title: D282, a non-nucleoside inhibitor of influenza virus infection that interferes with de novo pyrimidine biosynthesis.
abstract: BACKGROUND
  The discovery of novel influenza virus inhibitors remains an important priority in light of the emergence of drug-resistant viruses. Toward this end, a library of over 6,000 compounds was tested for antiviral activity.
  
  
  METHODS
  Strains of influenza virus were evaluated by cytopathic effect (CPE) inhibition and virus yield reduction assays. Intracellular nucleoside triphosphate pools were analysed by strong anion exchange HPLC. Dihydroorotate dehydrogenase inhibition assays were conducted. Influenza virus-infected mice were treated for 5 days with D282.
  
  
  RESULTS
  A non-nucleoside, 4-[(4-butylphenyl)amino]-2-methylene-4-oxo-butanoic acid (D282), was discovered that inhibited influenza A and B virus CPE by 50% at 6-31 μM (giving selectivity indices of >13 to >67, based on cytotoxicity of >400 µM in stationary cell cultures). Ribavirin (positive control) was active at 14-44 µM (yielding selectivity indices of >9 to >29, with >400 µM toxicity). D282 and ribavirin inhibited virus yield by 90% at 9.5 ±3.3 and 10.8 ±3.2 µM, respectively. The antiviral activity of D282 in vitro was reversed by addition of uridine, cytidine and orotic acid. D282 exhibited an uncompetitive inhibition of mouse liver dihydroorotate dehydrogenase (inhibitor constant [Ki] of 2.3 ±0.9 µM, Michaelis constant [Km] of 150 ±16 µM). Because cellular pyrimidine biosynthesis was inhibited, D282-treated cells had decreased uridine triphosphate and cytidine triphosphate levels. D282 (≤100 mg/kg/day) failed to prevent death of mice infected with influenza.
  
  
  CONCLUSIONS
  D282 was active against influenza A and B viruses by inhibiting de novo pyrimidine biosynthesis. Although effective in vitro, the compound, like others in its class, was devoid of antiviral activity in infected mice.
authors.affiliation: Institute for Antiviral Research, Utah State University, Logan, UT, USA. don.smee@usu.edu
authors.forename: Donald F
authors.initials: DF
authors.lastname: Smee
authors.forename: Brett L
authors.initials: BL
authors.lastname: Hurst
authors.forename: Craig W
authors.initials: CW
authors.lastname: Day
mesh_terms: D000813:Anilides / Q000037:antagonists & inhibitors / Q000494:pharmacology* / Q000633:toxicity; D000818:Animals; D000998:Antiviral Agents / Q000037:antagonists & inhibitors / Q000494:pharmacology* / Q000633:toxicity; D020148:Butyric Acid / Q000037:antagonists & inhibitors / Q000494:pharmacology* / Q000633:toxicity; D000090722:Dihydroorotate Dehydrogenase; D004285:Dogs; D005260:Female; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000502:physiology; D053122:Influenza A Virus, H3N2 Subtype / Q000187:drug effects / Q000502:physiology; D053124:Influenza A Virus, H5N1 Subtype / Q000187:drug effects / Q000502:physiology; D009980:Influenza A virus / Q000187:drug effects* / Q000502:physiology; D008099:Liver / Q000201:enzymology; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D009963:Orotic Acid / Q000494:pharmacology; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000037:antagonists & inhibitors; D011743:Pyrimidines / Q000096:biosynthesis*; D013386:Succinates / Q000037:antagonists & inhibitors / Q000494:pharmacology* / Q000633:toxicity
chemical_list: C579253:4-((4-butylphenyl)amino)-2-methylene-4-oxobutanoic acid; D000813:Anilides; D000998:Antiviral Agents; D000090722:Dihydroorotate Dehydrogenase; D011743:Pyrimidines; D013386:Succinates; D020148:Butyric Acid; D009963:Orotic Acid; D044925:Oxidoreductases Acting on CH-CH Group Donors; C030986:pyrimidine