Molecular Pharmacology of Adenosine Transport in <i>Trypanosoma brucei</i>: P1/P2 Revisited

Geiser, Federico; Lüscher, Alexandra; Koning, Harry P. de; Seebeck, Thomas; Mäser, Pascal

doi:10.1124/mol.104.010298

Cited by 52 publications

(61 citation statements)

References 32 publications

(40 reference statements)

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“…While the P1 transporters are redundant (30,46), P2 is encoded by a single gene, T. brucei AT1 (TbAT1) (33,35). Homozygous disruption of TbAT1 in T. brucei bloodstream forms caused resistance to melarsoprol, diamidines, cordyce-pin, and tubercidin (21,35). Cordycepin was found to be a potent and selective trypanocide in vitro (34,53) but not in vivo (40).…”

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confidence: 99%

Adenosine Kinase ofTrypanosoma bruceiand Its Role in Susceptibility to Adenosine Antimetabolites

Lüscher

Önal

Schweingruber

et al. 2007

Antimicrob Agents Chemother

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Trypanosoma brucei cannot synthesize purines de novo and relies on purine salvage from its hosts to build nucleic acids. With adenosine being a preferred purine source of bloodstream-form trypanosomes, adenosine kinase (AK; EC 2.7.1.20) is likely to be a key player in purine salvage. Adenosine kinase is also of high pharmacological interest, since for many adenosine antimetabolites, phosphorylation is a prerequisite for activity. Here, we cloned and functionally characterized adenosine kinase from T. brucei (TbAK). TbAK is a tandem gene, expressed in both procyclic-and bloodstream-form trypanosomes, whose product localized to the cytosol of the parasites. The RNA interference-mediated silencing of TbAK suggested that the gene is nonessential under standard growth conditions. Inhibition or downregulation of TbAK rendered the trypanosomes resistant to cordycepin (3-deoxyadenosine), demonstrating a role for TbAK in the activation of adenosine antimetabolites. The expression of TbAK in Saccharomyces cerevisiae complemented a null mutation in the adenosine kinase gene ado1. The concomitant expression of TbAK with the T. brucei adenosine transporter gene TbAT1 allowed S. cerevisiae ado1 ade2 double mutants to grow on adenosine as the sole purine source and, at the same time, sensitized them to adenosine antimetabolites. The coexpression of TbAK and TbAT1 in S. cerevisiae ado1 ade2 double mutants proved to be a convenient tool for testing nucleoside analogues for uptake and activation by T. brucei adenosine salvage enzymes.

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Adenosine Kinase ofTrypanosoma bruceiand Its Role in Susceptibility to Adenosine Antimetabolites

Lüscher

Önal

Schweingruber

et al. 2007

Antimicrob Agents Chemother

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“…To date, the antitrypanosomal adenosine analogues developed against T. brucei are taken up primarily by the P2 transporter (25,26,36). However, because mutations in this transporter are strongly associated with resistance to current treatments (37-40), we propose that adenosine analogues that are instead taken up by the P1 transporter, or by both the P1 and P2 transporters, should be prioritized in the search for new agents against T. brucei.…”

Section: Discussionmentioning

confidence: 99%

“…Most of the antitrypanosomal adenosine/deoxyadenosine analogues developed so far, including cordycepin, 2-fluorocordycepin, and tubercidin, are taken up mainly by the P2 nucleoside transporter (25,26), which poses a problem with respect to crossresistance to existing therapies. Our aim in this study was to identify novel antitrypanosomal adenosine analogues that are taken up by the P1 transporter and thus, due to the many genetic variants, are less likely to become ineffective as a result of gene mutation.…”

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“…FANA-A is taken up via the P1 nucleoside transporter family in T. brucei. Exhaustive characterization of purine transport activities in T. brucei has shown that the P1 and P2 transporters are the only transporters that facilitate the uptake of adenosine or its analogues in bloodstream forms of the parasite (4,5,25,32). By comparing the sensitivity of T. brucei cells (Lister 427, the parent strain) to FANA-A with those of multidrug-resistant cells (strain B48) that lack both the P2 transporter and the highaffinity pentamidine transporter (HAPT) (33) and the same cells carrying a plasmid for stable expression of the P2 gene (strain B48 ϩ P2) (17), it was possible to determine if the P2 nucleoside transporter plays a significant role in FANA-A uptake.…”

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confidence: 99%

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9-(2′-Deoxy-2′-Fluoro-β- d -Arabinofuranosyl) Adenine Is a Potent Antitrypanosomal Adenosine Analogue That Circumvents Transport-Related Drug Resistance

Ranjbarian

Vodnala

Alzahrani

et al. 2017

Antimicrob Agents Chemother

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Current chemotherapy against African sleeping sickness, a disease caused by the protozoan parasite Trypanosoma brucei, is limited by toxicity, inefficacy, and drug resistance. Nucleoside analogues have been successfully used to cure T. brucei-infected mice, but they have the limitation of mainly being taken up by the P2 nucleoside transporter, which, when mutated, is a common cause of multidrug resistance in T. brucei. We report here that adenine arabinoside (Ara-A) and the newly tested drug 9-(2=-deoxy-2=-fluoro-␤-D-arabinofuranosyl) adenine (FANA-A) are instead taken up by the P1 nucleoside transporter, which is not associated with drug resistance. Like Ara-A, FANA-A was found to be resistant to cleavage by methylthioadenosine phosphorylase, an enzyme that protects T. brucei against the antitrypanosomal effects of deoxyadenosine. Another important factor behind the selectivity of nucleoside analogues is how well they are phosphorylated within the cell. We found that the T. brucei adenosine kinase had a higher catalytic efficiency with FANA-A than the mammalian enzyme, and T. brucei cells treated with FANA-A accumulated high levels of FANA-A triphosphate, which even surpassed the level of ATP and led to cell cycle arrest, inhibition of DNA synthesis, and the accumulation of DNA breaks. FANA-A inhibited nucleic acid biosynthesis and parasite proliferation with 50% effective concentrations (EC 50 s) in the low nanomolar range, whereas mammalian cell proliferation was inhibited in the micromolar range. Both Ara-A and FANA-A, in combination with deoxycoformycin, cured T. brucei-infected mice, but FANA-A did so at a dose 100 times lower than that of Ara-A.KEYWORDS 9-(2=-deoxy-2=-fluoro-␤-D-arabinofuranosyl) adenine, FANA-A, Trypanosoma brucei, adenosine kinase, drug resistance, methylthioadenosine phosphorylase, nucleoside transporters, trypanosome T rypanosoma brucei is transmitted by tsetse flies and causes African sleeping sickness in humans and nagana in cattle (1, 2). The disease is characterized by two stages. Initially, the parasites are restricted to circulating in the blood and lymph systems, but in the advanced stages of the disease, they are also found in the cerebrospinal fluid, which leads to coma and death if the patient is not treated. There is no existing vaccine, and available treatments are specific to the disease stage and the parasite subspecies. High toxicity, complex administration protocols, and drug resistance all contribute to the urgent need for new therapies.

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“…Cordycepin (3Ј-deoxyadenosine) and tubercidin (7-deazaadenosine) are two such analogues that inhibit T. brucei growth with IC 50 values in the nanomolar range (13). Cordycepin has been shown to cure both stages of the disease in T. brucei-infected mice when given together with deoxycoformycin, which protects the drug from deamination by serum adenosine deaminase (14).…”

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Trypanosoma brucei Methylthioadenosine Phosphorylase Protects the Parasite from the Antitrypanosomal Effect of Deoxyadenosine

Vodnala

Ranjbarian

Павлова

et al. 2016

Journal of Biological Chemistry

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Trypanosoma brucei causes African sleeping sickness for which no vaccine exists and available treatments are of limited use due to their high toxicity or lack of efficacy. T. brucei cultivated in the presence of deoxyadenosine accumulates high levels of dATP in an adenosine kinase-dependent process and dies within a few hours. Here we show that T. brucei treated with 1 mM deoxyadenosine accumulates higher dATP levels than mammalian cells but that this effect diminishes quickly as the concentration of the deoxynucleoside decreases. Radioactive tracer studies showed that the parasites are partially protected against lower concentrations of deoxyadenosine by the ability to cleave it and use the adenine for ATP synthesis. T. brucei methylthioadenosine phosphorylase (TbMTAP) was found to be responsible for the cleavage as indicated by the phosphate dependence of deoxyadenosine cleavage in T. brucei cell extracts and increased deoxyadenosine sensitivity in TbMTAP knockdown cells. Recombinant TbMTAP exhibited higher turnover number (k cat ) and K m values for deoxyadenosine than for the regular substrate, methylthioadenosine. One of the reaction products, adenine, inhibited the enzyme, which might explain why TbMTAP-mediated protection is less efficient at higher deoxyadenosine concentrations. Consequently, T. brucei grown in the presence of adenine demonstrated increased sensitivity to deoxyadenosine. For deoxyadenosine/adenosine analogues to remain intact and be active against the parasite, they need to either be resistant to TbMTAP-mediated cleavage, which is the case with the three known antitrypanosomal agents adenine arabinoside, tubercidin, and cordycepin, or they need to be combined with TbMTAP inhibitors.

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Molecular Pharmacology of Adenosine Transport in Trypanosoma brucei: P1/P2 Revisited

Cited by 52 publications

References 32 publications

Adenosine Kinase ofTrypanosoma bruceiand Its Role in Susceptibility to Adenosine Antimetabolites

Adenosine Kinase ofTrypanosoma bruceiand Its Role in Susceptibility to Adenosine Antimetabolites

9-(2′-Deoxy-2′-Fluoro-β- d -Arabinofuranosyl) Adenine Is a Potent Antitrypanosomal Adenosine Analogue That Circumvents Transport-Related Drug Resistance

Trypanosoma brucei Methylthioadenosine Phosphorylase Protects the Parasite from the Antitrypanosomal Effect of Deoxyadenosine

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