| dc.description.abstracteng | 1-Hydroxyquinolones were recently described as effective inhibitors of Toxoplasma gondii replication. These compounds (e.g. HDQ and compound B) were previously shown to inhibit the alternative NADH dehydrogenase (NADH2-I) activity and thus the respiratory chain of T. gondii. However, the phenotype of ndh2-I knock-out mutants suggested the presence of additional target(s).
In this study, the fourth enzyme of de novo pyrimidine biosynthesis, the T. gondii dihydroorotate dehydrogenase (TgDHODH), was identified as a novel 1-hydroxyquinolone target. T. gondii mutants with a partial resistance phenotype towards 1-hydroxyquinolones were used to analyze the expression profile and the coding sequence of all ubiquinone reducing mitochondrial dehydrogenases, which are the likely targets of 1-hydroxyquinolones. Quantitative RT-PCR analysis revealed no significant differences in the mRNA expression level of these enzymes. However, DNA-sequencing identified a single point mutation in codon 302 of the TgDHODH coding sequence that changes a conserved asparagine into serine in the vicinity of the dihydroorotate binding site. This mutation is sufficient to confer the partial drug resistance phenotype, as shown by replacement of the wild type allele with the N302S allele, using a “knock-in” strategy. Enzyme kinetics of E. coli expressed and purified wild type and N302S TgDHODH revealed an inhibition of the wild type enzyme activity by nanomolar concentrations of 1-hydroxyquinolones, while the IC50s for the N302S mutant were significantly increased. Furthermore, inhibition kinetics demonstrated that 1-hydroxyquinolones act as competitive inhibitors for the electron acceptor QD, but as uncompetitive inhibitors for dihydroorotate.
TgDHODH is localized in the inner mitochondrial membrane, and is linking the pyrimidine de novo pathway to the mitochondrial respiratory chain by transferring electrons from dihydroorotate to ubiquinone. To distinguish whether the lack of TgDHODH-mediated electron transfer or the inhibition of de novo pyrimidine biosynthesis is responsible for growth inhibition, both pathways were uncoupled by expression of the ubiquinone independent Saccharomyces cerevisiae DHODH (ScDHODH). Heterologous expression of the 1-hydroxyquinolone-insensitive ScDHODH in T. gondii should lead to a restoration of de novo pyrimidine biosynthesis in the presence of HDQ and compound B. These parasites showed a strongly increased resistance towards 1-hydroxyquinolones, demonstrating that inhibition of pyrimidine de novo synthesis significantly contributes to the growth inhibitory potential of 1-hydroxyquinolones in T. gondii. 1-Hydroxyquinolones thus directly inhibit two important metabolic pathways: oxidative phosphorylation and pyrimidine de novo synthesis.
The growth kinetics of drug treated parasites is characterized by a biphasic pattern, with an initial phase (~ 0 to 24 hours) in which the parasites - independent of their resistance or sensitivity to 1-hydroxyquinolones - show an almost complete growth inhibition. In the later phase of treatment, drug-resistant mutants - and to a much lower extent also wild type parasites - start replicating again. This growth recovery is occurring in the absence of a detectable mitochondrial membrane potential. However, parasites possess a strong dependency on glucose metabolism. A decrease of glucose in the tissue culture medium, and a treatment with a glycolytic inhibitor both prevented the growth recovery. Thus, 1-hydroxyquinolone treatment seems to induce a metabolic shift from oxidative phosphorylation towards glycolysis, which can partly restore the energy production. | de |