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High throughput screening against the peroxidase cascade of African trypanosomes identifies antiparasitic compounds that inactivate tryparedoxin.

ABSTRACT: In African trypanosomes, the detoxification of broad spectrum hydroperoxides relies on a unique cascade composed of trypanothione (T(SH)(2)), trypanothione reductase, tryparedoxin (Tpx), and nonselenium glutathione peroxidase-type enzymes. All three proteins are essential for Trypanosoma brucei. Here, we subjected the complete system to a high throughput screening approach with nearly 80,000 chemicals. Twelve compounds inhibited the peroxidase system. All but one carried chloroalkyl substituents. The detailed kinetic analysis showed that two compounds weakly inhibited trypanothione reductase, but none of them specifically interacted with the peroxidase. They proved to be time-dependent inhibitors of Tpx-modifying Cys-40, the first cysteine of its active site WCPPC motif. Importantly, gel shift assays verified Tpx as a target in the intact parasites. T(SH)(2), present in the in vitro assays and in the cells in high molar excess, did not interfere with Tpx inactivation. The compounds inhibited the proliferation of bloodstream T. brucei with EC(50) values down to <1 ?M and exerted up to 83-fold lower toxicity toward HeLa cells. Irreversible inhibitors are traditionally regarded as unfavorable. However, a large number of antimicrobials and anticancer therapeutics acts covalently with their target protein. The compounds identified here also interacted with recombinant human thioredoxin, a distant relative of Tpx. This finding might even be exploited for thioredoxin-based anticancer drug development approaches reported recently. The fact that the T(SH)(2)/Tpx couple occupies a central position within the trypanosomal thiol metabolism and delivers electrons also for the synthesis of DNA precursors renders the parasite-specific oxidoreductase an attractive drug target molecule.

SUBMITTER: Fueller F

PROVIDER: S-EPMC3308743 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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High throughput screening against the peroxidase cascade of African trypanosomes identifies antiparasitic compounds that inactivate tryparedoxin.

Fueller Florian F Jehle Britta B Putzker Kerstin K Lewis Joe D JD Krauth-Siegel R Luise RL

The Journal of biological chemistry 20120123 12

In African trypanosomes, the detoxification of broad spectrum hydroperoxides relies on a unique cascade composed of trypanothione (T(SH)(2)), trypanothione reductase, tryparedoxin (Tpx), and nonselenium glutathione peroxidase-type enzymes. All three proteins are essential for Trypanosoma brucei. Here, we subjected the complete system to a high throughput screening approach with nearly 80,000 chemicals. Twelve compounds inhibited the peroxidase system. All but one carried chloroalkyl substituents ...[more]

PMID: 22275351

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Project description:Leishmaniasis is a neglected disease caused by Leishmania, an intracellular protozoan parasite which possesses a unique thiol metabolism based on trypanothione. Trypanothione is used as a source of electrons by the tryparedoxin/tryparedoxin peroxidase system (TXN/TXNPx) to reduce the hydroperoxides produced by macrophages during infection. This detoxification pathway is not only unique to the parasite but is also essential for its survival; therefore, it constitutes a most attractive drug target. Several forms of TXNPx, with very high sequence identity to one another, have been found in Leishmania strains, one of which has been used as a component of a potential anti-leishmanial polyprotein vaccine. The structures of cytosolic TXN and TXNPx from L. major (LmTXN and LmTXNPx) offer a unique opportunity to study peroxide reduction in Leishmania parasites at a molecular level, and may provide new tools for multienzyme inhibition-based drug discovery. Structural analyses bring out key structural features to elucidate LmTXN and LmTXNPx function. LmTXN displays an unusual N-terminal ?-helix which allows the formation of a stable domain-swapped dimer. In LmTXNPx, crystallized in reducing condition, both the locally unfolded (LU) and fully folded (FF) conformations, typical of the oxidized and reduced protein respectively, are populated. The structural analysis presented here points to a high flexibility of the loop that includes the peroxidatic cysteine which facilitates Cys52 to form an inter-chain disulfide bond with the resolving cysteine (Cys173), thereby preventing over-oxidation which would inactivate the enzyme. Analysis of the electrostatic surface potentials of both LmTXN and LmTXNPx unveils the structural elements at the basis of functionally relevant interaction between the two proteins. Finally, the structural analysis of TXNPx allows us to identify the position of the epitopes that make the protein antigenic and therefore potentially suitable to be used in an anti-leishmanial polyprotein vaccine.

Dataset Information

High throughput screening against the peroxidase cascade of African trypanosomes identifies antiparasitic compounds that inactivate tryparedoxin.

Publications

High throughput screening against the peroxidase cascade of African trypanosomes identifies antiparasitic compounds that inactivate tryparedoxin.

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