Identification of Resistance Determinants for 1-Hydroxy-2-Alkyl-4(1)quinolones in Toxoplasma gondii

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The increasing resistance of protozoan parasites against established drugs, particularly those of the malaria causative agent Plasmodium, emphasises the need for the identification of novel anti-parasitic compounds. We use Toxoplasma gondii as a model to elucidate the mode of action of anti-parasitic drugs in detail. The inhibition of the mitochondrial electron transport chain from apicomplexan parasites as T. gondii and P.falciparum is an effective anti-parasitic strategy. We recently identified 1-hydroxy-2-alkyl-4(1)quinolones, for example 1-hydroxy-2-dodecyl-4(1)quinolone (HDQ), as specific inhibitors of T. gondii oxidative phosphorylation. These compounds lead to a fast depolarization of the parasitic inner mitochondrial membrane potential and inhibit T. gondii and P. falciparum replication in the nano-molar range. HDQ possesses structural similarities to ubiquinone and it was shown by inhibition kinetics that HDQ is at least inhibiting the enzymatic activity of two enzymes, which possess ubiquinone binding sites (TgNDH2-I and PfDHODH). The T. gondii genome contains six ubiquinone reducing genes, which could be potential HDQ-targets. We propose to identify the relevant HDQ-target(s), whose inhibition is causing the replication inhibition, with the aid drug resistance T. gondii mutants. All six ubiquinone reducing genes will be amplified and sequenced from available HDQ resistant mutants, which were generated by chemical mutagenesis. Mutant alleles will be introduced into the wild type background in order to test whether a particular mutation is sufficient to confer resistance. Resistant mutants against two other HDQ derivatives will be generated and analyzed in the same way in order to investigate whether the individual 1-hydroxy-2-alkyl-4(1)quinolone derivatives target identical or different parasite molecules. Particular attention will be given to dihydroorotate dehydrogenase (DHODH) as a putative target, since we have preliminary evidence for a link between HDQ sensitivity and pyrimidine metabolism. We will uncouple pyrimidine de novo synthesis from respiratory chain function by expression of a ubiquinone independent DHODH, which uses fumarate instead of ubiquinone as an electron acceptor. Together, these studies will define relevant drug targets within the parasitic electron transport chain and will contribute to a detailed understanding on the mode of action of 1-hydroxy-2-alkyl-4(1) quinolones against T. gondii. Since P. falciparum possesses an identical set of ubiquinone reducing enzyme as T. gondii, these studies might also provide insights for novel drug targets in Plasmodium.