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Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods.

ABSTRACT: The bifunctional trypanothione synthetase-amidase (TRYS) comprises two structurally distinct catalytic domains for synthesis and hydrolysis of trypanothione (N(1),N(8)-bis(glutathionyl)spermidine). This unique dithiol plays a pivotal role in thiol-redox homeostasis and in defence against chemical and oxidative stress in trypanosomatids. A tetracycline-dependent conditional double knockout of TRYS (cDKO) was generated in bloodstream Trypanosoma brucei. Culture of cDKO parasites without tetracycline induction resulted in loss of trypanothione and accumulation of glutathione, followed by growth inhibition and cell lysis after 6 days. In the absence of inducer, cDKO cells were unable to infect mice, confirming that this enzyme is essential for virulence in vivo as well as in vitro. To establish whether both enzymatic functions were essential, an amidase-dead mutant cDKO line was generated. In the presence of inducer, this line showed decreased growth in vitro and decreased virulence in vivo, indicating that the amidase function is not absolutely required for viability. The druggability of TRYS was assessed using a potent small molecule inhibitor developed in our laboratory. Growth inhibition correlated in rank order cDKO, single KO, wild-type and overexpressing lines and produced the predicted biochemical phenotype. The synthetase function of TRYS is thus unequivocally validated as a drug target by both chemical and genetic methods.

SUBMITTER: Wyllie S

PROVIDER: S-EPMC2784880 | biostudies-other | 2009 Nov

REPOSITORIES: biostudies-other

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Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods.

Wyllie Susan S Oza Sandra L SL Patterson Stephen S Spinks Daniel D Thompson Stephen S Fairlamb Alan H AH

Molecular microbiology 20090624 3

The bifunctional trypanothione synthetase-amidase (TRYS) comprises two structurally distinct catalytic domains for synthesis and hydrolysis of trypanothione (N(1),N(8)-bis(glutathionyl)spermidine). This unique dithiol plays a pivotal role in thiol-redox homeostasis and in defence against chemical and oxidative stress in trypanosomatids. A tetracycline-dependent conditional double knockout of TRYS (cDKO) was generated in bloodstream Trypanosoma brucei. Culture of cDKO parasites without tetracycli ...[more]

PMID: 19558432

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Project description:Trypanosoma brucei gambiense is the main causative agent of Human African Trypanosomiasis (HAT), also known as sleeping sickness. Because of limited alternatives and treatment toxicities, new therapeutic options are urgently needed for patients with HAT. Sterol 14alpha-demethylase (CYP51) is a potential drug target but its essentiality has not been determined in T. brucei. We used a tetracycline-inducible RNAi system to assess the essentiality of CYP51 in T. brucei bloodstream form (BSF) cells and we evaluated the effect of posaconazole, a well-tolerated triazole drug, within a panel of virulent strains in vitro and in a murine model. Expression of CYP51 in several T. brucei cell lines was demonstrated by western blot and its essentiality was demonstrated by RNA interference (CYP51RNAi) in vitro. Following reduction of TbCYP51 expression by RNAi, cell growth was reduced and eventually stopped compared to WT or non-induced cells, showing the requirement of CYP51 in T. brucei. These phenotypes were rescued by addition of ergosterol. Additionally, CYP51RNAi induction caused morphological defects with multiflagellated cells (p<0.05), suggesting cytokinesis dysfunction. The survival of CYP51RNAi Doxycycline-treated mice (p = 0.053) and of CYP51RNAi 5-day pre-induced Doxycycline-treated mice (p = 0.008) were improved compared to WT showing a CYP51 RNAi effect on trypanosomal virulence in mice. The posaconazole concentrations that inhibited parasite growth by 50% (IC50) were 8.5, 2.7, 1.6 and 0.12 μM for T. b. brucei 427 90-13, T. b. brucei Antat 1.1, T. b. gambiense Feo (Feo/ITMAP/1893) and T. b. gambiense Biyamina (MHOM/SD/82), respectively. During infection with these last three virulent strains, posaconazole-eflornithine and nifurtimox-eflornithine combinations showed similar improvement in mice survival (p≤0.001). Our results provide support for a CYP51 targeting based treatment in HAT. Thus posaconazole used in combination may represent a therapeutic alternative for trypanosomiasis.

Project description:BACKGROUND:The search for novel chemical entities targeting essential and parasite-specific pathways is considered a priority for neglected diseases such as trypanosomiasis and leishmaniasis. The thiol-dependent redox metabolism of trypanosomatids relies on bis-glutathionylspermidine [trypanothione, T(SH)2], a low molecular mass cosubstrate absent in the host. In pathogenic trypanosomatids, a single enzyme, trypanothione synthetase (TryS), catalyzes trypanothione biosynthesis, which is indispensable for parasite survival. Thus, TryS qualifies as an attractive drug target candidate. METHODOLOGY/PRINCIPAL FINDING:A library composed of 144 compounds from 7 different families and several singletons was screened against TryS from three major pathogen species (Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum). The screening conditions were adjusted to the TryS´ kinetic parameters and intracellular concentration of substrates corresponding to each trypanosomatid species, and/or to avoid assay interference. The screening assay yielded suitable Z' and signal to noise values (?0.85 and ~3.5, respectively), and high intra-assay reproducibility. Several novel chemical scaffolds were identified as low ?M and selective tri-tryp TryS inhibitors. Compounds displaying multi-TryS inhibition (N,N'-bis(3,4-substituted-benzyl) diamine derivatives) and an N5-substituted paullone (MOL2008) halted the proliferation of infective Trypanosoma brucei (EC50 in the nM range) and Leishmania infantum promastigotes (EC50 = 12 ?M), respectively. A bis-benzyl diamine derivative and MOL2008 depleted intracellular trypanothione in treated parasites, which confirmed the on-target activity of these compounds. CONCLUSIONS/SIGNIFICANCE:Novel molecular scaffolds with on-target mode of action were identified as hit candidates for TryS inhibition. Due to the remarkable species-specificity exhibited by tri-tryp TryS towards the compounds, future optimization and screening campaigns should aim at designing and detecting, respectively, more potent and broad-range TryS inhibitors.

Dataset Information

Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods.

Publications

Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods.

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