Allopurinol and xanthine use different translocation mechanisms and trajectories in the fungal UapA transporter

Biochimie 95 (2013) 1755e1764

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Research paper

Allopurinol and xanthine use different translocation mechanisms and trajectories in the fungal UapA transporter George Diallinas* Faculty of Biology, Department of Botany, University of Athens, Panepistimioupolis, Athens 15784, Greece

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 April 2013 Accepted 31 May 2013 Available online 18 June 2013

In Aspergillus nidulans UapA is a Hþ-driven transporter specific for xanthine, uric acid and several analogues. Here, genetic and physiological evidence is provided showing that allopurinol is a high-affinity, low-capacity, substrate for UapA. Surprisingly however, transport kinetic measurements showed that, uniquely among all recognized UapA substrates, allopurinol is transported by apparent facilitated diffusion and exhibits a paradoxical effect on the transport of physiological substrates. Specifically, excess xanthine or other UapA substrates inhibit allopurinol uptake, as expected, but the presence of excess allopurinol results in a concentration-dependent enhancement of xanthine binding and transport. Flexible docking approaches failed to detect allopurinol binding in the major UapA substrate binding site, which was recently identified by mutational analysis and substrate docking using all other UapA substrates. These results and genetic evidence suggest that the allopurinol translocation pathway is distinct from, but probably overlapping with, that of physiological UapA substrates. Furthermore, although the stimulating effect of allopurinol on xanthine transport could, in principle, be rationalized by a cryptic allopurinol-specific allosteric site, evidence was obtained supporting that accelerated influx of xanthine is triggered through exchange with cytoplasmically accumulated allopurinol. Our results are in line with recently accumulating evidence revealing atypical and complex mechanisms underlying transport systems. Ó 2013 Published by Elsevier Masson SAS.

Keywords: Purine Uric acid Influx Reflux Gating

1. Introduction Transporters are proteins mediating the translocation of solutes and drugs across the cell membrane of all cells, a biological process implicated in nutrition, signalling and cell communication and survival. Similarly to most enzymes, and unlike channels, transporters have a single major binding site interacting specifically with a single substrate molecule in each transport cycle, and consequently are characterized by MichaeliseMenten hyperbolic kinetics. The current dogma also states that the binding site of transporters is accessible either from the extracellular environment or from the cytoplasm. The mechanism of exposing alternate outward- and inward-facing conformers of transporters, often simplified as a rocking-switch mechanism, has gained significant support from structural studies and a plethora of genetic, biochemical or biophysical data [1e5]. Noticeably however, functional and crystallographic studies have also supported the view that substrate binding and transport * Tel.: þ30 210 7274649; fax: þ30 210 7274702. E-mail address: [email protected]. 0300-9084/$ e see front matter Ó 2013 Published by Elsevier Masson SAS. http://dx.doi.org/10.1016/j.biochi.2013.05.013

might not simply be determined by the molecular interactions of a given solute in a single binding site. For example, based on genetic and kinetic studies of the UapA-xanthineeuric acid/Hþ symporter of the filamentous ascomycete Aspergillus nidulans, we have provided strong evidence for the existence of dynamic gating domains, or selectivity filters, operating at both the outward- and inwardface of the transporter for controlling the access of substrates to the major substrate binding site and thus contributing to transporter specificity and transport kinetics [6e10]. The role of complex and highly specific interdomain synergy between these gates and the major substrate binding site in determining transporter specificity has been supported by several combinations of mutations [8,9]. Most importantly, in the last years, direct structural evidence has been obtained for the existence of domains putatively acting as dynamic gates in several transporters. More specifically, several crystal structures of different transporters have been resolved showing the existence of occluded or open states, in both inward- or outward-facing transporter conformations [[11] and references therein]. In the handful of cases where the structure of a single transporter has been resolved in various conformational snapshots, a mechanism of transport was proposed and also used as a

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framework for understanding several other structurally similar transporters [11,12]. The existence of gates in transporters does not seem to violate the generally accepted model of the rocking-switch mechanism. It rather adds a further degree of complexity, related to how substrate specificity is determined, in some transporters [13]. However, entirely alternative mechanisms on how transporters function have also been proposed. The most provocative of these models states that substrates ‘slide down’ through several docking points in a channel-like trajectory, rather than inducing abrupt alternating conformational changes exposing a single major substrate binding site extra- or intracellularly [14,15]. This model is mostly supported through docking studies and thermodynamic approaches performed with specific facilitators, but also gains genetic support in cases where mutations mapping outside the major substrate binding site modify substrate specificity. Allopurinol, a structural isomer of hypoxanthine (4-hydroxy [3,4-d]-pyrimidine), is a purine analogue which acts as substrate and non-competitive inhibitor of xanthine oxidase/dehydrogenase, a key enzyme for purine catabolism [16,17]. This enzyme is responsible for the successive oxidation of hypoxanthine and xanthine, resulting in the production of uric acid, which is the final product of purine catabolism in primates [18]. Allopurinol blocks xanthine oxidase/dehydrogenase and uric acid production and is thus used for gout treatment [19]. Importantly, allopurinol has been used to treat Leishmania infections and also displays activity against the related parasite Trypanosoma brucei [20,21]. In contrast to other antiprotozoan drugs, which are associated with severe side effects and emerging resistance, allopurinol either alone or in combination with other drugs, has proved to be more effective against cutaneous. Consequently, transporters specific for allopurinol have been identified at the molecular level in Leishmania and Trypanosoma species. All transporters characterized are high-affinity (mM range) Hþ symporters and exhibit a rather broad specificity for most purines, pyrimidines and several analogues [21e25]. Interestingly, allopurinol has not been used as an antifungal or antibacterial agent and very little is known on the transporters responsible for its uptake in bacteria, fungi or their mammalians hosts. In A. nidulans early genetic evidence suggested that the uric acid/xanthine transporter UapA contributes to allopurinol uptake [26]. UapA belongs to the NucleobaseeAscorbate Transporter (NAT/NCS2) family, which is evolutionary, structurally and kinetically distinct from the purineeallopurinol transporters of protozoa, which all belong to the Equilibrative Nucleoside Transporter (ENT) family [27e29] (see also http://www.tcdb.org/). In this work, using genetic and physiological evidence, we show that UapA is the major gateway for high-affinity, low-capacity, allopurinol uptake. Interestingly however, in the course of identifying the transport mechanism of allopurinol transport, we came across of two unexpected kinetic observations; first, UapA-mediated radiolabelled allopurinol transport is non-saturable and Hþ gradient independent, and secondly, the uptake of radiolabelled xanthine, rather than being inhibited, as expected, by excess allopurinol, it is stimulated. The effect of allopurinol on xanthine uptake was not reciprocal, as excess xanthine or other substrates of UapA inhibit allopurinol uptake. Further kinetic measurements, the use of specific mutants, microscopic evidence and substrate docking approaches strongly suggested that allopurinol is transported by facilitated diffusion through a substrate translocation trajectory, which is different from that of xanthine and other substrates of UapA. Based on our observations, we propose that accelerated influx of xanthine is triggered by exchange with allopurinol accumulated within cells.

2. Experimental procedures 2.1. Growth media, strains, chemicals Standard media for A. nidulans were used. Supplemented auxotrophies were at the concentrations given in http://www.fgsc.net. The A. nidulans strains used are shown in Table 1. All mutants and control strains were isogenic. In strains expressing different UapA alleles (e.g. wild-type UapA, UapA-Q408E and UapA-F528S), the transporter was C-terminally tagged with GFP in order to ensure, by western blot and epifluorescence analyses, that expression levels were identical. UapA tagged with GFP is fully functional showing Km and V values identical to the untagged protein [6e8]. Previously constructed or genetically selected mutations used in this work [6e 8] were introduced in the context of an azgAþ background (AzgA is the major purine transporter; Results and discussion) through standard genetic crossing with an azgAþuapAD uapCD strain. Mutant hxAD hxnSD (herein called hx) cannot catabolize hypoxanthine and allopurinol due to genetic blocks in Purine Hydroxylases I (hypoxanthine dehydrogenase) and II, and shows only minimal xanthine catabolism, the latter taking place through the Table 1 A. nidulans strains used in this work. Strain

Genotype

Reference

Wild-type 1 (hxþ) Wild-type 2 (hxþ) hx

pabaA1

a

riboB2 yA2

a

hxAD hxnSD pabaA1

UapAD UapCD UapAD UapCD UapA UapAD UapCD yellow

uapAD pabaA1 uapCD pabaA1 uapAD uapCD pabaA1 pAN510-UapA uapAD uapCD pabaA1 yA2 uapAD uapCD riboB2

UapA-Q408E

pAN510-UapA-Q408E uapAD uapCD azgAD pabaA1 pAN510-UapA-F528S uapAD uapCD azgAD pabaA1 pAN510-UapA-F528A uapAD uapCD azgAD pabaA1 pAN510-UapA-T526M uapAD uapCD azgAD pabaA1 pAN510-UapA-T526L uapAD uapCD azgAD pabaA1 pAN510-UapA-Q408E/T526M uapAD uapCD azgAD pabaA1 pAN510-UapA-Q408E/T526L uapAD uapCD azgAD pabaA1 pAN510-UapA-Q408E uapAD uapCD pabaA1 pAN510-UapA-F528A uapAD uapCD pabaA1 pAN510-UapA-T526M uapAD uapCD pabaA1 pAN510-UapA-T526L uapAD uapCD pabaA1 pAN510-UapA-Q408E/T526M uapAD uapCD pabaA1 pAN510-UapA-Q408E/T526L uapAD uapCD pabaA1

This work [11,35] [11,35] [11,35] [11,35] This work [11,35]

UapA-F528S UapA-F528A UapA-T526M UapA-T526L UapA-Q408E/T526M UapA-Q408E/T526L UapA-Q408E UapA-F528A UapA-T526M UapA-T526L UapA-Q408E/T526M UapA-Q408E/T526L

[11,35] [11,35] [11,35] [11,35] [11,35] [11,35] This work This work This work This work This work This work

yA2, riboB2 and pabaA1 are loss-of-function mutations leading to yellow conidiospores or riboflavin or p-aminobenzoic acid requirements. pAN510 [42] is a pBluescript vector carrying the argB gene as a selection marker for transformation and the uapA alleles described in the text. hxAD and hxnSD are null mutants (total deletions) of the hypoxanthine dehydrogenase (also called purine hydroxylase I) and purine hydroxylase II. The double mutant is totally incapable for hypoxanthine and allopurinol catabolism and severely blocked in xanthine catabolism (residual xanthine catabolism operates through an alternative pathway [31,32]). a Standard A. nidulans strains carrying genetic markers.

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activity of XanA, an a-ketoglutarate Fe2þ-dependent dioxygenase (alternative purine catabolic pathway) [30e32]. Growth tests of mutants and control strains were performed at both 25 and 37  C, at pH 6.8. 0.5 mM is the standard purine concentration used for growth tests in all previous studies concerning purine catabolism in A. nidulans. Allopurinol in growth tests was used at 3 mM final concentration in minimal media with 0.5 mM hypoxanthine as sole nitrogen source. Media and chemical reagents were obtained from SigmaeAldrich (Life Science Chemilab SA, Hellas) and from AppliChem (Bioline Scientific SA, Hellas). 2.2. Transport assays [3H]-xanthine (33.4 Ci/mmol, Moravek Biochemicals, CA, USA) or [3H]-allopurinol (1.2 Ci/mmol, Moravek Biochemicals, CA, USA) uptake in minimal media (MM) was assayed in germinating conidiospores of A. nidulans concentrated at 107 conidiospores/100 mL, at 37  C, pH 6.8, as previously described [6,7,33]. Initial velocities were measured at 1 min of incubation with concentrations of 0.1 mM of [3H]-xanthine or 2 mM of [3H]-allopurinol at the polarity maintenance stage (3e4 h, 130 rpm). Steady state xanthine accumulation is obtained after 5 min of [3H]-xanthine uptake. Allopurinol uptake in response to time (30 s, 1, 2, 4 and 8 min) was performed with 2 mM of [3H]-allopurinol. Allopurinol uptake in response to concentration was performed for 1 min at 0.5, 1.0, 2.5, 5.0, 10.0, 20.0 and 50.0 mM concentrations of [3H]-allopurinol. The allopurinol effect on [3H]-xanthine was measured at various concentrations (0.1e400 mM) of non-labelled allopurinol. CCCP at 30 mM or DCCD at 100 mM were added 10 min before performing uptake assays [8]. Km values were obtained directly by performing and analyzing uptakes (Prism 3.02: GraphPad Software), using labelled xanthine at 0.1 mM at various concentrations (0.1e100 mM) of non-labelled substrates. Km values of xanthine were also determined at three constant concentrations of allopurinol (5, 20 and 100 mM). Reactions were terminated with addition of equal volumes of ice-cold MM containing 1000-fold excess of non-radiolabelled substrate (xanthine). Unless otherwise stated, background uptake values were corrected by subtracting values measured in the uapAD uapCD null mutants. In experiments investigating the effect of internally accumulated xanthine or allopurinol on [3H]xanthine uptake, cells were preloaded with non-radiolabelled allopurinol or xanthine (10, 20, 50 or 100 mM) for 5 min, at 37  C, washed thrice with 10 volumes MM at 4  C, and then [3H]-xanthine uptake measurements were performed for the desired period of time (1e5 min). For the experiments investigating directly the efflux of xanthine, [3H]-xanthine (0.2 mM) was allowed to accumulate for 5 min at 37  C, cells were washed thrice with 10 volumes MM at 4  C, then loaded with 100 mM of non-labelled allopurinol or xanthine and incubated for 5 min at 37  C, washed again, before radioactivity accumulated in the cells or in the supernatant was measured. As a control, cells without loading with non-labelled allopurinol or xanthine were also measured for [3H]-xanthine efflux. All transport assays were carried out in at least three independent experiments, with three replicates for each concentration or time point. Standard deviation was <20%. 2.3. Docking approaches Molecular docking was performed using the Induced Fit Docking (IFD) protocol (Schrödinger Suite 2011 Induced Fit Docking protocol), which is intended to circumvent the inflexible binding site and accounts for the side chain or backbone movements, or both, upon ligand binding. In the first stage of the IFD protocol, softened-potential docking step, 20 poses per ligand were retained. In the second step, for each docking pose, a full cycle of protein

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refinement was performed, with Prime 1.6 (Prime, version 3.0, Schrödinger, LLC, NY, 2011) on all residues having at least one atom within 8.0  A of an atom in any of the 20 ligand poses. The Prime refinement starts with a conformational search and minimization of the side chains of the selected residues and after convergence to a low-energy solution, an additional minimization of all selected residues (side chain and backbone) is performed with the truncated-Newton algorithm using the OPLS parameter set and a surface Generalized Born implicit solvent model. The obtained complexes are ranked according to Prime calculated energy (molecular mechanics and solvation), and those within 30 kcal/mol of the minimum energy structure are used in the last step of the process, re-docking with Glide 5.7 (Glide, version 5.7, Schrödinger, LLC, NY, 2011) using standard precision, and scoring. In the final round, the ligands used in the first docking step are re-docked into each of the receptor structures retained from the refinement step. The final ranking of the complexes is done by a composite score, which accounts for the receptoreligand interaction energy (GlideScore) and receptor strain and solvation energies (Prime energy). 3. Results and discussion 3.1. Genetic and physiological evidence show that allopurinol is UapA substrate Growth of a wild-type A. nidulans strain on hypoxanthine, a relatively good nitrogen source, is inhibited in the presence of allopurinol [34,35]. The inhibitory effect of allopurinol on the growth of A. nidulans on hypoxanthine media is seen at very low concentrations (3e5 mM) of allopurinol. This concentration corresponds approximately to the IC50 value (w2.5 mM) of xanthine dehydrogenase for allopurinol [17]. This finding suggests that there should be high-affinity uptake system(s) mediating allopurinol binding and transport at concentrations as low as 3e5 mM. Early genetic evidence suggested that uapA might be the major transporter mediating allopurinol uptake in A. nidulans, as apparent lossof-function mutations led to considerable allopurinol resistance on media containing hypoxanthine as the sole nitrogen source [36]. In addition, Scazzocchio et al. [17] reported competition of allopurinol uptake by xanthine analogues by growth tests (see also below). Here, undisputable genetic evidence was obtained showing that cellular uptake of allopurinol in A. nidulans is specifically mediated by UapA (Fig. 1). A single knock-out mutant strain carrying a total deletion of uapA (uapAD) was fully resistant to 3 mM of allopurinol in media containing hypoxanthine as the sole nitrogen source. In contrast, a single knock-out mutant strain carrying a total deletion of uapC (uapCD), uapC being a gene similar (paralogous) to uapA encoding a secondary uric acidexanthine transporter with additional, but very low-capacity to bind other purines [[37], and unpublished results], was sensitive to allopurinol similarly to a wildtype isogenic control strain. The double knock-out mutant strain carrying total deletions of both uapA and uapC (uapAD uapCD) was resistant to allopurinol, similarly to the uapAD single mutant (Fig. 1A). Knock-out strains related to other transporters known to catalyze the uptake of purines, pyrimidines or nucleosides (e.g AzgA, FurD, FcyB, CntA) [38e40] were also tested and showed that none of them is critical for apparent allopurinol uptake (results not shown). Thus, UapA seems to be the major allopurinol transporter operating at low concentrations of this analogue (<20 mM). Another unknown allopurinol transporter of lower affinity is however genetically suggested by the observation that the uapAD mutant strain becomes sensitive to allopurinol concentrations >20 mM (not shown). We have previously obtained, through classical or reverse genetics, and functionally analyzed a plethora of UapA mutants with

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Fig. 1. UapA is the major transporter mediating sensitivity at low concentrations of allopurinol. (A) Growth tests of isogenic uapAþ uapCþ (wt), uapAD, uapCD and uapAD uapCD strains on nitrate, uric acid (ua), hypoxanthine (hx) or hypoxanthine þ allopurinol (hx þ al) at 37  C. (B) Growth tests of isogenic strains expressing wild-type UapA, UapA-F528A, UapA-T526M, UapA-T526L, UapA-Q408E, UapA-Q408E/T526M, UapA-Q408E/T526L and a negative control strain (uapAD) on uric acid (ua), hypoxanthine (hx), hypoxanthine þ allopurinol (hx þ al), hypoxanthine þ allopurinol þ 3-methylxanthine (hx þ al-3mx) and hypoxanthine þ allopurinol þ 2-thioxanthine (hx þ alþ2tx). Allopurinol concentration is at 3 mM. Hypoxanthine and uric acid are used at 400 mM and sodium nitrate at 10 mM. Competing substrates 3-MX or 2-TX are at 400 mM. Notice that in strains capable of significant UapA-mediated 2-thioxanthine uptake the colour of conidiospores becomes yellow/yellowish. The rationale for this is unrelated to the present work and is described in Ref. [23].

altered specificities [6e10,41,42]. These were obtained in a genetic background lacking UapC, as well as, AzgA which is a major adenine/hypoxanthine/guanine transporter in A. nidulans. This genetic background (uapCD azgAD) allows for the direct assessment of UapA function and specificity by simple growth testing and direct uptake studies. For the present work, selected major specificity mutations of UapA were introduced by standard genetic crossing in an azgAþ background (see Materials and methods) in order to assess their ability to mediate allopurinol uptake by simple growth tests on media containing allopurinol and hypoxanthine as the sole nitrogen source. Results are summarized in Fig. 1B. One of the mutants analyzed, UapA-Q408E, proved of particular interest as it was resistant to allopurinol. Resistance to allopurinol suggested an inability for allopurinol uptake. This mutant could however mediate significant (w30% of wild-type)

uptake of uric acid or xanthine, the two physiological substrates of UapA, and noticeably, could also bind, but could not transport, hypoxanthine, guanine and uracil. This is evident from growth tests, direct uptake measurements and uptake competition assays [6,41]. Mutation Q408E also conferred cryosensitivity to UapA activity [41]. In other words, UapA-Q408E is a modified version of UapA which i) conserves a significant capacity for transporting its physiological substrates (uric acid and xanthine), ii) has acquired the novel property of binding hypoxanthine, guanine and uracil, and iii) has lost its ability to transport allopurinol. Interestingly, amino acid Gln408 has been shown to be one of the two residues directly implicated in strong polar interactions with substrates, the other residue being Glu356. Evidence for this comes through genetic and biochemical studies [8], but also through recent UapA structural modelling and docking approaches [9,10]. Noticeably, all

G. Diallinas / Biochimie 95 (2013) 1755e1764

purine-specific homologues of UapA in the NAT family conserve a Gln/Glu residue and a Glu residue at positions 408 and 356 of UapA, respectively. In contrast, in NAT homologues specific for Lascorbate, the Gln/Glu residue corresponding at position 408 is replaced by a Pro residue, which modifies significantly the architecture and thus the specificity of the substrate binding site [9,10]. These observations suggest that allopurinol requires elements of the major substrate binding site of UapA (the importance of this observation is also discussed later). Other specificity mutants showing altered growth behaviour in respect to allopurinol resistance/sensitivity, were those concerning substitutions of residues T526 and F528. Such mutations (e.g. T526M, T526L, F528A, F528S) have been shown to enlarge the specificity of UapA so that it is able to transport not only its natural substrates, but also all other purines, albeit with low affinity [8,42]. These mutants proved to be hypersensitive to allopurinol (see Fig. 1B and results on allopurinol concentrations <3 mM, not shown). Interestingly, strong genetic, biochemical and structural modelling evidence has suggested that residues T526 and F528 are part of an outward-facing dynamic gate which controls the access of substrates into the major substrate binding site embedded deeper in the transporter body [8,10,42]. These findings suggest that allopurinol translocation might also be controlled directly or indirectly by the outward-facing gate of UapA defined by residues T526 and F528. Last but not least, further evidence for allopurinol being a highaffinity substrate of UapA comes from in vivo competition assays performed in growth tests. In these tests, the negative effect of allopurinol on A. nidulans growth is examined in the presence of excess of non-metabolizable UapA substrates, such as 2thioxanthine or 3-methylxanthine, which cannot be used as nitrogen sources. UapA has a relatively high-affinity for these xanthine analogues (Km 28e63 mM), as previously established by direct kinetic analyses [33]. Excess of both these analogues (400 mM) rescued a wild-type strain from the toxicity of 3 mM allopurinol, in the presence of hypoxanthine as sole nitrogen source (Fig. 1B, two lower panels). A similar observation was also reported for the analogues 2-thiouric acid and 8-thiouric acid, which at concentrations of 500 mM reverse the inhibitory effect of allopurinol when the latter is present at concentrations of the order of 5 mM [35]. These results further support that allopurinol and 2thioxanthine, 2-thiouric acid, 8-thiouric acid and 3methylxanthine (and thus very possibly xanthine and uric acid), all share elements of single or converging substrate translocation pathway(s) in UapA (see also later). In summary, genetic and in vivo transport assays provided unequivocal evidence that high-affinity allopurinol uptake is mediated by UapA and that allopurinol transport requires elements (e.g. Gln408) of the major substrate binding site of UapA [9,10]. However, as it will be shown in the next sections, allopurinol is transported differently from other substrates of UapA. 3.2. UapA-mediated allopurinol uptake occurs through lowcapacity facilitated diffusion Allopurinol uptake was measured in a strain expressing wildtype uapAþ in a genetic background that lack all other carriers mediating purine or pyrimidine transport. An isogenic strain also lacking a functional UapA was used as a negative control (see Materials and methods, Transport assays). Radiolabelled [3H]-allopurinol (10 mM) uptake was measured after 0.5, 1, 2, 4 and 8 min. Extremely low UapA-mediated allopurinol could be detected in the wild-type uapAþ strain, compared to the negative control strain lacking UapA (Fig. 2A). This increase in allopurinol uptake was linear for 2 min. Surprisingly, low level radiolabelled allopurinol

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uptake was not inhibited by excess non-radiolabelled allopurinol or by Hþ uncouplers, such as CCCP or DCCP (Fig. 2B and C). In contrast, radiolabelled allopurinol uptake was poorly inhibited by excess xanthine or uric acid (40e55% competition, Fig. 2C). These results are best accounted by low-capacity UapA-mediated transport of allopurinol through facilitated diffusion. Interestingly, all other known substrates of UapA are transported by a concentration saturable Hþ symport mechanism, rather than facilitated diffusion. Unfortunately, the very low levels of UapA-mediated allopurinol uptake detected herein and the low specific activity of commercially available allopurinol (w30-fold lower than the usual specific activity of xanthine or other purines) did not allow further kinetic analysis of this transport activity, despite efforts employing specific UapA mutants, such as UapA-F528S or UapA-F528A, which are hypersensitive to allopurinol in growth tests. 3.3. Allopurinol transport does not elicit UapA turnover by endocytosis We have recently established that substrate transport elicits stimulation of UapA ubiquitination, endocytosis and turnover by sorting into the MVB/vacuolar pathway [43,44]. This phenomenon is elicited by all true substrates of UapA, including those of moderate affinities, but is not triggered by analogues that act only as ligands (e.g. 3-methylxanthine). We have provided compelling genetic and biochemical evidence that the substrate-elicited endocytosis is due to enhanced exposure of a C-terminal domain of UapA to the ubiquitination machinery, through conformational changes associated with substrate transport [43,44]. Here we investigated whether allopurinol produces the same effect using a strain expressing UapA tagged with GFP (see Table 1). Fig. 2D shows that the presence of allopurinol does not elicit UapA endocytosis and turnover. This result suggests that the mechanism of allopurinol transport by UapA is not the same as that for all other substrates. 3.4. The presence of external allopurinol stimulates UapA-mediated xanthine transport Radiolabelled xanthine (0.2 mM) initial transport rates were measured in the presence or absence of 1000-fold (200 mM) excess allopurinol in isogenic strains expressing wild-type UapA, UapA-Q408E and UapA-F528S, as well as, in strain expressing wild-type UapA in the hx genetic background. All strains expressed UapA at the same level as this was measured by western blots. The hx mutant lacks the major enzymes necessary for hypoxanthine or allopurinol catabolism (HxA and HxnS) and shows only minimal xanthine catabolism through XanA (see Materials and methods, Growth media, strains, chemicals and [30e32]). Fig. 3A shows two unexpected results. First, excess allopurinol did not compete with radiolabelled xanthine uptake. Second, allopurinol led to an increase in radiolabelled xanthine uptake in strains expressing wild-type UapA (both in hx and hxþ backgrounds) or mutant UapA-F528S, but not in mutant UapAQ408E. The actual xanthine transport capacities were: 174% in UapA, 202% in UapA-F528S, 201% in UapA/hx and 85e86% in UapA-Q408E, when the corresponding rate for each strain in the absence of allopurinol is taken as 100%. The fact that an increase in UapA-mediated xanthine transport by allopurinol was also observed in a strain incapable of metabolizing allopurinol (hx) dismissed the possibility that the increase in UapA-mediated xanthine transport is through an indirect metabolic effect of allopurinol catabolism. In addition, the fact that mutant UapAQ408E, which apparently has lost the ability to bind and transport allopurinol is the only mutant tested not showing an

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Fig. 2. UapA-mediated allopurinol uptake. (A) Time course of [3H]-allopurinol (2 mM) uptake in uapAþ and uapAD isogenic strains. (B) Concentration dependence of [3H]-allopurinol initial uptake rate (1 min) in the uapAþ strain (uapCD). (C) UapA-mediated allopurinol uptake rate (1 min) in the presence of specific inhibitors/competitors. UapA-dependent allopurinol uptake in the absence of inhibitors/competitors is taken as 100%. Al, 400 mM allopurinol; Xa, 400 mM xanthine; Ua, 400 mM uric acid; Hx, 400 mM hypoxanthine; DCCP, 100 mM dinitrophenol; CCCP, 30 mM carbonylcyanide m-chlorophenylhydrazone. (D) Epifluorescence microscopy showing the effect of xanthine or allopurinol (200 mM, 4 h) on UapA-GFP localization the in the plasma membrane. Notice that only xanthine leads to disappearance of UapA-GFP from the plasma membrane and appearance of GFP-labelled vacuoles and endosomes (for details see Ref. [44]).

increase in xanthine transport in the presence of allopurinol, provided strong evidence that the allopurinol effect is UapAdependent. 3.5. The allopurinol effect on xanthine uptake is concentrationdependent and saturable In order to examine further the effect of excess allopurinol on UapA-mediated xanthine transport, radiolabelled xanthine (0.2 mM) uptake rates were measured in UapA and UapA-F528S strains under different allopurinol concentrations ranging from 0.1 mM to 400 mM. Fig. 3B shows only the result with wild-type UapA, but for both strains a similar result was obtained. An increase in the initial uptake rate of radiolabelled xanthine reached a max plateau (174e189% of the rate in the absence of allopurinol) only at concentrations of allopurinol approaching 100 mM, but became apparent at 5 mM of allopurinol (128%). Interestingly, 5 mM is close to the standard concentration of allopurinol used in growth tests to score the effect of this analogue (3 mM). To further examine the effect of allopurinol on xanthine transport, Km values of UapA for xanthine were measured in the absence and presence of three allopurinol concentrations, 5, 20 or 50 mM (for details see Materials and methods). The measurements obtained, summarized in Fig. 3C, led to the estimation of Km values for xanthine shown in an insert in the same figure. It is thus shown that increasing concentrations of allopurinol led to a progressive reduction of Km values for xanthine binding. The maximum reduction in the Km for xanthine obtained at 100 mM allopurinol was 2.8-fold (from 6.7 to 2.4 mM).

3.6. Internally accumulated allopurinol or xanthine stimulates influx of radiolabelled xanthine through an apparent exchange mechanism The concentration-dependent allopurinol stimulation of the xanthine uptake might be rationalized by considering that allopurinol binds to a cryptic site and elicits a specific, previously unnoticed, allosteric effect on UapA structure and activity. Alternatively, the phenomenon observed might be explained by considering that internally accumulated allopurinol effluxes back, thus stimulating a hetero-exchange process that accelerates external xanthine influx. Whilst an allosteric conformational change elicited by allopurinol binding in a cryptic site cannot be ruled out, no further rigorous evidence could be obtained in favour of this hypothesis (see also substrate docking studies described later). In contrast, the alternative explanation of accelerated influx of xanthine elicited through an exchange with allopurinol accumulating cytoplasmically could be easily tested. Conidiospores of a wild-type strain (hxþ) or a purine/allopurinol non-metabolizing mutant (hx) were preloaded with 100 mM of allopurinol or xanthine and tested for stimulation of radiolabelled xanthine uptake, in the same way as with externally added allopurinol. Fig. 4A shows that preloading of the cells with either 100 mM xanthine or allopurinol led to a significant stimulating effect on radiolabelled xanthine uptake, especially in the non-metabolizing mutant (250%), but also in the wildtype (200%). In fact, the stimulation of preloaded internal allopurinol is even higher than that elicited by external allopurinol (compare results in Figs. 3A and 4A). Finally, as with external allopurinol, the effect of internally accumulated allopurinol was also concentration-dependent. UapA-dependent xanthine uptake

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Fig. 4. Cytoplasmically accumulated allopurinol or xanthine elicit a concentrationdependent increase in UapA-mediated xanthine transport. (A) Radiolabelled [3H]xanthine (0.2 mM) uptake capacity, expressed as % uptake rate, in cells preloaded with either allopurinol (Al) or xanthine (Xa) at 100 mM, or cells not preloaded with substrates (for details see Materials and methods, Transport assays). Measurements were performed in hxþ and hx isogenic strains. hx cells are blocked in purine or allopurinol catabolism (see text). (B) Concentration dependence of the effect of cytoplasmically accumulated allopurinol on UapA-mediated radiolabelled [3H]-xanthine (0.2 mM) uptake. Results shown represent averages of three experiments with practically no standard deviation.

stimulation was evident at 20 mM, but reached its maximum at concentrations close to 100 mM of allopurinol (Fig. 4B). These results provided strong evidence in favour of an influx/efflux exchange model rather than an explanation based on allostery. 3.7. Direct evidence that UapA is capable of performing substrate exchange Fig. 3. External allopurinol elicits a concentration-dependent increase in UapAmediated xanthine transport. (A) Radiolabelled [3H]-xanthine (0.2 mM) uptake capacity, expressed as % initial uptake rate, in the absence or presence of 1000-fold excess non-labelled xanthine (Xa), uric acid (Ua) or allopurinol (Al). Measurements were performed in isogenic strains expressing wild-type UapA, UapA-F528S, UapAQ408E and in a strain lacking xanthine dehydrogenase (hxAD). (B) Radiolabelled [3H]xanthine (0.2 mM) uptake capacity measured in the presence of increasing concentrations of non-labelled allopurinol. For details see Materials and methods. (C) Radiolabelled xanthine initial uptake rates measured in the absence or presence of 5, 20 or 100 mM allopurinol. From these measurements, Km values of UapA for xanthine were estimated, as described in Materials and methods, and shown within an inserted table. Results shown represent averages of several experiments with standard deviation <20%.

In an independent experiment, and in order to directly show that there is an exchange between external and internal substrates when these are present in trans, hx cells were preloaded with radiolabelled xanthine (0.2 mM), washed, xanthine or allopurinol (100 mM) was added for 5 min, and radioactivity was measured in both the cells and the external medium. Fig. 5 shows that excess external xanthine led to a significant (20%) efflux of radiolabelled xanthine back in the medium. However, excess external allopurinol did not lead to an analogous reflux of radiolabelled xanthine. Interestingly, samples not loaded with xanthine or allopurinol also did not show any reflux of preloaded internal [3H]-xanthine. This result shows that UapA can efflux back its major physiological

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Fig. 5. Cytoplasmically accumulated non-radiolabelled xanthine elicits [3H]-xanthine efflux. Cells preloaded with radiolabelled [3H]-xanthine (0.2 mM) and washed were loaded with 100 mM xanthine or allopurinol, or not loaded with substrates, and subsequently allowed 5 min at 37  C before measuring radioactivity in the supernatant (sup) and in the cells (for details see Materials and methods, Transport assays).

substrate (xanthine) only when this is accumulated in relative excess, as seems to be the case in the hx mutant. A similar but much lower reflux was measured in the wild-type strain (results not shown). In contrast however, allopurinol influx and its hypothesized efflux seemed not to trigger detectable co-efflux of radiolabelled xanthine, even in the hx genetic background. This might be due to very low affinity internal xanthine binding site, given that radiolabelled xanthine is used at rate limiting concentration. This explanation is independent of whether there are distinct internal efflux gates for allopurinol and xanthine. An apparent substrate exchange process, as that observed with UapA, can be mechanistically rationalized if we consider that efflux of excess substrates increases the ratio of outward/inward-facing conformers of UapA, and thus more UapA molecules become transport-ready, as is the case with typical antiporters. Alternatively, a bona fidae exchange process might occur within overlapping channel-like trajectories made of several secondary substrate biding sites [14,15]. This work does not intend to discriminate between these two mechanisms. 3.8. Docking approaches show that allopurinol transport by UapA occurs through a non-canonical trajectory We have recently constructed a 3D model of UapA, based on the crystal structure of the uracil transporter UraA of Escherichia coli,

and used this model to identify a major substrate binding site of xanthine, using Molecular Mechanics (MM) simulations, quantitative structureeactivity relationship (SAR) and docking approaches [9,10]. The model proposed for UapA-xanthine binding is strongly supported by re-evaluation of a plethora of available UapA mutations [10]. Furthermore, the UapA-xanthine model is also fully compatible with the binding of several moderate affinity substrates of UapA and rationalizes why other purines, such as hypoxanthine, guanine or adenine are not recognized by UapA [10]. Here, similar approaches for identifying possible UapA-allopurinol interactions were employed. No evidence was obtained for allopurinol docking, either within the major substrate binding site or in the extended substrate translocation trajectory, as this was defined with xanthine [10]. To test the validity of the negative results obtained with allopurinol docking in UapA, oxypurinol docking was also tested. Oxypurinol is an allopurinol analogue (see Fig. 6), which is also a UapA substrate with a Ki value of 103 mM [33]. Interestingly, unlike allopurinol, excess oxypurinol competes xanthine uptake and does not elicit a co-operative effect on xanthine uptake. In other words, oxypurinol, despite its similarity to allopurinol, behaves like a typical UapA substrate. Fig. 6 shows that oxypurinol binds within the major substrate binding site of UapA, in an orientation that is similar, but not identical to xanthine. In particular, groups N1eH, C2]O, C6]O, N3eH and N8eH of oxypurinol interact with the polar side chains of residues Q408 and E356, and the backbone of A407 and F155, that is, with the same residues interacting with xanthine and other xanthine analogues which are substrates of UapA. This result shows that the in silico approaches used herein and previously [10] are sufficiently sensitive to detect docking poses of all ‘canonical’ UapA substrates, such as oxypurinol, or even those with moderate affinities, such as 6-thioxanthine [10]. The differences in the allopurinol and oxypurinol chemical structures concern the presence of ]O2 group at C2 in oxypurinol and the protonation state of N3, N7 and N8 at neutral pH (see Fig. 6). The preferred tautomeric form of allopurinol contains non-protonated N3, N8 and protonated N9eH, whereas that of oxypurinol the opposite (N3eH, N8eH and N9) [33]. These differences seem to trigger an alternative translocation pathway for allopurinol transport. The absence of in silico detectable allopurinol docking sites in the modelled structure of UapA obviously does not mean that allopurinol does not bind to UapA in vivo. This apparent negative result rather supports that allopurinol binds differently from all other UapA substrates tested, including the very similar analogue oxypurinol. In line with his idea, allopurinol seems to be transported by facilitated diffusion mechanism employing a ‘sliding down’ trajectory through several docking points, rather than a Hþ symport active mechanism characterized by abrupt alternating

Fig. 6. Relative docking of oxypurinol (OXY) and xanthine (XAN) in UapA. The right panel shows the superimposition of docking poses of Xan and Oxy. Notice the slight shift in the orientation of the oxypurinol ring compared to that of xanthine, which results in partially different interactions with the same four amino acid residues constituting the UapA substrate binding site [13]. Xanthine interacts with UapA through H bonds of N1eH, C2]O, N7eH and N9, whereas oxypurinol interacts through H bonds of N1eH, C2]O, C6]O, N8 and possibly N3eH. For details on the docking method used see Materials and methods.

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conformational changes, exposing a single major substrate binding site extra- or intracellularly [14,15]. The fact that we have not detected any site for allopurinol binding through in silico docking, although in vivo evidence is suggestive for binding at a concentration range >5 mM, might also be due to the fact the docking approaches have been performed with a cytoplasm-facing UapA topology based on the crystal structure of UraA, obtained in the presence of its physiological substrate (uracil). If UapA functions through a sequential substrate-induced fit mechanism, nonphysiological substrates or substrate analogues such as allopurinol, which seem to bind through a non-canonical manner, might elicit the formation of alternative substrate translocation trajectories, not evident in structural models built with physiological substrates. The crystal or modelled structure of UapA in an outward-facing topology obtained in the absence of substrates might prove ideal as a template for searching allopurinol binding site(s). This however is not available at present. 4. Conclusions Six major facts are described in this work. First, UapA can function as an allopurinol transporter. This is strongly supported not only by the null allele, but also by specific functional alleles like Q408E, which alter the effect of allopurinol in vivo. Second, UapA-mediated allopurinol transport is of low-capacity, non-saturable and Hþ-independent, strongly suggesting that it occurs via facilitated diffusion. Third, excess allopurinol does not compete with UapAmediated uptake of all other canonical substrates. Fourth, both external or internally accumulated allopurinol leads to accelerated xanthine binding and transport in a concentration-dependent manner. Fifth, allopurinol docking cannot be detected. Lastly, allopurinol des not elicit accelerated UapA turnover by endocytosis. The most reasonable explanations for these findings, some of which are atypical for Hþ symporters, can be summarized as follows (see also Fig. 7). Xanthine and allopurinol use different but, functionally and/or structurally, overlapping translocation trajectories. The existence of different trajectories and mechanisms of transport are supported by the fact that, first, allopurinol does not dock in the canonical binding site of all other UapA substrates, including those of moderate affinity, and second, the observation that allopurinol does not elicit UapA endocytosis. The convergence of the two trajectories is supported by genetics and non-reciprocal

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inhibitions. The non-reciprocity of substrate inhibitions probably also reflects the different mechanisms of translocation of xanthine (Hþ symport) versus allopurinol (diffusion-leakage), the former requiring critical conformational changes that the latter might not employ. Stimulation of [3H]-xanthine binding and transport thus occurs through an exchange mechanism, when non-labelled allopurinol or xanthine accumulate cytoplasmically. Allostery has been shown to operate in monomeric enzymes [45e47] (cation symporters are believed to function as monomers) and moreover a recent report has proposed that the human serotonin transporter (hSERT) is allosterically stimulated by a chemically synthesized ligand (ATM7), especially when serotonin was at rate limiting concentrations [48]. In that case, docking studies identified the binding of this ligand in a so-called allosteric pocket, where mutations can mimic the effect of chemical allostery. Interestingly, ATM7 also elicited an ‘ecstasy’-dependent reflux of serotonin. The authors of this work proposed that the ligand mechanistically stabilizes an outward-facing conformation of SERT. In their work, however, there are no experiments addressing whether the apparent ‘allosteric’ ligand ATM7 is acting analogously to allopurinol stimulating the uptake of physiological substrates by an exchange process. Although the allopurinol effect reported in this work could also, in principle, be explained by assuming a cryptic allosteric binding site specific for allopurinol, the preloading experiments performed herein and the lack of direct evidence for allostery, make this assumption less probable. The atypical phenomenon described herein is basically rationalized by proposing the existence of substrate-specific alternative mechanisms and partially overlapping translocation trajectories in a single transporter. Interestingly, the existence of alternative and partially overlapping substrate trajectories has also been shown in the mammalian glucose transporter GLUT1 [14,15,49]. In the case of UapA, where a plethora of functional and altered specificity mutations are already available or new mutations can be easily selected or constructed, it will be interesting and feasible to try to understand the details of the exchange mechanism and identify the molecular elements of the putative alternative substrate translocation pathways. In this direction further more flexible docking studies, particularly under the light of expected novel crystal structures of the NATs, and the employment of models simulating the exchanges mechanism, will be invaluable. Apart from its obvious

Fig. 7. Schematic outline of putative allopurinol and xanthine translocation trajectories and mechanisms of transport. External xanthine (X) and allopurinol (Al) use different gates but follow converging pathways for translocation. Genetic support for convergence of the two translocation pathways comes from the simultaneous effect of mutation Q408E, a mutation concerning a major residue of the substrate binding site, on xanthine transport and on the allopurinol effect. (A) Xanthine, when present at concentrations approaching saturation, is transported (thick green arrow) via an active Hþ symport mechanism, which blocks allopurinol transport via diffusion. Low level xanthine reflux (fine dashed arrow) is observed only when the internal concentration is significantly higher than the external. (B) When allopurinol is in excess and xanthine at rate limiting concentrations, allopurinol diffuses (blue arrow) through ‘xanthine free’ UapA molecules and accumulates cytoplasmically. Internal allopurinol refluxes (dashed blue arrow) and accelerates the influx of rate limiting xanthine (green arrow), probably by increasing the ratio of outward- to inward-facing conformers of UapA, or by direct a bona fidae exchange within the transporter. The scheme also shows distinct internal efflux gates which are speculative.

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importance in understanding the details of a basic biological function related to solute transport, identifying and mechanistically understanding atypical and complex phenomena in transporters should have an important impact in the area of molecular pharmacology and in the screening of large chemical libraries. Acknowledgements I am mostly grateful to the reviewer who suggested the explanation based on an exchange process rather than allostery, which eventually proved to be fully supported by the results presented in this work. I thank Marina Koukaki for performing the initial kinetic experiments and Vasso Kosti for help in docking studies, performed at the facilities of Emmanuel Mikros laboratory in the School of Pharmacy. Finally, I thank Claudio Scazzocchio, George Zacharioudakis, Richard Naftalin and Ron Kaback for several discussions concerning this work, Emilia Krypotou, Mayia Karachaliou and Sotiris Amillis for their comments and help in preparing and correcting the manuscript. References [1] I. Smirnova, V. Kasho, H.R. Kaback, Lactose permease and the alternating access mechanism, Biochemistry 50 (2011) 9684e9693. [2] L.R. Forrest, R. Krämer, C. Ziegler, The structural basis of secondary active transport mechanisms, Biochim. Biophys. Acta 1807 (2011) 167e188. [3] H.R. Kaback, I. Smirnova, V. Kasho, Y. Nie, Y. Zhou, The alternating access transport mechanism in LacY, J. Membr. Biol. 239 (2011) 85e93. [4] X. Jiang, L. Guan, Y. Zhou, W.X. Hong, Q. Zhang, H.R. Kaback, Evidence for an intermediate conformational state of LacY, Proc. Natl. Acad. Sci. U.S.A. 109 (2012) 698e704. [5] M.G. Madej, S.N. Soro, H.R. Kaback, Apo-intermediate in the transport cycle of lactose permease (LacY), Proc. Natl. Acad. Sci. U.S.A. 109 (2012) E2970eE2978. [6] M. Koukaki, A. Vlanti, S. Goudela, A. Pantazopoulou, H. Gioule, S. Tournaviti, G. Diallinas, The nucleobaseeascorbate transporter (NAT) signature motif in UapA defines the function of the purine translocation pathway, J. Mol. Biol. 350 (2005) 499e513. [7] A. Vlanti, S. Amillis, M. Koukaki, G. Diallinas, A novel-type substrate-selectivity filter and ER-exit determinants in the UapA purine transporter, J. Mol. Biol. 357 (2006) 808e819. [8] I. Papageorgiou, C. Gournas, A. Vlanti, S. Amillis, A. Pantazopoulou, G. Diallinas, Specific interdomain synergy in the UapA transporter determines its unique specificity for uric acid among NAT carriers, J. Mol. Biol. 382 (2008) 1121e 1135. [9] V. Kosti, I. Papageorgiou, G. Diallinas, Dynamic elements at both cytoplasmically and extracellularly facing sides of the UapA transporter selectively control the accessibility of substrates to their translocation pathway, J. Mol. Biol. 397 (2010) 1132e1143. [10] V. Kosti, G. Lambrinidis, V. Myrianthopoulos, G. Diallinas, E. Mikros, Identification of the substrate recognition and transport pathway in a eukaryotic member of the nucleobaseeascorbate transporter (NAT) family, PLoS One 7 (2012) e41939. [11] G. Rudnick, Cytoplasmic permeation pathway of neurotransmitter transporters, Biochemistry 50 (2011) 7462e7475. [12] T. Shimamura, S. Weyand, O. Beckstein, N.G. Rutherford, J.M. Hadden, D. Sharples, M.S. Sansom, S. Iwata, P.J. Henderson, A.D. Cameron, Molecular basis of alternating access membrane transport by the sodiumehydantoin transporter Mhp1, Science 328 (2010) 470e473. [13] G. Diallinas, Biochemistry. An almost-complete movie, Science 322 (2008) 1644. [14] R.J. Naftalin, Alternating carrier models of asymmetric glucose transport violate the energy conservation laws, Biophys. J. 95 (2008) 4300e4314. [15] R.J. Naftalin, Reassessment of models of facilitated transport and cotransport, J. Membr. Biol. 234 (2010) 75e112. [16] P. Pacher, A. Nivorozhkin, C. Szabó, Therapeutic effects of xanthine oxidase inhibitors: renaissance half a century after the discovery of allopurinol, Pharmacol. Rev. 58 (2006) 87e114. [17] C. Scazzocchio, F.B. Holl, A.I. Foguelman, The genetic control of molybdoflavoproteins in Aspergillus nidulans, allopurinol-resistant mutants constitutive for xanthine-dehydrogenase, Eur. J. Biochem. 36 (1973) 428e445. [18] M.S. Lipkowitz, Regulation of uric acid excretion by the kidney, Curr. Rheumatol. Rep. 14 (2012) 179e188. [19] J.S. Cameron, F. Moro, H.A. Simmonds, Gout, uric acid and purine metabolism in paediatric nephrology, Pediatr. Nephrol. 7 (1993) 105e118. [20] J. Mishra, A. Saxena, S. Singh, Chemotherapy of leishmaniasis: past, present and future, Curr. Med. Chem. 14 (2007) 1153e1169. [21] M.J. Natto, L.J. Wallace, D. Candlish, M.I. Al-Salabi, S.E. Coutts, H.P. de Koning, Trypanosoma brucei: expression of multiple purine transporters prevents the development of allopurinol resistance, Exp. Parasitol. 109 (2005) 80e86.

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