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Validation of spermidine synthase as a drug target in African trypanosomes.

ABSTRACT: The trypanocidal activity of the ODC (ornithine decarboxylase) inhibitor DFMO (difluoromethylornithine) has validated polyamine biosynthesis as a target for chemotherapy. As DFMO is one of only two drugs used to treat patients with late-stage African trypanosomiasis, the requirement for additional drug targets is paramount. Here, we report the biochemical properties of TbSpSyn (Trypanosoma brucei spermidine synthase), the enzyme immediately downstream of ODC in this pathway. Recombinant TbSpSyn was purified and shown to catalyse the formation of spermidine from putrescine and dcSAM (decarboxylated S-adenosylmethionine). To determine the functional importance of TbSpSyn in BSF (bloodstream form) parasites, we used a tetracycline-inducible RNAi (RNA interference) system. Down-regulation of the corresponding mRNA correlated with a decrease in intracellular spermidine and cessation of growth. This phenotype could be complemented by expressing the SpSyn (spermidine synthase) gene from Leishmania major in cells undergoing RNAi, but could not be rescued by addition of spermidine to the medium due to the lack of a spermidine uptake capacity. These results therefore genetically validate TbSpSyn as a target for drug development and indicate that in the absence of a functional biosynthetic pathway, BSF T. brucei cannot scavenge sufficient spermidine from their environment to meet growth requirements.

SUBMITTER: Taylor MC

PROVIDER: S-EPMC2427175 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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Validation of spermidine synthase as a drug target in African trypanosomes.

Taylor Martin C MC Kaur Harparkash H Blessington Bernard B Kelly John M JM Wilkinson Shane R SR

The Biochemical journal 20080101 2

The trypanocidal activity of the ODC (ornithine decarboxylase) inhibitor DFMO (difluoromethylornithine) has validated polyamine biosynthesis as a target for chemotherapy. As DFMO is one of only two drugs used to treat patients with late-stage African trypanosomiasis, the requirement for additional drug targets is paramount. Here, we report the biochemical properties of TbSpSyn (Trypanosoma brucei spermidine synthase), the enzyme immediately downstream of ODC in this pathway. Recombinant TbSpSyn ...[more]

PMID: 17916066

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Project description:Bloodstream forms of four populations of the livestock pathogen Trypanosoma congolense, isolated from different natural infections, have been shown to exhibit a wide range of sensitivities to the trypanocide isometamidium chloride (Samorin(R)). In mice the 50% curative doses (CD50) for Samorin range from 0.007 to 20 mg/kg body weight. Uptake of isometamidium chloride demonstrated Michaelis-Menten-type kinetics in all the populations, with Km values in the range 0.35-0.87 microM, and Vmax varied from 17 to 216 pmol/min per 10(8) cells. The magnitude of Vmax was correlated with sensitivity to the drug. In contrast, no correlation was observed between Km values and drug sensitivity. Pulse-chase experiments indicated two compartments for accumulation of drug. The first consists of freely diffusible drug that is invariant between populations; the other consists of retained isometamidium, which is of variable magnitude between the populations and is correlated with drug sensitivity. Autoradiography and fluorescence microscopy demonstrated initial, rapid accumulation of the drug within the mitochondrion, specifically the kinetoplast. In a drug-sensitive population of T. congolense, agents affecting mitochondrial function were shown to produce dose-dependent inhibition of mitochondrial membrane potential (DeltaPsimito), as measured by the accumulation of the lipophilic cations [3H]methyltriphenylphosphonium iodide or rhodamine 123. The agents also produced parallel inhibition of isometamidium uptake, suggesting an involvement of DeltaPsimito in the accumulation of the drug. When characterized in each of the four populations, the spontaneous DeltaPsimito was shown to be characteristic of each population and was correlated with Vmax for drug uptake and sensitivity to the drug in vitro and in vivo. We therefore conclude that in T. congolense DeltaPsimito is an important determinant of the rate and accumulation of the trypanocide isometamidium chloride. Populations of this trypanosome species vary with respect to DeltaPsimito, which is correlated with sensitivity to isometamidium. We suggest that when exposed to drug, the selection of such populations represents a novel mechanism of drug resistance in protozoan parasites.

Dataset Information

Validation of spermidine synthase as a drug target in African trypanosomes.

Publications

Validation of spermidine synthase as a drug target in African trypanosomes.

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