Dataset Information

Exploring the mode of action of ebselen in Trypanosoma brucei hexokinase inhibition.

ABSTRACT: Glycolysis is essential to Trypanosoma brucei, the causative agent of African sleeping sickness, suggesting enzymes in the pathway could be targets for drug development. Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one, EbSe) was identified in a screen as a potent inhibitor of T. brucei hexokinase 1 (TbHK1), the first enzyme in the pathway. EbSe has a history of promiscuity as an enzyme inhibitor, inactivating proteins through seleno-sulfide conjugation with Cys residues. Indeed, dilution of TbHK1 and inhibitor following incubation did not temper inhibition suggesting conjugate formation. Using mass spectrometry to analyze EbSe-based modifications revealed that two Cys residues (C327 and C369) were oxidized after treatment. Site-directed mutagenesis of C327 led to enzyme inactivation indicating that C327 was essential for catalysis. C369 was not essential, suggesting that EbSe inhibition of TbHK1 was the consequence of modification of C327 via thiol oxidation. Additionally, neither EbSe treatment nor mutation of the nine TbHK1 Cys residues appreciably altered enzyme quaternary structure.

SUBMITTER: Joice AC

PROVIDER: S-EPMC3862409 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Exploring the mode of action of ebselen in Trypanosoma brucei hexokinase inhibition.

Joice April C AC Harris Michael T MT Kahney Elizabeth W EW Dodson Heidi C HC Maselli Andrew G AG Whitehead Daniel C DC Morris James C JC

International journal for parasitology. Drugs and drug resistance 20130912

Glycolysis is essential to Trypanosoma brucei, the causative agent of African sleeping sickness, suggesting enzymes in the pathway could be targets for drug development. Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one, EbSe) was identified in a screen as a potent inhibitor of T. brucei hexokinase 1 (TbHK1), the first enzyme in the pathway. EbSe has a history of promiscuity as an enzyme inhibitor, inactivating proteins through seleno-sulfide conjugation with Cys residues. Indeed, dilution of TbH ...[more]

PMID: 24533305

Similar Datasets

Project description:Elucidating the mechanism of action of trypanocidal compounds is an important step in the development of more efficient drugs against Trypanosoma brucei. In a screening approach using an RNAi library in T. brucei bloodstream forms, we identified a member of the mitochondrial carrier family, TbMCP14, as a prime candidate mediating the action of a group of anti-parasitic choline analogs. Depletion of TbMCP14 by inducible RNAi in both bloodstream and procyclic forms increased resistance of parasites towards the compounds by 7-fold and 3-fold, respectively, compared to uninduced cells. In addition, down-regulation of TbMCP14 protected bloodstream form mitochondria from a drug-induced decrease in mitochondrial membrane potential. Conversely, over-expression of the carrier in procyclic forms increased parasite susceptibility more than 13-fold. Metabolomic analyses of parasites over-expressing TbMCP14 showed increased levels of the proline metabolite, pyrroline-5-carboxylate, suggesting a possible involvement of TbMCP14 in energy production. The generation of TbMCP14 knock-out parasites showed that the carrier is not essential for survival of T. brucei bloodstream forms, but reduced parasite proliferation under standard culture conditions. In contrast, depletion of TbMCP14 in procyclic forms resulted in growth arrest, followed by parasite death. The time point at which parasite proliferation stopped was dependent on the major energy source, i.e. glucose versus proline, in the culture medium. Together with our findings that proline-dependent ATP production in crude mitochondria from TbMCP14-depleted trypanosomes was reduced compared to control mitochondria, the study demonstrates that TbMCP14 is involved in energy production in T. brucei. Since TbMCP14 belongs to a trypanosomatid-specific clade of mitochondrial carrier family proteins showing very poor similarity to mitochondrial carriers of mammals, it may represent an interesting target for drug action or targeting.

Dataset Information

Exploring the mode of action of ebselen in Trypanosoma brucei hexokinase inhibition.

Publications

Exploring the mode of action of ebselen in Trypanosoma brucei hexokinase inhibition.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets