African sleeping sickness is caused by Trypanosoma brucei. This extracellular parasite lacks de novo purine biosynthesis, and it is therefore dependent on exogenous purines such as adenosine that is taken up from the blood and other body fluids by high affinity transporters. The general belief is that adenosine needs to be cleaved to adenine inside the parasites in order to be used for purine nucleotide synthesis. We have found that T. brucei also can salvage this nucleoside by adenosine kinase (AK), which has a higher affinity to adenosine than the cleavagedependent pathway. The recombinant T. brucei AK (TbAK) preferably used ATP or GTP to phosphorylate both natural and synthetic nucleosides in the following order of catalytic efficiencies: adenosine > cordycepin > deoxyadenosine > adenine arabinoside (Ara-A) > inosine > fludarabine (F-Ara-A). TbAK differed from the AK of the related intracellular parasite Leishmania donovani by having a high affinity to adenosine (K m ‫؍‬ 0.04 -0.08 M depending on [phosphate]) and by being negatively regulated by adenosine (K i ‫؍‬ 8 -14 M). These properties make the enzyme functionally related to the mammalian AKs, although a phylogenetic analysis grouped it together with the L. donovani enzyme. The combination of a high affinity AK and efficient adenosine transporters yields a strong salvage system in T. brucei, a potential Achilles' heel making the parasites more sensitive than mammalian cells to adenosine analogs such as Ara-A. Studies of wild-type and AK knockdown trypanosomes showed that Ara-A inhibited parasite proliferation and survival in an AK-dependent manner by affecting nucleotide levels and by inhibiting nucleic acid biosynthesis.Trypanosoma brucei is an extracellular parasite that is transmitted by tsetse flies and lives in the blood, lymph, and central nervous system of its mammalian hosts (1, 2). The parasite causes African sleeping sickness in humans and nagana in cattle. There are two variants of African sleeping sickness, a chronic form caused by the subspecies Trypanosoma brucei gambiense and an acute form caused by Trypanosoma brucei rhodesiense. Both variants are fatal, but the chronic form has a slower progress. Current treatment is unsatisfactory because of low efficacy and high toxicity. Therefore, there is a great need of new drugs to treat the disease, especially at later stages when the parasites infect the brain. Promising results with the adenosine analog cordycepin (3Ј-deoxyadenosine) on T. brucei-infected mice with brain infection suggest that adenosine analogs can be developed into new antitrypanosomal agents (3).Unlike mammalian cells, trypanosomes lack de novo purine biosynthesis, and they are therefore totally dependent on purine salvage (4). The major purine source in human blood is a matter of controversy; when the blood was directly mixed with an adenosine deaminase inhibitor to prevent purine degradation, adenosine was present at 2 M concentration, whereas hypoxanthine (0.7 M) and inosine (0.2 M) were minor sources (5). However, other r...

BackgroundHuman African trypanosomiasis (HAT), a major parasitic disease spread in Africa, urgently needs novel targets and new efficacious chemotherapeutic agents. Recently, we discovered that 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (compound 1) exhibits specific antitrypanosomal activity with an IC50 of 1.0 µM on Trypanosoma brucei rhodesiense (T. b. rhodesiense), the causative agent of the acute form of HAT.Methodology/Principal FindingsIn this work we show adenosine kinase of T. b. rhodesiense (TbrAK), a key enzyme of the parasite purine salvage pathway which is vital for parasite survival, to be the putative intracellular target of compound 1 using a chemical proteomics approach. This finding was confirmed by RNA interference experiments showing that down-regulation of adenosine kinase counteracts compound 1 activity. Further chemical validation demonstrated that compound 1 interacts specifically and tightly with TbrAK with nanomolar affinity, and in vitro activity measurements showed that compound 1 is an enhancer of TbrAK activity. The subsequent kinetic analysis provided strong evidence that the observed hyperactivation of TbrAK is due to the abolishment of the intrinsic substrate-inhibition.Conclusions/SignificanceThe results suggest that TbrAK is the putative target of this compound, and that hyperactivation of TbrAK may represent a novel therapeutic strategy for the development of trypanocides.

The molecular mechanisms of K ؉ homeostasis are only poorly understood for protozoan parasites. Trypanosoma brucei subsp. parasites, the causative agents of human sleeping sickness and nagana, are strictly extracellular and need to actively concentrate K ؉ from their hosts' body fluids. The T. brucei genome contains two putative K ؉ channel genes, yet the trypanosomes are insensitive to K ؉ antagonists and K ؉ channelblocking agents, and they do not spontaneously depolarize in response to high extracellular K ؉ concentrations. However, the trypanosomes are extremely sensitive to K ؉ ionophores such as valinomycin. Surprisingly, T. brucei possesses a member of the Trk/HKT superfamily of monovalent cation permeases which so far had only been known from bacteria, archaea, fungi, and plants. The protein was named TbHKT1 and functions as a Na ؉ -independent K ؉ transporter when expressed in Escherichia coli, Saccharomyces cerevisiae, or Xenopus laevis oocytes. In trypanosomes, TbHKT1 is expressed in both the mammalian bloodstream stage and the Tsetse fly midgut stage; however, RNA interference (RNAi)-mediated silencing of TbHKT1 expression did not produce a growth phenotype in either stage. The presence of HKT genes in trypanosomatids adds a further piece to the enigmatic phylogeny of the Trk/HKT superfamily of K ؉ transporters. Parsimonial analysis suggests that the transporters were present in the first eukaryotes but subsequently lost in several of the major eukaryotic lineages, in at least four independent events.

Summary