Differential Kinetic Activities of Glycerol Kinase among African Trypanosome Species: Phylogenetic and Therapeutic Implications

Ohashi-Suzuki, Mitsuko; Yabu, Yoshisada; Ohshima, Shigeru; Nakamura, Kôsuke; Kido, Yasutoshi; Sakamoto, Kimitoshi; Kita, Kiyoshi; Ohta, Nobuhiro; Suzuki, Takashi

doi:10.1292/jvms.10-0481

Cited by 14 publications

(11 citation statements)

References 17 publications

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“…In anaerobiosis, BSF produce equivalent amounts of pyruvate and glycerol from glucose breakdown and consequently need the GK activity to maintain the glycosomal redox balance required to sustain the glycolytic flow [ 21 ]. Interestingly, in vitro and in vivo studies have shown that the glycerol kinase activity considerably reduced the lethal effect of TAO inhibitors, which mimics anaerobiosis [ 46 , 47 ]. This highlights the essential role of the GK for BSF during anaerobiosis.…”

Section: Discussionmentioning

confidence: 99%

Glycerol supports growth of the Trypanosoma brucei bloodstream forms in the absence of glucose: Analysis of metabolic adaptations on glycerol-rich conditions

et al. 2018

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The bloodstream forms of Trypanosoma brucei (BSF), the parasite protist causing sleeping sickness, primarily proliferate in the blood of their mammalian hosts. The skin and adipose tissues were recently identified as additional major sites for parasite development. Glucose was the only carbon source known to be used by bloodstream trypanosomes to feed their central carbon metabolism, however, the metabolic behaviour of extravascular tissue-adapted parasites has not been addressed yet. Since the production of glycerol is an important primary function of adipocytes, we have adapted BSF trypanosomes to a glucose-depleted but glycerol-rich culture medium (CMM_Glyc/GlcNAc) and compared their metabolism and proteome to those of parasites grown in standard glucose-rich conditions (CMM_Glc). BSF were shown to consume 2-folds more oxygen per consumed carbon unit in CMM_Glyc/GlcNAc and were 11.5-times more sensitive to SHAM, a specific inhibitor of the plant-like alternative oxidase (TAO), which is the only mitochondrial terminal oxidase expressed in BSF. This is consistent with (i) the absolute requirement of the mitochondrial respiratory activity to convert glycerol into dihydroxyacetone phosphate, as deduced from the updated metabolic scheme and (ii) with the 1.8-fold increase of the TAO expression level compared to the presence of glucose. Proton NMR analysis of excreted end products from glycerol and glucose metabolism showed that these two carbon sources are metabolised through the same pathways, although the contributions of the acetate and succinate branches are more important in the presence of glycerol than glucose (10.2% versus 3.4% of the excreted end products, respectively). In addition, metabolomic analyses by mass spectrometry showed that, in the absence of glucose, 13C-labelled glycerol was incorporated into hexose phosphates through gluconeogenesis. As expected, RNAi-mediated down-regulation of glycerol kinase expression abolished glycerol metabolism and was lethal for BSF grown in CMM_Glyc/GlcNAc. Interestingly, BSF have adapted their metabolism to grow in CMM_Glyc/GlcNAc by concomitantly increasing their rate of glycerol consumption and decreasing that of glucose. However, the glycerol kinase activity was 7.8-fold lower in CMM_Glyc/GlcNAc, as confirmed by both western blotting and proteomic analyses. This suggests that the huge excess in glycerol kinase that is not absolutely required for glycerol metabolism, might be used for another yet undetermined non-essential function in glucose rich-conditions. Altogether, these data demonstrate that BSF trypanosomes are well-adapted to glycerol-rich conditions that could be encountered by the parasite in extravascular niches, such as the skin and adipose tissues.

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Section: Discussionmentioning

confidence: 99%

Glycerol supports growth of the Trypanosoma brucei bloodstream forms in the absence of glucose: Analysis of metabolic adaptations on glycerol-rich conditions

et al. 2018

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“…net production of 1 mole of ATP per glucose molecule consumed) and glycerol. [40][41][42][43] Hence, the co-administration of TAO inhibitors and glycerol is known to effectively kill the parasites 44 because the added glycerol competes with G3P as GK substrate, and thus inhibits anaerobic ATP production by mass action. 40 Co-incubation with glycerol significantly (P<0.05) increased the trypanocidal activities of 6e, 6f, 10d, 10f, and SHAM, whereas it had no effect on the efficacy of control drugs pentamidine and diminazene ( Table 2).…”

Section: Biologymentioning

confidence: 99%

Conjugates of 2,4-Dihydroxybenzoate and Salicylhydroxamate and Lipocations Display Potent Antiparasite Effects by Efficiently Targeting the Trypanosoma brucei and Trypanosoma congolense Mitochondrion

et al. 2017

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We investigated a chemical strategy to boost the trypanocidal activity of 2,4dihydroxybenzoic acid (2,4-DHBA)-and salicylhydroxamic acid (SHAM)-based trypanocides with triphenylphosphonium and quinolinium lipophilic cations (LC). Three series of LC conjugates were synthesized that were active in the submicromolar (5a-d and 10d-f) to low nanomolar (6a-f) range against wild-type and multi-drug resistant strains of African trypanosomes (Trypanosoma brucei brucei and T. congolense). This represented an improvement in trypanocidal potency of at least 200fold, and up to >10,000-fold, compared with the non-LC coupled parent compounds 2,4-DHBA and SHAM. Selectivity over human cells was >500 and reached >23,000 for 6e. Mechanistic studies showed that 6e did not inhibit the cell cycle but affected parasite respiration in a dose-dependent manner. Inhibition of the trypanosome alternative oxidase (TAO) and the mitochondrial membrane potential was also studied for selected compounds. We conclude that effective mitochondrial targeting greatly potentiated the activity of these compound series.

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“…In the context of AT, as is the case for most infectious diseases, a good drug target should be essential for the parasite survival (or proliferation) in the host and, preferably, absent or not essential to the host (although the latter obstacle can be overcome with agents of sufficient specificity for the pathogen target). TAO fulfils these criteria, as it is central to ATP synthesis in the BSFs of African trypanosomes and no homolog of the gene is present in mammals …”

Section: Validation Of Tao As Drug Target In African Trypanosomesmentioning

confidence: 99%

Alternative oxidase inhibitors: Mitochondrion‐targeting as a strategy for new drugs against pathogenic parasites and fungi

Ebiloma

Balogun

Cueto-Díaz

et al. 2019

Medicinal Research Reviews

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The alternative oxidase (AOX) is a ubiquitous terminal oxidase of plants and many fungi, catalyzing the four‐electron reduction of oxygen to water alongside the cytochrome‐based electron transfer chain. Unlike the classical electron transfer chain, however, the activity of AOX does not generate adenosine triphosphate but has functions such as thermogenesis and stress response. As it lacks a mammalian counterpart, it has been investigated intensely in pathogenic fungi. However, it is in African trypanosomes, which lack cytochrome‐based respiration in their infective stages, that trypanosome alternative oxidase (TAO) plays the central and essential role in their energy metabolism. TAO was validated as a drug target decades ago and among the first inhibitors to be identified was salicylhydroxamic acid (SHAM), which produced the expected trypanocidal effects, especially when potentiated by coadministration with glycerol to inhibit anaerobic energy metabolism as well. However, the efficacy of this combination was too low to be of practical clinical use. The antibiotic ascofuranone (AF) proved a much stronger TAO inhibitor and was able to cure Trypanosoma vivax infections in mice without glycerol and at much lower doses, providing an important proof of concept milestone. Systematic efforts to improve the SHAM and AF scaffolds, aided with the elucidation of the TAO crystal structure, provided detailed structure‐activity relationship information and reinvigorated the drug discovery effort. Recently, the coupling of mitochondrion‐targeting lipophilic cations to TAO inhibitors has dramatically improved drug targeting and trypanocidal activity while retaining target protein potency. These developments appear to have finally signposted the way to preclinical development of TAO inhibitors.

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Differential Kinetic Activities of Glycerol Kinase among African Trypanosome Species: Phylogenetic and Therapeutic Implications

Cited by 14 publications

References 17 publications

Glycerol supports growth of the Trypanosoma brucei bloodstream forms in the absence of glucose: Analysis of metabolic adaptations on glycerol-rich conditions

Glycerol supports growth of the Trypanosoma brucei bloodstream forms in the absence of glucose: Analysis of metabolic adaptations on glycerol-rich conditions

Conjugates of 2,4-Dihydroxybenzoate and Salicylhydroxamate and Lipocations Display Potent Antiparasite Effects by Efficiently Targeting the Trypanosoma brucei and Trypanosoma congolense Mitochondrion

Alternative oxidase inhibitors: Mitochondrion‐targeting as a strategy for new drugs against pathogenic parasites and fungi

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