Adenosine Kinase Mediates High Affinity Adenosine Salvage in Trypanosoma brucei

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 cleavage-dependent 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 (Km = 0.04–0.08 μm depending on [phosphate]) and by being negatively regulated by adenosine (Ki = 8–14 μm). 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. 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 cleavage-dependent 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 (Km = 0.04–0.08 μm depending on [phosphate]) and by being negatively regulated by adenosine (Ki = 8–14 μm). 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 (1Barrett M.P. Lancet. 2006; 367: 1377-1378Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 2Brun R. Karger-Gazette. 1999; 63: 5-7Google Scholar). 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 (3′-deoxyadenosine) on T. brucei-infected mice with brain infection suggest that adenosine analogs can be developed into new anti-trypanosomal agents (3Rottenberg M.E. Masocha W. Ferella M. Petitto-Assis F. Goto H. Kristensson K. McCaffrey R. Wigzell H. J. Infect. Dis. 2005; 192: 1658-1665Crossref PubMed Scopus (55) Google Scholar). Unlike mammalian cells, trypanosomes lack de novo purine biosynthesis, and they are therefore totally dependent on purine salvage (4El Kouni M.H. Pharmacol. Ther. 2003; 99: 283-309Crossref PubMed Scopus (140) Google Scholar). 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 μm concentration, whereas hypoxanthine (0.7 μm) and inosine (0.2 μm) were minor sources (5Slowiaczek P. Tattersall M.H.N. Anal. Biochem. 1982; 125: 6-12Crossref PubMed Scopus (22) Google Scholar). However, other reports claim that the adenosine concentration is much lower and that hypoxanthine is the major source (6Ontyd J. Schrader J. J. Chromatogr. 1984; 307: 404-409Crossref PubMed Scopus (128) Google Scholar, 7Jabs C.M. Neglen P. Eklöf B. Thomas E.J. Clin. Chem. 1990; 36 (81): 81Crossref PubMed Scopus (21) Google Scholar). T. brucei has high affinity adenosine transporters driven by the protonmotive force that are able to concentrate adenosine inside the cells (8Carter N.S. Fairlamb A.H. Nature. 1993; 361: 173-176Crossref PubMed Scopus (311) Google Scholar, 9De Koning H.P. Watson C.J. Jarvis S.M. J. Biol. Chem. 1998; 273: 9486-9494Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Mammalian cells have both facilitated diffusion carriers and Na+/adenosine cotransporters (10Thorn J.A. Jarvis S.M. Gen. Pharmacol. 1996; 27: 613-620Crossref PubMed Scopus (204) Google Scholar), but the affinities for adenosine are 1–2 orders of magnitude lower than the adenosine affinities of the trypanosome transporters. The current belief is that adenosine is salvaged by a two-step process in T. brucei where intracellular cleavage to adenine is followed by phosphoribosylation to AMP (4El Kouni M.H. Pharmacol. Ther. 2003; 99: 283-309Crossref PubMed Scopus (140) Google Scholar). The adenosine cleavage is performed by inosine-adenosine-guanosine-nucleoside hydrolase (IAG-NH), 2The abbreviations used are:IAG-NHinosine-adenosine-guanosine-nucleoside hydrolaseAKadenosine kinaseTbAKT. brucei adenosine kinaseAra-Aadenine arabinoside (vidarabine)TrxAthioredoxin ATEVtobacco etch virusDTTdithiothreitolTBSTTris-buffered saline Tween 20BiPhomolog of immunoglobulin heavy-chain binding proteinEHNA9-erythro-(2-hydroxy-3-nonyl) adenineF-Ara-AfludarabineDFMOdifluoromethyl ornithine (eflornithine)ADAadenosine deaminaseBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolHPLChigh pressure liquid chromatographyMICminimal inhibitory concentration. which has a low adenosine affinity (Km ∼ 15 μm (11Parkin D.W. J. Biol. Chem. 1996; 271: 21713-21719Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 12Pellé R. Schramm V.L. Parkin D.W. J. Biol. Chem. 1998; 273: 2118-2126Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar)) as compared with the blood level. It is therefore important for the parasites to have efficient transporters that concentrate adenosine inside the cells. However, because of the great uncertainty about the blood adenosine concentration, it is not known whether this transport is efficient enough to saturate the enzyme activity. The AMP formed by the cleavage-dependent pathway can be used for the synthesis of all purines needed (GMP is made from AMP via IMP and XMP (4El Kouni M.H. Pharmacol. Ther. 2003; 99: 283-309Crossref PubMed Scopus (140) Google Scholar)). inosine-adenosine-guanosine-nucleoside hydrolase adenosine kinase T. brucei adenosine kinase adenine arabinoside (vidarabine) thioredoxin A tobacco etch virus dithiothreitol Tris-buffered saline Tween 20 homolog of immunoglobulin heavy-chain binding protein 9-erythro-(2-hydroxy-3-nonyl) adenine fludarabine difluoromethyl ornithine (eflornithine) adenosine deaminase 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol high pressure liquid chromatography minimal inhibitory concentration. The cleavage-dependent pathway is not the to salvage adenosine because the T. brucei M. H. B. L. J. L. 2005; PubMed Scopus Google two adenosine kinase and The are to by it was that T. brucei sensitive to cordycepin when AK is by P. P. PubMed Scopus Google Scholar). We have and the T. brucei AK to its in adenosine salvage and of nucleoside The T. brucei AK (TbAK) orders of magnitude higher adenosine affinity catalytic than (11Parkin D.W. J. Biol. Chem. 1996; 271: 21713-21719Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 12Pellé R. Schramm V.L. Parkin D.W. J. Biol. Chem. 1998; 273: 2118-2126Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), which the in the cleavage-dependent pathway. The salvage system of a high affinity AK and efficient transporters to make T. brucei to It is also from a of because nucleoside analogs need to be by TbAK to The adenine arabinoside (Ara-A) was taken up by the parasites and by the pathway. The inhibited trypanosome proliferation and survival by into nucleic and by affecting nucleotide levels in the and of AK was from T. brucei used were and The was into the by and The of the AK was by analysis and The a with thioredoxin A and the also a followed by a tobacco etch virus the two to and of the and of was into cells and The cells were in of and at with at an of at was to a concentration of and the was for an at The cells were for and in of A 20 The cells were by and and at for The was a with of The was with of in A and with of in The TbAK was with of 20 in The were and with at and for 20 at The was in of and and on a with B. The protein concentration was by the protein and compared with a made from The of was to the protein and the from TbAK by of protein in 2 of was cleaved by of for at The cleaved protein was a with B. TbAK was in the whereas the and were to the The in the were by a in liquid and at The on enzyme not TbAK were performed with or inosine from to the enzyme TbAK was in which of was used in the Tween and The protein was mixed with in a of The nucleoside or and high nucleoside and enzyme were kinase and were to the to of ATP the and kinase on enzyme where ATP levels were enzyme and The were mixed on and for the were at for of the the were on and 20 of the was the were for by The was from the by as Anal. Biochem. PubMed Scopus Google Scholar). The of enzyme from of TbAK protein is in AK was by to the of the and to TbAK with adenosine analogs that were not of the the were in and 20 was a The was and were with a of and was to with The was also used to from whereas the concentration was to to adenosine from Ara-A from and from of and Mammalian of the T. brucei brucei H. K. PubMed Scopus Google were in H. K. J. PubMed Scopus Google and but with at in a This of the has on trypanosome proliferation L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). or human were as in with and at in a of T. brucei were by at at for and the was in The trypanosomes were by as Biochem. B. Scopus Google and at at for The was in liquid and at The was performed as that 2 ATP was used of and that was to prevent ATP concentration was by the protein and compared with a made from were from and TbAK knockdown T. brucei cells. were by with and in of the cells, the were mixed with of and for 15 were on The were for and the were used for analysis was performed with in Tris-buffered 20 and The was with an developed with the system and by The was and a was made with from J. of is a T. brucei homolog of immunoglobulin binding protein and is used as in The TbAK was by of and trypanosomes or mammalian cells from a were and and were by a as L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The were by P. L. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google a A from The was The was made up from and and was to with were from by chromatography to the analysis L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The were by the to a of of or Mammalian in the of brucei cells were for in a in the of of of Ara-A combination with μm 9-erythro-(2-hydroxy-3-nonyl) adenine or trypanosomes that were with were in the at The cells were as B. M. R. R. PubMed Scopus Google the of 20 of and a the were by at and at The used of an and an and cells were also for in the of drugs and by the cells with were at were with of Ara-A μm or for cells a of at this were by and as the concentration of where trypanosomes with were was was as the concentration where more than of the cells were than of into were at for 2 and in of of with μm of or was together with of and the cells were for The and were from with acid was in and at of of this the of in nucleic be from the nucleic by acid of the and on as B. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The in was by the from the The of into nucleic acid was the not and of TbAK was into The a protein to in the of this in of The protein was to by chromatography The protein was by and by the and the they to the and TbAK be in the with a of from a of of protein The and TbAK were in with of and analysis showed in with the Leishmania donovani and C.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.A. B. Biochem. 1999; PubMed Scopus Google Scholar), the TbAK protein was in the and of ATP not of was μm adenosine is inhibited at higher and ATP as enzyme was at The enzyme was by The on be a general of because it has also in from L. and B. PubMed Scopus Google Scholar). The human enzyme can also be by such as J. B. J. PubMed Scopus Google Scholar), but this on the trypanosome enzyme TbAK was dependent on whereas and on enzyme not TbAK was with and protein concentration not The of the enzyme was and depending on the concentration. This is higher than for the and recombinant human and J. N.S. K. M. T. 1996; PubMed Scopus Google Scholar)) but in the as for the recombinant AK B. J. J.A. J. Biochem. 271: PubMed Scopus Google Scholar)). ATP and GTP for enzyme or as ATP as the enzyme a of The other higher but they needed to be present at higher to saturate the enzyme as by high the catalytic with that ATP and GTP are the ATP is present at higher than GTP in T. brucei cells L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), it is the major of TbAK is the the adenosine concentration was used at is the the adenosine concentration was used at is the the adenosine concentration was used at is the the adenosine concentration was used at The is the the adenosine concentration was used at in a new TbAK by at adenosine showed an enzyme up to a at μm adenosine in the of The and followed other at low adenosine but the to when the in higher and high adenosine the enzyme was inhibited as in where a of is with a the of by the which has also used to for the mammalian adenosine M. B. PubMed Scopus Google Scholar), where A = a was for all when the concentration was μm or The from this are in the and and TbAK from at high adenosine TbAK to mammalian B. PubMed Scopus Google Scholar, C.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and B. PubMed Scopus Google and from of L. donovani R. J. Biol. Chem. Full Text PDF PubMed Google Scholar), which to the of as trypanosomes A phylogenetic of from that the T. brucei enzyme has developed the of the mammalian and that to a TbAK is grouped together with the L. donovani enzyme that lacks It was not which or protein are for the in T. brucei and of brucei high intracellular levels of when in the of deoxyadenosine in the L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, to whether TbAK is for this high salvage activity. The recombinant AK was able to phosphorylate not adenosine but also deoxyadenosine and inosine However, the catalytic with were orders of magnitude lower than with adenosine was by that the and as with adenosine as A and was also in T. brucei protein to whether AK is for the high deoxyadenosine salvage in T. brucei cells L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). to the two variants of kinase is the nucleoside kinase known from the T. brucei and not more than when making with of known nucleoside in T. brucei was inhibited by adenosine and AMP which are the and of TbAK therefore to be for the of deoxyadenosine in The enzyme was not by of or The of TbAK in intracellular deoxyadenosine was in knockdown trypanosomes where the TbAK can be by knockdown cells were P. P. PubMed Scopus Google Scholar)). analysis showed that the TbAK protein was in taken from cells as compared with cells the of the to a made with the recombinant it was to that the cells of TbAK of the protein and that the of with of the TbAK levels was at not Studies of deoxyadenosine salvage in the knockdown cells showed that the cells high intracellular levels of when in the of whereas this was in the cells μm) was in the in to prevent of and of adenosine cordycepin and the and survival of T. brucei (3Rottenberg M.E. Masocha W. Ferella M. Petitto-Assis F. Goto H. Kristensson K. McCaffrey R. Wigzell H. J. Infect. Dis. 2005; 192: 1658-1665Crossref PubMed Scopus (55) Google and T. J. PubMed Scopus Google Scholar), The is in both dependent on AK P. P. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). Studies of the recombinant TbAK showed that it phosphorylate cordycepin and The enzyme with cordycepin and Ara-A as was higher in the of than with but there was in catalytic because the was by an A of catalytic showed that cordycepin was a much TbAK than Ara-A or However, the as a is dependent on the of TbAK to the mammalian than the catalytic Ara-A T. brucei and in a was for the which were by 2 for and The used for of the is in Studies on TbAK knockdown trypanosomes showed that Ara-A was dependent on The of cells was also the trypanosomes in a but the and cells was from showed that the and cells was for = in a of is that Ara-A trypanosome and survival in a manner whereas to be dependent on of wild-type T. brucei AK knockdown T. brucei and mammalian with Ara-A μm or brucei brucei in a new Ara-A by T. brucei than Mammalian and and in the is a of TbAK but nucleoside transporters to make its salvage were with of Ara-A μm of the intracellular was The salvage of Ara-A was at 20 μm and at this of the major in the Ara-A salvage in mammalian cells was much lower and not in the concentration The in the mammalian cells made up of the although the concentration of Ara-A in the was up to from showed the as the salvage T. brucei was more sensitive to Ara-A than and human The of the was to has for cells where the was μm in the of adenosine deaminase M. Google Scholar). The Ara-A salvage in the human was efficient and they were not sensitive to the in the concentration Ara-A treatment of T. brucei cells caused a general in GTP that was the levels The be a of a of the for biosynthesis. the of into in trypanosomes was The with Ara-A concentration. were from with This is to and by and it can therefore be used to both and biosynthesis. at μm of the synthesis of was inhibited whereas a much higher concentration was needed to the synthesis of of has also in mammalian cells where it was that the analog as a D.W. M. Google Scholar). T. brucei is an extracellular parasite that needs to salvage purines from the in order to The salvage of adenosine can be performed by the cleavage-dependent pathway or by TbAK TbAK is not an enzyme in because T. brucei is able to salvage adenosine the cleavage-dependent and it also has a of to salvage and adenine (4El Kouni M.H. Pharmacol. Ther. 2003; 99: 283-309Crossref PubMed Scopus (140) Google Scholar). However, the purine concentration is low in the blood and other body and the concentration when the purine at high which in the cleavage-dependent has a low adenosine affinity (Km ∼ 15 μm (11Parkin D.W. J. Biol. Chem. 1996; 271: 21713-21719Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 12Pellé R. Schramm V.L. Parkin D.W. J. Biol. Chem. 1998; 273: 2118-2126Abstract Full Text Full Text PDF PubMed Scopus (49) Google whereas the adenosine affinity of TbAK is orders of magnitude of having TbAK is to The high affinity pathway ATP to AMP from whereas the cleavage-dependent pathway salvage of other purines than which is made from and TbAK is able to low of adenosine but is inhibited when the concentration is high to prevent of high intracellular purine nucleotide The AK from the related parasite L. donovani is from TbAK by having a lower affinity to adenosine (Km = depending on concentration B. PubMed Scopus Google Scholar)) and R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, B. PubMed Scopus Google Scholar). The properties of the of the parasites in mammalian L. donovani is intracellular whereas T. brucei is to other adenosine the TbAK was by but in to the other B. PubMed Scopus Google the affinity for adenosine was not the L. donovani AK higher affinity to adenosine when is to the B. PubMed Scopus Google Scholar). in T. brucei have that AK is Biochem. PubMed Scopus Google or low Biochem. B. Scopus Google Scholar, PubMed Scopus Google Scholar). is that it is important to the to AK from T. brucei. are by and AK was whereas not the of was on of the uncertainty of it is to whether AK or is the major from in However, the high of L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google and in trypanosomes that TbAK is high in There is also a for adenosine salvage with of adenosine by adenosine deaminase and cleavage of the formed inosine by It is known that the parasite has an but it is whether adenosine deaminase is in Studies of adenosine deaminase in T. brucei showed results Biochem. B. Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). We have not able to in T. brucei of The results be by with mammalian adenosine which is an enzyme in The catalytic of TbAK was high in with of the other AKs, the enzyme which has and in a as TbAK B. J. J.A. J. Biochem. 271: PubMed Scopus Google Scholar). having the combination of a high affinity AK and T. brucei to be to with the mammalian cells for the purine TbAK also drugs such as and as by enzyme and of TbAK knockdown cells. It is important that Ara-A is not by the cleavage-dependent pathway because that the and adenine is in T. brucei that Ara-A is to such cleavage not T. brucei was more sensitive to Ara-A than mammalian cells. results that the of the the trypanosomes was because of high salvage The high salvage can be by both efficient nucleoside transport and by the Ara-A affinity to mammalian cells, Ara-A is by adenosine kinase and which have lower affinities to Ara-A than TbAK of human and adenosine are in the J. Google Scholar, J. PubMed Scopus Google Scholar, B. J. Biol. Chem. Full Text PDF PubMed Google Scholar)). The of the two to Ara-A salvage in mammalian cells is cells and cells, adenosine kinase is for Ara-A salvage M. Google Scholar, V.L. P. PubMed Scopus Google Scholar), whereas in human cells, the two are J. Google Scholar). the is by kinase in mammalian cells Clin. PubMed Scopus Google Scholar). The in the trypanosomes caused of and biosynthesis. The of can also be an caused by that are needed as for We not the It be that Ara-A the for nucleotide in the parasites or that the in the cells the is because Ara-A ATP or other mammalian cells, also by as an J. Biol. Chem. Full Text PDF PubMed Google Scholar). However, the T. brucei lacks L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, L. PubMed Scopus Google Scholar), and of was analogs have both of are and for into anti-trypanosomal A major is that of Ara-A R. PubMed Scopus Google and cordycepin (3Rottenberg M.E. Masocha W. Ferella M. Petitto-Assis F. Goto H. Kristensson K. McCaffrey R. Wigzell H. J. Infect. Dis. 2005; 192: 1658-1665Crossref PubMed Scopus (55) Google Scholar), to the that is a to treat African sleeping is also for the adenosine deaminase inhibitor that was into the in J. R. J. 1982; Google Scholar). there are two drugs that can be used sleeping sickness, and (1Barrett M.P. Lancet. 2006; 367: 1377-1378Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 2Brun R. Karger-Gazette. 1999; 63: 5-7Google Scholar). is and of the a whereas of the two subspecies the human brucei treatment is not in of because it for Promising results with cordycepin on T. brucei-infected mice with brain infection (3Rottenberg M.E. Masocha W. Ferella M. Petitto-Assis F. Goto H. Kristensson K. McCaffrey R. Wigzell H. J. Infect. Dis. 2005; 192: 1658-1665Crossref PubMed Scopus (55) Google that adenosine analogs can be developed into drugs African sleeping However, the of cordycepin be it can be as a on and have when they were as drugs and Clin. PubMed Scopus Google Scholar, R. PubMed Scopus Google Scholar). is a great because of and properties are results on T. brucei-infected mice with a combination of Ara-A and that the trypanosome levels in the blood the a not results were with the However, the needs to be in both because the trypanosomes a the treatment was the of an efficient TbAK and high affinity transporters the trypanosomes sensitive to AK analogs that be developed into new African sleeping The of analog is dependent on such as transport TbAK and the on We for T. brucei with