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

Abstract

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.

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

“…2B). The concomitant reduction in the ATP pool, which was previously observed with deoxyadenosine-treated and Ara-A-treated trypanosomes (11,12), was only modest in the case of FANA-A, indicating that this is probably not a significant factor contributing to the growth inhibition, which occurs at FANA-A concentrations much lower than those needed to reduce ATP levels. A radioactive tracer experiment showed that FANA-A blocked nucleic acid biosynthesis in the parasites (Fig.…”

“…TbMTAP assays were performed as described previously with the recombinantly expressed and purified enzyme (11). Briefly, the enzyme was diluted in a buffer containing 50 mM potassium acetate, 50 mM Tris-HCl (pH 7.4), and 0.05% Tween 20 prior to use in the enzyme assay.…”

“…Thymidine kinase phosphorylates thymidine and deoxyuridine (7), whereas adenosine kinase is able to salvage adenosine and deoxyadenosine (9). It has been shown that higher levels of dATP accumulate in T. brucei than in mammalian cells after treatment with high concentrations of deoxyadenosine in the growth medium and that this accumulation is followed by T. brucei growth inhibition and lysis (11,12). The dATP increase is less prominent at lower concentrations of deoxyadenosine, because T. brucei methylthioadenine phosphorylase (TbMTAP) cleaves this substrate into adenine and deoxyribose-1-phosphate, thereby rescuing the parasite from the cytotoxicity of deoxyadenosine (11).…”

“…It has been shown that higher levels of dATP accumulate in T. brucei than in mammalian cells after treatment with high concentrations of deoxyadenosine in the growth medium and that this accumulation is followed by T. brucei growth inhibition and lysis (11,12). The dATP increase is less prominent at lower concentrations of deoxyadenosine, because T. brucei methylthioadenine phosphorylase (TbMTAP) cleaves this substrate into adenine and deoxyribose-1-phosphate, thereby rescuing the parasite from the cytotoxicity of deoxyadenosine (11). At higher concentrations of deoxyadenosine, this protective system is inhibited by its reaction product, adenine, which allows the deoxynucleoside to be phosphorylated by adenosine kinase instead, leading to the subsequent buildup of toxic levels of dATP in the cell (11).…”

“…The dATP increase is less prominent at lower concentrations of deoxyadenosine, because T. brucei methylthioadenine phosphorylase (TbMTAP) cleaves this substrate into adenine and deoxyribose-1-phosphate, thereby rescuing the parasite from the cytotoxicity of deoxyadenosine (11). At higher concentrations of deoxyadenosine, this protective system is inhibited by its reaction product, adenine, which allows the deoxynucleoside to be phosphorylated by adenosine kinase instead, leading to the subsequent buildup of toxic levels of dATP in the cell (11). TbMTAP is relevant for drug development, because adenosine kinase substrate analogues need to be resistant to TbMTAP cleavage in order to be effective against the parasites.…”