title: Identification of dihydroorotate dehydrogenase as a relevant drug target for 1-hydroxyquinolones in Toxoplasma gondii.
abstract: 1-Hydroxyquinolones as for example 1-hydroxy-2-dodecyl-4(1)quinolone (HDQ) are effective growth inhibitors for Toxoplasma gondii. These compounds were shown to interfere with the respiratory chain function by inhibition of type II NADH dehydrogenase activity. With the aid of partial drug resistant mutants we identified in this study the fourth enzyme of the de novo pyrimidine synthesis pathway, the T. gondii dihydroorotate dehydrogenase (TgDHODH), as an additional 1-hydroxyquinolone target. A single point mutation was found in the TgDHODH coding sequence of drug resistant clones that change a conserved Asn into Ser in the vicinity of the dihydroorotate binding site. This mutation is sufficient to confer the partial drug resistance phenotype as shown by allele replacement. Enzyme kinetics revealed that 1-hydroxyquinolones inhibit wild type TgDHODH with IC50s in the nanomolar range, while the IC50s for the N302S mutant were significantly increased. Furthermore, inhibition kinetics revealed that 1-hydroxyquinolones act as competitive inhibitors for the electron acceptor QD, but as uncompetitive inhibitors for dihydroorotate. Moreover, heterologous expression of the ubiquinone independent DHODH from Saccharomyces cerevisiae in T. gondii also leads to partial 1-hydroxyquinolone resistance. Our data suggest that inhibition of TgDHODH activity significantly contributes to the growth inhibiting potential of 1-hydroxyquinolones in T. gondii.
authors.affiliation: Institute of Medical Microbiology, University Medical Center Göttingen, Kreuzbergring 57, Göttingen D-37075, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Hegewald
authors.forename: Uwe
authors.initials: U
authors.lastname: Gross
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Bohne
mesh_terms: D001665:Binding Sites; D000090722:Dihydroorotate Dehydrogenase; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015363:Quinolones / Q000737:chemistry / Q000494:pharmacology*; D014122:Toxoplasma / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D014123:Toxoplasmosis / Q000469:parasitology
chemical_list: D000090722:Dihydroorotate Dehydrogenase; D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D015363:Quinolones; C004768:4,5-dihydroorotic acid; D009963:Orotic Acid; D044925:Oxidoreductases Acting on CH-CH Group Donors
keywords: 1-Hydroxyquinolone; Dihydroorotate dehydrogenase (DHODH); Drug resistance; HDQ; Toxoplasma gondii