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 (kcat) and Km 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.
Highlights
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
We show that T. brucei methylthioadenosine phosphorylase (TbMTAP) normally protects T. brucei cells against deoxyadenosine, but product inhibition by adenine sets an upper level on the capacity of the enzyme
Results dATP Accumulates in Deoxyadenosine-treated T. brucei bloodstream forms (BSFs) and Mammalian Cells—Generally, the levels of dNTPs are much lower than NTPs in T. brucei cells [12]
Summary
One of the reaction products, adenine, inhibited the enzyme, which might explain why TbMTAP-mediated protection is less efficient at higher deoxyadenosine concentrations. 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. Knowledge of the TbMTAP-mediated protection system is relevant for drug discovery because adenosine kinase substrate analogues need to be resistant to cleavage by TbMTAP (and TbIAG-NH) or be combined with cleavage enzyme inhibitors to be active against the parasite. We found that cordycepin, tubercidin, and Ara-A were all resistant to TbMTAP activity and that the presence of the TbMTAP inhibitor adenine in the culture medium could increase deoxyadenosine-induced growth inhibition of T. brucei bloodstream forms (BSFs)
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