the step of virion assembly/ release. Taken together, the results indicate that (i) brequinar blocks DENV RNA synthesis through depletion of intracellular pyrimidine pools and (ii) the compound may also exert its antiviral activity through inhibition of virion assembly/release.

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

“…Brequinar is an inhibitor of dihydroorotate dehydrogenase, the fourth enzyme of pyrimidine biosynthesis pathway, with a potent antiviral activity in vitro 30,31 . When looking at IC50 values, less than one order of magnitude separated this reference molecule from most active hits identified by screening.…”

Broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis

“…Our attempts to generate A3-resistant influenza viruses were most likely unsuccessful due to the fact that a lack of nucleosides is hard to overcome. Nevertheless, Qing et al (15) recently reported a brequinar-resistant dengue virus in which a mutation in the NS5 gene (RNA-dependent RNA polymerase) conferred resistance through enhancement of viral RNA synthesis.…”

“…It does not affect the synthesis of cellular DNA or RNA (13,14), and may therefore target viral RNAdependent RNA polymerases directly or may be preferentially incorporated into viral RNA, thereby causing hypermutations. Brequinar was described as a broad-spectrum antiviral agent capable of inhibiting both negative-and positive-strand RNA viruses (15), and leflunomide and a derivative, FK778, have been reported to inhibit human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1) (16)(17)(18)(19). This group of compounds all target the cellular enzyme dihydroorotate dehydrogenase (DHODH) (20)(21)(22), although leflunomide shows the weakest activity against this enzyme (23).…”

Inhibition of Dengue Virus through Suppression of Host Pyrimidine Biosynthesis

“…We previously selected brequinar-resistant viruses by serial passaging of DENV-2 in the presence of increasing concentrations of brequinar. We found that mutation M260V in Env alone, E802Q in NS5 alone, or Env M260V plus NS5 E802Q conferred resistance of DENV-2 to brequinar (39). Given the fact that DHODH is the target of both brequinar and NITD-982, we hypothesized that the brequinar-resistant viruses would be insensitive to the inhibition of NITD-982.…”

“…The cholesterol biosynthesis pathway has been linked to DENV entry and replication as well as to the host immune response (29,42). We recently showed that brequinar inhibits DENV through depletion of the intracellular level of pyrimidine (39). Brequinar was originally developed as an antimetabolite in cancer and immunosuppression therapies through inhibition of dihydroorotate dehydrogenase (DHODH), the fourth enzyme in the de novo pyrimidine biosynthesis pathway (25,30).…”