Identification of dihydroorotate dehydrogenase as a relevant drug target for 1-hydroxyquinolones in Toxoplasma gondii

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 nova 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 IC(50)s in the nanomolar range, while the IC(50)s for the N302S mutant were significantly increased. Furthermore, inhibition kinetics revealed that 1-hydroxyquinolones act as competitive inhibitors for the electron acceptor Q(D), 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. (C) 2013 Elsevier B.V. All rights reserved.