Purine Nucleobase Transport in Amastigotes of Leishmania mexicana: Involvement in Allopurinol Uptake

Nucleobase and nucleoside transporters play central roles inthe biochemistry of parasitic protozoa, as they lack the abilityto synthesize purines de novo and are absolutely reliant uponpurine salvage from their hosts. Furthermore, such transportersare potentially critical to the pharmacology of these importanthuman pathogens, because they mediate the uptake of purine analogues,as well as some nonpurine drugs, that can be selectively cytotoxicto the parasites. We here report the first identification andcharacterization of a purine nucleobase transporter in Leishmaniaamastigotes. Uptake of [³H]hypoxanthine by Leishmania mexicanaamastigotes was mediated by a single high-affinity transporter,LmexNBT1, with a K_m of 1.6 ± 0.4 µM and high affinityfor adenine, guanine, and xanthine but low affinity for nucleosidesand pyrimidine nucleobases. Allopurinol, an antileishmanialhypoxanthine analogue, was apparently taken up by the same transporter.Using [³H]allopurinol, a K_m value of 33.6 ± 6.0 µMwas obtained. All evidence was compatible with a model of asingle purine nucleobase transporter being expressed in amastigotes.Using various purine nucleobase analogues, a model for the interactionsbetween hypoxanthine and the transporter's permeant bindingsite was constructed. The binding interactions were comparedwith those of the LmajNBT1 transporter in Leishmania major promastigotesand found to be very similar.

INTRODUCTION

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Introduction

Parasitic protozoa of the genus Leishmania are the etiologicalagents of leishmaniasis. Some 12 million people throughout theworld suffer from leishmaniasis, ranging from the disfiguringcutaneous form to often-fatal visceral leishmaniasis. Leishmaniaspecies are sandfly-transmitted protozoan parasites. The lifecycle is divided into promastigotes and metacyclics in the insectvectors and amastigotes in their mammalian hosts, which areresponsible for all clinical manifestations.

Most of the currently available antileishmanial drugs have beendiscovered empirically, as until recently insufficient informationwas known about the biochemistry, physiology, and molecularbiology of these parasites and the interactions with their hosts(23). The limitations of the current treatment for leishmaniasis,as a result of drug resistance and the severe side effects ofmost of the existing therapeutic agents, and the urgent needfor new therapeutic approaches, are well documented (12, 56,60).

The development of a rational therapeutic strategy for the treatmentand prevention of parasitic disease depends on exploitationof fundamental biochemical disparities between parasite andhost, such as the inability of protozoan parasites to synthesizepurines de novo (5). Current antiprotozoal agents often deriveselectivity from selective accumulation by the parasite ratherthan the host cell (15). The selectivity and the efficacy ofpurine antimetabolites can be achieved by the cell surface transportersthat mediate access to the cell, as substrate recognition bynucleobase transporters is strikingly different in humans andkinetoplastids such as Trypanosoma brucei (58) and Leishmaniamajor promastigotes (2). Purine and pyrimidine antimetaboliteshave been highly successful against many viral infections aswell as malignancies (27) and show great promise against protozoalinfections as well (reviewed in references 14, 17, and 23).Allopurinol, a purine nucleobase analogue, is clinically usedagainst various manifestations of leishmaniasis (1, 13, 39).

Whereas purine transporters in promastigotes have now been studiedin detail (see references 17 and 29 for recent reviews), verylittle is known about such transporters in the infective amastigoteforms. The only study of purine transporters in amastigotesdemonstrated that there are at least two high-affinity adenosinetransporters in Leishmania donovani amastigotes, T1 and T2 (24).Purine nucleobase transport in this intracellular form has yetto be described, despite a clear interest from physiologicaland pharmacological perspectives.

In a previous study, we have shown that allopurinol is efficientlytaken up by a high-affinity purine nucleobase transporter inL. major promastigotes, LmajNBT1 (2). We now report the firstcharacterization of nucleobase transport in Leishmania mexicanaamastigotes, including the transport of allopurinol. Our dataare consistent with a single broad-specificity nucleobase transportermediating the uptake of all natural purine nucleobases and allopurinol.The functional characterization of this transporter, LmexNBT1,showed it to be virtually identical to LmajNBT1.

MATERIALS AND METHODS

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Materials and Methods

Materials. Nonradioactive nucleobases and nucleosides were obtained asindicated in the footnotes to Table 1. [2, 8-³H]adenine (1.19TBq/mmol) was bought from NEN (Boston, MA), [8-³H]hypoxanthine(1.04 TBq/mmol) and [2-³H]adenosine (0.92 TBq/mmol) were purchasedfrom Amersham Pharmacia Biotech (Buckinghamshire, United Kingdom),and [³H(G)]allopurinol (44.4 GBq/mmol) was obtained from Moravek(Brea, CA).

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TABLE 1. Kinetic constants of purine nucleobase uptake in L. mexicana amastigotes and L. major promastigotes^a

Leishmania culture. Axenic cultures of the MNYC/BZ/62/M379 strain of Leishmaniamexicana were used throughout. Amastigotes were maintained bytwice-weekly serial passage in Schneider's Drosophila medium(Gibco), including 20% heat-inactivated fetal calf serum (Gibco)and 0.3% gentamicin. The pH of the medium was adjusted to 5.5with 1.0 M HCl. Cultures were maintained at 33°C in a CO₂incubator. Amastigotes were harvested by centrifugation (10min, 2,500 rpm), washed twice with a modified CBSS buffer (33mM MES [morpholineethanesulfonic acid], 98 mM NaCl, 4.6 mM KCl,0.3 mM CaCl₂, 0.07 mM MgSO4, 5.8 mM NaH₂PO4, 0.3 mM MgCl₂, 14mM D-glucose, pH 6.0), and resuspended at 10⁸ cells/ml in thesame buffer (24).

Transport assays. Transport of radiolabeled purine nucleosides and nucleobases(adenosine, adenine, hypoxanthine, and allopurinol) into L.mexicana amastigotes was performed using the oil stop technique.The amastigotes were harvested by centrifugation (2,500 x g,10 min) and washed twice in modified CBSS buffer. Cells werewashed and resuspended in the same buffer at 32°C and usedat approximately 10⁷ cells/ml. Transport was measured at 32°C.One hundred microliters of cell suspension was added to a microcentrifugetube containing 200 µl oil (7:1 [vol/vol] dibutyl-phthalate/mineraloil; d = 1.018 g/ml) and 100 µl [³H]adenine, [³H]hypoxanthine,[³H]adenosine, or [³H]allopurinol (concentrations as indicatedbelow) and incubated for the indicated times. Incubations wereterminated by adding 1 ml ice-cold 4 mM unlabeled permeant inCBSS buffer, and cells were separated from extracellular labelby centrifugation through the oil (12,000 x g, 30 s). The tubeswere frozen in liquid nitrogen and the tips containing the cellpellet cut off. The pellet was dissolved in 250 µl 2%sodium dodecyl sulfate. Scintillation fluid (3 ml) (OptiphaseHiSafe III; Perkin-Elmer) was added, and radioactivity was determinedin a 1450 MicroBeta Trilax scintillation counter. Zero-uptakevalues were obtained in the presence of 1 mM unlabeled permeantat 0°C. Nonmediated transport was determined in the presenceof 1 mM unlabeled permeant at 32°C. Transport values werecalculated after subtraction of the zero-uptake values. In inhibitionstudies, test compounds were mixed with ³H-labeled permeantprior to addition of cells.

Data analysis. The Prism 3 software package (GraphPad Software, San Diego,CA) was used to calculate the kinetic parameters, given as meansand standard errors, by using nonlinear regression. Inhibitiondata were plotted to a sigmoid curve with a variable slope andused to determine 50% inhibitory concentrations (IC₅₀s) (basedon a minimum of six points in triplicate). K_i values were thenobtained using the Cheng-Prusoff equation (10), K_i = IC₅₀/[1+ (L/K_m)], where L is the permeant concentration and K_m is thevalue obtained for this permeant. The Gibbs free energy of thetransporter-ligand interaction was then calculated from ${Delta}$ G°= –RTln(Ki), in which R is the gas constant and T theabsolute temperature. Errors given in tables and shown as barsin figures are standard errors. It should be noted that theseequations are valid only for competitive inhibition, as describedpreviously (19, 58).

Toxicity test. The drug sensitivity assay with Alamar blue was performed asdescribed for T. brucei and Leishmania (38, 51) in 96-well plates,with some modifications. Briefly, 100 µl of Schneider'sDrosophila medium (Gibco), used for culturing amastigotes, wasadded to wells, leaving the first column empty. Two hundredmicroliters of different test compounds was added, at doublethe final concentration, to the first column. Doubling dilutionswere performed, leaving one well drug free as negative control.L. mexicana amastigotes were inoculated into each well at afinal density of 1 x 10⁶/ml culture medium. Twenty microlitersof Alamar blue reagent (TREK Diagnostic Systems, Ltd.) was addedafter 48 h and fluorescence development determined after a furtherincubation time of 72 h. The plates were read using a LS55 luminescencespectrometer (Perkin-Elmer) at 530-nm excitation and 590-nmemission wavelengths.

RESULTS

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Results

Purine transport in axenic amastigotes of Leishmania. Total uptake of adenosine in axenic amastigote forms of L. mexicanawas determined using a rapid stop oil spin protocol adaptedfrom similar protocols with various other protozoa (2, 16, 18-20).Figure 1A demonstrates that the transport of 1 µM [³H]adenosinewas linear for at least 120 s at 0.021 ± 0.001 pmol 10⁷cells^–1 s^–1 (linear regression; r² = 0.97). Theaddition of 1 mM unlabeled adenosine inhibited the uptake of1 µM [³H] adenosine by 87% (r² = 0.99), indicating thatthe vast majority of adenosine transport occurs via a mediatedpathway (Fig. 1A). These observations are consistent with anearlier report for adenosine transport in amastigote forms ofL. donovani (24).

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FIG. 1. Linear purine transport in axenic amastigotes of Leishmania mexicana. A. Time course of 1 µM [³H]adenosine uptake by L. mexicana amastigotes in the presence ( ${square}$ ) or absence ( ${blacksquare}$ ) of 1 mM unlabeled adenosine. B. Uptake of 1 µM [³H]hypoxanthine was linear over 120 s (r² = 0.97), as calculated by linear regression, in the presence ( ${square}$ ) or the absence ( ${blacksquare}$ ) of 1 mM unlabeled hypoxanthine. C. Uptake of 1 µM [³H]adenine was linear over 120 s (r² = 0.94), as calculated by linear regression, in the presence ( ${square}$ ) or the absence ( ${blacksquare}$ ) of 1 mM unlabeled adenine.

Uptake of [³H]hypoxanthine at 1 µM by the axenic amastigotesof L. mexicana was linear for up to 120 s (Fig. 1B) and fasterthan [³H]adenosine uptake, at 0.031 ± 0.001 pmol 10⁷cells^–1 s^–1 (r² = 0.98). In the presence of 1 mMunlabeled hypoxanthine, [³H]hypoxanthine transport was not significantlydifferent from zero (P = 0.7, F test), indicating high-affinitytransport that is completely saturated at 1 mM. [³H]adenineuptake (1 µM) was likewise linear over 120 s, and thiswas the least efficiently accumulated of the three purines tested,with a rate of 0.012 ± 0.001 pmol 10⁷ cells^–1 s^–1,which was almost 95% inhibited by 1 mM unlabeled adenine (Fig.1C). Subsequent experiments were performed with [³H]hypoxanthineand 60-s incubations, which are well within the linear phaseof uptake and therefore measure true initial rates of transport.

A high-affinity purine nucleobase transporter in amastigotes. [³H]hypoxanthine transport (0.1 µM) measured in the presenceof up to 1 mM of unlabeled hypoxanthine was monophasic and compliedwith simple Michaelis-Menten kinetics, yielding a K_m of 1.6± 0.4 µM and a V_max of 0.092 ± 0.057 pmol10⁷ parasites^–1 s^–1 (n = 4) (Fig. 2). The transportof [³H]hypoxanthine was inhibited by a range of purine nucleobasesand nucleosides (Table 1) but not significantly by various pyrimidinesat up to 1 mM (P > 0.05 by a paired Student's t test againsta no-inhibitor control; n = 3). The hypoxanthine transporter,designated L. mexicana nucleobase transporter 1 (LmexNBT1),appears to be mildly selective for oxopurines over aminopurines,judging from the K_i values for guanine and adenine (1.7 ±0.1 and 4.2 ± 0.8 µM, respectively). None of theinhibition plots suggested the presence of more than one hypoxanthinetransporter in L. mexicana amastigotes, as Hill slopes wereconsistently near –1 and complete inhibition to controlvalues (transport at 0°C, 0 s) was generally observed.

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FIG. 2. Transport of 0.1 µM [³H]hypoxanthine over 60 s was inhibited by the indicated concentrations of unlabeled hypoxanthine, with an IC₅₀ of 0.81 µM for this experiment. The inset depicts the conversion of the hypoxanthine inhibition data to a Michaelis-Menten plot of total hypoxanthine uptake, with a K_m of 1.6 µM and a V_max of 0.29 pmol 10⁷ cells^–1 s^–1 for this experiment, showing total hypoxanthine transport as opposed to [³H]hypoxanthine transport only.

[³H]allopurinol transport by L. mexicana amastigotes. Allopurinol is a close structural analogue of hypoxanthine andin L. major promastigotes is indeed accumulated through thehypoxanthine transporter LmajNBT1 (2). We therefore investigatedwhether a similar situation exists in amastigotes. As L. majoramastigotes cannot be kept in axenic culture, we used amastigotesfrom L. mexicana. Using these cells, transport of allopurinolwas clearly less efficient than that of hypoxanthine, with arate for 5 µM [³H]allopurinol of 0.0061 ± 0.0002pmol 10⁷ cells^–1 s^–1 (linear regression of six pointsover 4 min; r² = 0.99), which was completely inhibited by 1mM hypoxanthine (uptake not significantly different from zero;P = 0.33 by F test). Transport of [³H]allopurinol (5 µM)was still detectable, however, in the presence of 1 mM unlabeledallopurinol (0.00052 ± 0.00014 pmol 10⁷ cells^–1s^–1) (linear regression of five points over 10 min; P= 0.03 by F test), although it was greatly reduced. These resultsindicate that the allopurinol transporter has a higher affinityfor hypoxanthine than for allopurinol.

Competition experiments showed that transport of 2 µM[³H]allopurinol over 120 s was indeed inhibited more stronglyby hypoxanthine than by allopurinol, with a K_i of 3.8 ±0.6 µM for hypoxanthine and a K_m of 33.6 ± 6.0µM for allopurinol (n = 3) (Fig. 3). These data are allconsistent with a single high-affinity transporter for purinenucleobases, including allopurinol. While the V_max values ofLmexNBT1 for hypoxanthine and allopurinol are similar (Table1), the efficiency of uptake, expressed as K_m/V_max, is considerablyhigher for hypoxanthine.

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FIG. 3. Transport of [³H]allopurinol by L. mexicana amastigotes. A. Inhibition of 2 µM [³H]allopurinol transport over 120 s by up to 100 µM of unlabeled allopurinol ( ${blacksquare}$ ) or hypoxanthine ( ${circ}$ ). B. Michaelis-Menten plot of allopurinol uptake, derived from the data in panel A.

Structure-activity relationships of purine analogues and the LmexNBT1 transporter. The inhibitory effects of purine analogues for LmexNBT1 weretested by measuring initial uptake rates of 0.1 µM [³H]hypoxanthinein the presence of various concentrations of the test compound.Results were plotted as log inhibitor concentration versus hypoxanthineuptake, and K_i values, calculated from the IC₅₀s, were usedto calculate the estimated Gibbs free energy of binding, ${Delta}$ G°(reviewed in reference 17). The results, summarized in Tables1 and 2, show that the LmexNBT1 transporter shows broad specificityfor purine nucleobases but displays low affinity for the correspondingnucleosides.

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TABLE 2. Gibbs free energies of substrates interacting with the LmexNBT1 transporter^a

LmexNBT1 displayed equal affinity for guanine (Fig. 4A) andhypoxanthine (Fig. 2), showing that the 2-amino group of guaninedid not contribute to binding or pose a steric hindrance tobinding. However, affinity for xanthine (Fig. 4A) was markedlyreduced [ ${delta}$ ( ${Delta}$ G°) = 5.5 kJ/mol compared to hypoxanthine]. Asthis is unlikely to be the result of steric hindrance at position2, this must be the result of protonation of the pyrimidinenitrogen at position 3 of the purine ring that occurs in xanthine.A comparison between guanine and 3-deazaguanine (Fig. 4A) showsthat the unprotonated N-3 contributes 11.2 kJ/mol to the bindingenergy of guanine (Table 2). As only 5.5 kJ/mol is lost in xanthine,it can be postulated that weak hydrogen bonds totaling $~$ 6 kJ/molcan be established between the LmexNBT1 binding pocket and N(3)Hand/or the 2-keto group of xanthine. From inhibition plots with9-deazaguanine (Fig. 4B), the Gibbs free energy of H bonds withN(9)H was similarly estimated at –12.3 kJ/mol (Table 2).The K_i for 7-deazaguanine could not be clearly determined, dueto very low affinity and solubility limitations, but was shownto be over 1 mM, which would indicate a ${delta}$ ( ${Delta}$ G°) of >15 kJ/molfor N-7. Furthermore, the substitution of the 6-keto group fora thione group (6-thioguanine) reduced the ${Delta}$ G° by 4.5 kJ/mol(6-thioguanine versus guanine) (Fig. 4B). This could be an indicationof a hydrogen bond interacting with the keto group, as thionesare much weaker H bond acceptors (50), although the possibilityof a steric effect by the larger sulfur atom provides an alternativeexplanation. Indeed, the ${delta}$ ( ${Delta}$ G°) between hypoxanthine and purine,which lacks both the 6-keto and lactam hydrogen on N-1, is only1.9 kJ/mol (Table 2). However, this needs to take into accountthe strong hydrogen bond with the pyrimidine N-1, estimatedat 7.2 kJ/mol from the comparison between purine and 1-deazapurine(Fig. 4C), bringing the actual contribution of the 6-keto/N(1)Hcombination to 9.1 kJ/mol, of which 4.5 kJ/mol could be tentativelyassigned to the keto group from the ${delta}$ ( ${Delta}$ G°) of 6-thioguanine.Finally, adenine (Fig. 4C) and purine proved to have the sameaffinity for LmexNBT1 (Table 1), confirming the preference fora hydrogen bond acceptor at position 6 of the purine ring. Theabove observations were combined into a model for the bindingof purine substrates by LmexNBT1 and compared to the model forsubstrate binding by the equivalent transporter of L. major(Fig. 5).

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FIG. 4. Inhibition of 0.1 µM [³H]hypoxanthine uptake in axenic amastigotes of L. mexicana by guanine (Gua), xanthine (Xan), and 3-deazaguanine (3-DG) (A); by 6-thioguanine (6-TG), 7-deazaguanine, and 9-deazaguanine (B); and by purine, adenine (Ade), and 1-deazapurine (1-DPur) (C). Data are expressed as percentages of control values (uptake in the absence of any inhibitor, typically 0.005 to 0.02 pmol/10⁷ cells/s).

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FIG. 5. Schematic representation of the LmajNBT1 (A) and LmexNBT1 (B) transporters and the interactions with their permeants, adenine and hypoxanthine. Estimated Gibbs free energies for proposed bonds are indicated, in negative kilojoules per mole. The model for LmajNBT1 was adapted from reference 2, with permission.

Antileishmanial activity of some nucleobase analogues. Allopurinol is well known to possess antileishmanial activity,but we previously noted that, at least in promastigotes, aminopurinoland the pyrimidine nucleobase 5-fluorouracil displayed substantiallyhigher activity (46). We therefore tested the same compoundson the human-infective amastigote stage, using pentamidine asa positive control and the same technique based on the fluorophoreAlamar blue (Fig. 6). The method was validated using cell countsin parallel to the fluorescence assay, yielding the same IC₅₀s(Fig. 6, inset). As with L. major promastigotes, aminopurinoland 5-fluorouracil were 10- to 20-fold more active against axenicL. mexicana amastigotes than allopurinol. IC₅₀s (in micromolar;n = 3) were 5.6 ± 1.0, 630 ± 87, 65 ± 5,and 36 ± 6 for pentamidine, allopurinol, aminopurinol,and 5-fluorouracil, respectively.

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FIG. 6. Effects of various drugs on L. mexicana amastigotes in axenic culture. Amastigotes (2 x 10⁵) were incubated with doubling dilutions of pentamidine ( ${blacksquare}$ ), allopurinol ( ${square}$ ), aminopurinol ( ${blacktriangledown}$ ), and 5-fluorouracil ( ${circ}$ ) for 72 h and for an additional 48 h in the presence of Alamar blue reagent as described in Materials and Methods. Fifty percent effective doses for pentamidine as determined by fluorescence or cell counts (inset) were identical.

DISCUSSION

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Discussion

Purine and pyrimidine antimetabolites are widely used to combata variety of infectious diseases and other pathologies (14).Current evidence indicates that purine analogues alone or incombination with other chemotherapeutic agents are very effectiveagainst almost any fast-growing cells (22, 41). Allopurinol,a purine analogue, has been used to treat leishmaniasis alone(34, 48) or combined with other drugs (21, 35, 36, 39, 47).It should be pointed out, however, that the efficacy of allopurinolmonotherapy remains unclear. A large-scale randomized trialsponsored by the World Health Organization showed no differencebetween allopurinol- and placebo-treated groups (57). It maybe that the value of allopurinol in the treatment of leishmaniasisis solely by synergistically enhancing the action of other treatments,and there is much, although largely anecdotal, evidence forthis. One such report, by Chunge et al. (11), describes thetreatment of five patients with visceral leishmaniasis unresponsiveto sodium stibogluconate who were cured by a combination ofthe same drug plus additional allopurinol. In vitro as well,allopurinol has been reported to augment the antileishmanialeffects of pentavalent antimonials (7). Other pyrazolopyrimidines,such as 9-deazainosine, displayed favorable therapeutic indexesin L. donovani-infected hamsters (6) and also offer promiseas potential chemotherapeutic agents (32). However, it is widelyacknowledged that the potential for damaging side effects canpose a challenge to the development of purine-based drugs (23).

Selectivity and efficacy of purine antimetabolites can be achievedthrough the enzymes of the purine metabolic pathways that convertthe prodrug to the cytotoxic metabolite, usually a nucleotideanalogue, and understanding of the substrate specificities ofthe enzymes involved is important in both drug efficacy andselectivity. The metabolism of allopurinol in Leishmania speciesto the active metabolite 4-aminopyrazolo(3,4-d)pyrimidine ribonucleosidetriphosphate, which is incorporated into RNA, has been wellstudied (30, 31, 33, 43, 45). The drug is selective, becausemammalian cells do not show this conversion or incorporation(44).

In addition, many antiprotozoal drugs derive their selectiveactivity through uptake by cell surface transporters on thetarget cell rather than the host's cells (15). The main humannucleobase transporter, hFNT1, displays very low affinity forallopurinol (52, 58), whereas allopurinol uptake in Trypanosomabrucei was mediated by high-affinity nucleobase transporters(18, 42). We have demonstrated that a strategy of purine-basedchemotherapy, designed for specific uptake by protozoan parasites,is feasible and depends on detailed knowledge of the substraterecognition motifs of the respective transporters (17, 58, 59).

We recently reported the characterization of [³H]hypoxanthine,[³H]allopurinol, and [³H]uracil transport in L. major promastigotes(2, 46), but the presence of nucleobase transporters in amastigoteshas not yet been reported. The issue is important in understandingthe selectivity of the drug as well as the potential for thedevelopment of resistance, as allopurinol-resistant Leishmanialines have been described (9, 26) and resistance appears tobe easily inducible (K. Soteriadou, personal communication).We consider that this resistance might be related to loss oftransporter function, as allopurinol was taken up by a singlenucleobase transporter in L. major promastigotes (2), in sharpcontrast to the situation in T. brucei brucei, where the presenceof multiple allopurinol transporters appears to preclude transport-relatedresistance (42). We have therefore conducted a study of purinenucleobase uptake in Leishmania mexicana amastigotes, with particularemphasis on allopurinol uptake. We found that hypoxanthine andallopurinol are taken up by a single plasma membrane transporter,LmexNBT1, and we characterized its substrate profile in detail.

LmexNBT1 proved to be extremely similar to LmajNBT1. A comparisonof their substrate binding models (Fig. 5) reveals that thepositions and even the relative strengths of the hydrogen bondsare completely conserved between the two transporters, suggestingvery similar architectures for the two transporters. This levelof conservation is remarkable in that it signifies conservationboth throughout the life cycle and between Leishmania speciesfrom different continents. Furthermore, the binding site architectureof these transporters appears to be very similar to that ofthe H2 hypoxanthine/allopurinol transporter in bloodstream T.brucei brucei but very different from the binding motif of hFNT1(2, 58). This suggests that these related kinetoplastids wouldaccumulate similar purine antimetabolites, which could be designedto be excluded from human cells. At present, none of the genesfor these involved transporters have been identified with anycertainty, precluding an assessment of whether the high levelof functional conservation between the various T. brucei andLeishmania transporters is matched by genetic conservation.However, all protozoan purine transporter genes cloned to dateare members of the equilibrative nucleoside transporter family(17), and the percent identity between the T. brucei bruceiTbNBT1/H4 or TbNT8.1 nucleobase transporters and the L. majorLmNT3 nucleobase transporter is only 50% (8, 25, 53).

LmexNBT1 showed only moderate affinity for allopurinol and aminopurinol.Yet, it seems unlikely that transport rates are a limiting factorin the efficacy of pyrazolopyrimidines against leishmaniasis,the conversion to the aminopurinol riboside triphosphate beinga more likely bottleneck: aminopurinol, despite having loweraffinity for LmexNBT1, was 10-fold more active against axenicamastigotes in culture. Nelson and coworkers reported the directincorporation of aminopurinol into the aminopurine nucleotidepool, and subsequently into the RNA, of both Leishmania andTrypanosoma cruzi (33, 43) by means of phosphoribosylation,a conversion that does not happen in humans (44).

This begs the question as to why aminopurinol has not been usedagainst leishmaniasis, particularly as it has been long knownthat aminopurinol is more potent than allopurinol against Leishmaniapromastigotes in vitro (4) as well as against T. cruzi in mice(3). In mammals, aminopurinol is used as an experimental drug,which reduces the release of lipoproteins by the liver, loweringplasma cholesterol levels (55). Chronic use, however, inducesa "fatty liver" (40) and adrenal cell hypertrophy by depletingadrenal cholesterol (28, 37). Aminopurinol given to mice for10 days at 1 mg/kg induced no toxic effect, but an increaseto 10 mg/kg caused high mortality rates and hepatomegaly (3).A toxicological study with rodents, dogs, and cats found toleranceto high single doses of aminopurinol but mouse 50% lethal dosesof 25 mg/kg when five consecutive daily doses were given (49).The hepatotoxic effects led to the abandonment of aminopurinolas a potential antileukemia drug (54), but levels active againstT. cruzi in mice (3) were found to be nontoxic to humans (54).

In summary, we have undertaken the first study of nucleobasetransport in Leishmania amastigotes and identified a high-affinitynucleobase transporter that is responsible for the uptake ofallopurinol and other purine bases. As the evidence is consistentwith a model for a single hypoxanthine/allopurinol transporter,the potential for the rapid development of allopurinol resistanceby loss of such a transporter would appear to be a potentialconcern. However, the use of purine analogues in combinationchemotherapy with another antileishmanial agent may well besynergistic and prevent or even overcome early onset of resistanceto either drug.

ACKNOWLEDGMENTS

M.I.A. was supported by a personal studentship from the Libyangovernment.

FOOTNOTES

* Corresponding author. Mailing address: Institute of Biomedical and Life Sciences, Division of Infection and Immunity, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, United Kingdom. Phone and fax: 44 141 330 3753. E-mail: H.de-Koning{at}bio.gla.ac.uk.

REFERENCES

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