Transport of methionine in Trypanosoma brucei brucei

Hasne, Marie Pierre; Barrett, Michael P.

doi:10.1016/s0166-6851(00)00321-2

Cited by 18 publications

(17 citation statements)

References 24 publications

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“…This suggests a crucial role for hydrophobic amino acids in the provision of amino groups, possibly for methionine recycling (45). However, methionine depletion from the medium suggests that methionine acquisition occurs by uptake from the medium to a significant degree (46), while accumulation of the corresponding keto acid in spent medium suggests that methionine itself is an amino donor for as-yet-uncharacterized substrates. The significant accumulation of alanine in the spent medium suggests that transamination of pyruvate to alanine may be a primary role of amino acid-derived nitrogen.…”

Section: Discussionmentioning

confidence: 99%

Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

Creek

Nijagal

Kim

et al. 2013

Antimicrob Agents Chemother

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134

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dIn vitro culture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra-and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream form Trypanosoma brucei. The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supports in vitro growth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (<3-fold change; ␣ ‫؍‬ 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole-cell phenotypic screenings and in the metabolomic mode of action assays. Four-hundred-fold 50% inhibitory concentration decreases were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomic studies and drug activity screening.T rypanosoma brucei is the kinetoplastid parasite responsible for human African trypanosomiasis (HAT), a potentially fatal infection of sub-Saharan Africa. Current treatments for HAT are inadequate because of high toxicity and complex administration regimens, and new drugs are urgently required to ensure ongoing control of the disease in the face of emerging reports of drug resistance (1). Newly invigorated efforts to bring novel compounds forward as drugs have led to several promising candidates emerging through screening against parasites in culture (2-5). In vitro continuous culture methods enable the routine screening of compounds for trypanocidal activity against bloodstream form trypanosomes, for example, with resazurin fluorescence-based assays (5-7).Culture methods for bloodstream form T. brucei were first developed in the 1970s and required feeder layers to support continuous growth (8). This requirement could be overcome by regular supplementation with cysteine (9) and supplementation with mammalian serum (10). Continuous culture was successfully maintained by the addition of reducing and chelating agents (2-mercaptoethanol and bathocuproine sulfonate) to modified Iscove medium with 10% fetal bovine serum (HMI11) (11). HMI11 is now widely used for routine cell culture of laboratory strains of T. brucei and underpins many of the approaches employed in the search for new trypanocidal compounds. Nevertheless, HMI11 is a very rich medium compared to the natural in vivo environment of bloodstream form T. brucei, which may give...

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Section: Discussionmentioning

confidence: 99%

Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

Creek

Nijagal

Kim

et al. 2013

Antimicrob Agents Chemother

Self Cite

134

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“…Uptake involved the oil stop transport assay as described (37,38) with 100 l of the test solution cushioned above 90 l of oil mixture (di-n-butylphthalate and mineral oil at a ratio of 7:1). 100 l of cells (at 10 8 cells⅐ml Ϫ1 ) were added to each tube and incubated for times ranging between 3 s and 3 h depending on the experiment.…”

Section: Methodsmentioning

confidence: 99%

Proline Metabolism in Procyclic Trypanosoma brucei Is Down-regulated in the Presence of Glucose

Lamour

Rivière

Coustou

et al. 2005

Journal of Biological Chemistry

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202

265

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Proline metabolism has been studied in procyclic form Trypanosoma brucei. These parasites consume six times more proline from the medium when glucose is in limiting supply than when this carbohydrate is present as an abundant energy source. The sensitivity of procyclic T. brucei to oligomycin increases by three orders of magnitude when the parasites are obliged to catabolize proline in medium depleted in glucose. This indicates that oxidative phosphorylation is far more important to energy metabolism in this latter case than when glucose is available and the energy needs of the parasite can be fulfilled by substrate level phosphorylation alone. A gene encoding proline dehydrogenase, the first enzyme of the proline catabolic pathway, was cloned. RNA interference studies revealed the loss of this activity to be conditionally lethal. Proline dehydrogenase defective parasites grew as wild-type when glucose was available, but, unlike wild-type cells, they failed to proliferate using proline. In parasites grown in the presence of glucose, proline dehydrogenase activity was markedly lower than when glucose was absent from the medium. Proline uptake too was shown to be diminished when glucose was abundant in the growth medium. Wild-type cells were sensitive to 2-deoxy-D-glucose if grown using proline as the principal carbon source, but not in glucose-rich medium, indicating that this non-catabolizable glucose analogue might also stimulate repression of proline utilization. These results indicate that the ability of trypanosomes to use proline as an energy source can be regulated depending upon the availability of glucose.

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“…In this regard, biochemical pathways general to parasites but absent from mammalian hosts have long been an attractive source of molecular targets for anti-infective drug development [23,27]. Additionally, biochemical peculiarities of the parasites such as (i) turnover rates [42,43]; (ii) structure of biological membranes [44][45][46][47]; (iii) cell signaling [48] and (iv) protein expression and regulation [49] have also been investigated as potential targets for drug intervention [50].…”

Section: Molecular Targets For Drug Designmentioning

confidence: 99%

Structure-Based Drug Discovery for Tropical Diseases

Güido

Oliva

2009

CTMC

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Parasitic diseases are amongst the foremost threats to human health and welfare around the world. In tropical and subtropical regions of the world, the consequences of parasitic infections are devastating both in terms of human morbidity and mortality. The current available drugs are limited, ineffective, and require long treatment regimens. To overcome these limitations, the identification of new macromolecular targets and small-molecule modulators is of utmost importance. The advances in genomics and proteomics have prompted drug discovery to move toward more rational strategies. The increasing understanding of the fundamental principles of protein-ligand interactions combined with the availability of compound libraries has facilitated the identification of promising hits and the generation of high quality lead compounds for tropical diseases. This review presents the current progresses and applications of structure-based drug design (SBDD) for the discovery of innovative chemotherapy agents for a variety of parasitic diseases, highlighting the challenges, limitations, and future perspectives in medicinal chemistry.

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Transport of methionine in Trypanosoma brucei brucei

Cited by 18 publications

References 24 publications

Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

Proline Metabolism in Procyclic Trypanosoma brucei Is Down-regulated in the Presence of Glucose

Structure-Based Drug Discovery for Tropical Diseases

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