A Molecular Mechanism for Eflornithine Resistance in African Trypanosomes

Abstract

Human African trypanosomiasis, endemic to sub-Saharan Africa, is invariably fatal if untreated. Its causative agent is the protozoan parasite Trypanosoma brucei. Eflornithine is used as a first line treatment for human African trypanosomiasis, but there is a risk that resistance could thwart its use, even when used in combination therapy with nifurtimox. Eflornithine resistant trypanosomes were selected in vitro and subjected to biochemical and genetic analysis. The resistance phenotype was verified in vivo. Here we report the molecular basis of resistance. While the drug's target, ornithine decarboxylase, was unaltered in resistant cells and changes to levels of metabolites in the targeted polyamine pathway were not apparent, the accumulation of eflornithine was shown to be diminished in resistant lines. An amino acid transporter gene, TbAAT6 (Tb927.8.5450), was found to be deleted in two lines independently selected for resistance. Ablating expression of this gene in wildtype cells using RNA interference led to acquisition of resistance while expression of an ectopic copy of the gene introduced into the resistant deletion lines restored sensitivity, confirming the role of TbAAT6 in eflornithine action. Eflornithine resistance is easy to select through loss of a putative amino acid transporter, TbAAT6. The loss of this transporter will be easily identified in the field using a simple PCR test, enabling more appropriate chemotherapy to be administered.

Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth

“…The only drug for which there is a well-defi ned molecular target is efl ornithine, an analog of ornithine which blocks spermidine synthesis and the formation of trypanothione through irreversible inhibition of ornithine decarboxylase; administered with nifurtimox, the combination therapy has enjoyed rapid implementation ( 52 ). Based on previous successes with substrate analogs containing nitrogen or sulfur on the lateral sterol side chain proving them to be potent antifungal agents ( 5, 49-51 ), we studied two of them, AZA and 25-thialanosterol sulfonium salt, as reversible tight-binding inhibitors of Tb-SMT.…”

Metabolomic-Based Strategies for Anti-Parasite Drug Discovery

“…34 This was verified using standard molecular techniques, and an amino acid transporter, AAT6, was shown to import the drug, and its absence was responsible for resistance. 34 The MOAs of novel drugs have also been analyzed using metabolomics. In a recent study, fluoropyrimidine analogues were synthesized, and their action on T. brucei was investigated by LC-MS.…”

Antiprotozoal glutathione derivatives with flagellar membrane binding activity against T. brucei rhodesiense

“…Genetic approaches have identified some key gene products responsible for resistance 8 in trypanosomes these include the trypanosome P2 adenosine transporter, AT1 9 , responsible for the uptake of the front-line drugs melarsoprol and pentamidine used to treat HAT, which was found to be mutated or absent in a number of drug resistant strains 10 . The upregulation of the melarsoprol-trypanothione (Mel-T) transporter, multidrug resistance protein A (MRPA) and the loss of the amino acid transporter, AAT6 in resistant strains 12 , involved in the uptake of the drug eflornithine used to treat HAT.…”