Trypanosoma brucei: A survey of pyrimidine transport activities

Experimental Parasitology 114 (2006) 118–125 www.elsevier.com/locate/yexpr

Trypanosoma brucei: A survey of pyrimidine transport activities Simon Gudin a,1, Neils B. Quashie a, Denise Candlish a, Mohammed I. Al-Salabi a, Simon M. Jarvis b, Lisa C. Ranford-Cartwright a, Harry P. de Koning a,¤ a

Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Biomedical Research Center, Glasgow G12 8TA, UK b University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK Received 24 November 2005; received in revised form 23 February 2006; accepted 24 February 2006 Available online 18 April 2006

Abstract Purine uptake has been studied in many protozoan parasites in the last few years, and several of the purine transporters have been cloned. In contrast, very little is known about the salvage of preformed pyrimidines by protozoa, and no pyrimidine transporters have been cloned, yet chemotherapy based on pyrimidine nucleobases and nucleosides has been as eVective as purine antimetabolites in the treatment of infectious and neoplastic disease. Here, we surveyed the presence of pyrimidine transporters in Trypanosoma brucei brucei. We could not detect any mediated uptake of thymine, thymidine or cytidine, but identiWed a very high-aYnity transporter for cytosine, designated C1, with a Km value of 0.048 § 0.009 M. We also conWrmed the presence of the previously reported U1 uracil transporter and found it capable of mediating uridine uptake as well, with a Km of 33 § 5 M. A higher-aYnity U2 uridine transporter (Km D 4.1 § 2.1 M) was also identiWed, but eYciency of the C1 and U2-mediated transport was low. Pyrimidine antimetabolites were tested as potential trypanocidal agents and only 5-Xuorouracil was found to be eVective. This drug was eYciently taken up by bloodstream forms of T. b. brucei. © 2006 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Uridine transporter; Pyrimidine salvage; Cytosine transporter; Uracil transporter; 5-Fluorouracil

1. Introduction Because protozoa lack the ability to synthesize the purine ring de novo, they have developed a variety of eYcient salvage pathways to acquire preformed purines from their host. These purine salvage pathways are essential for the survival of the parasite and therefore have been studied extensively (De Koning et al., 2005). It has been well documented that some of these salvage pathways are also essential for the internalization and activity of various antiparasitic agents (De Koning, 2001; el Kouni, 2003; Mäser et al., 2003). To date, eight plasma membrane purine transporters have been identiWed in the various stages of Trypanosoma brucei brucei, three in the procyclic form, four in the long-slender bloodstream form (De Koning et al., *

1

Corresponding author. Fax: +44 141 3303753. E-mail address: [email protected] (H.P. de Koning). Present address: Université Joseph Fourier, Grenoble, France.

0014-4894/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2006.02.018

2005), and one in the short-stumpy form (Sanchez et al., 2004). Many purine analogues with antiparasitic activity have been identiWed (e.g., Bhattacharya et al., 1990; Bressi et al., 2001; Kicska et al., 2002; Rodenko et al., 2006), but translation into clinical use has, to date, been poor. Allopurinol, a nucleobase analogue, is sometimes used against human leishmaniasis (Das et al., 2001; Momeni et al., 2002) and has also demonstrated some activity against T. b. brucei (Natto et al., 2005). However, there has been a spate of recent patents on purine and pyrimidine-based antiparasitics that indicates renewed interest in this Weld (Lawton, 2005). In marked contrast to purines, most protozoa, with the exception of Trichomonas vaginalis, Tritrichomonas foetus, and Giardia lamblia, are able to synthesize pyrimidines (Randolph et al., 1995) and few studies have addressed pyrimidine uptake in protozoa, except where pyrimidines and purines share the same transporters: many purine transporters are competitively inhibited by some pyrimidines.

S. Gudin et al. / Experimental Parasitology 114 (2006) 118–125

For example, the adenosine transporters NT1 of Crithidia fasciculata, TgAT2 of Toxoplasma gondii, and T1 of Leishmania donovani amastigotes are competitively inhibited by uridine, thymidine, and cytidine (De Koning et al., 2003, 2000; Ghosh and Mukherjee, 2000), but, in most cases, transport of the potential pyrimidine permeants was not further investigated. However, Vasudevan et al. (1998) established a Km for [3H]uridine for the NT1.1 adenosine/ uridine transporter of L. donovani promastigotes. In addition, some broad-speciWcity nucleoside and nucleobase transporters are also able to transport pyrimidines. Giardia intestinalis have been reported to possess three facilitative transporters for purines and/or pyrimidines: the type 1 thymidine transporter (Davey et al., 1991), the type 2 deoxycytidine/adenosine transporter (Davey et al., 1992), and the type 3 adenine/thymine transporter (Ey et al., 1992). A Wrst exclusive pyrimidine transporter in the kinetoplastidae, designated TbU1, was characterized in 1998 and displayed a very high level of selectivity for uracil, as well as submicromolar aYnity (De Koning and Jarvis, 1998). Recently a similar transporter, LmU1, was found in Leishmania major promastigotes (Papageorgiou et al., 2005). Interestingly, the well-known anticancer drug 5-Xuorouracil, a competitive inhibitor of these uracil transporters, demonstrated promising anti-leishmanial activity and further characterization of pyrimidine uptake could provide new leads for drug development. TbU1 is the only speciWc pyrimidine transporter identiWed in T. b. brucei and the aim of this work is to investigate the uptake of all the other pyrimidine nucleobases and nucleosides to build a complete picture of pyrimidine uptake and metabolism in T. b. brucei. We Wnd that not all pyrimidines are salvaged by this parasite, and pyrimidine uptake in procyclic trypanosomes is generally much less eYcient than purine uptake. 2. Materials and methods 2.1. Materials Biochemicals, mineral oil, and diminazene acetutate were all from Sigma, as were most non-radioactive nucleosides and nucleobases, except 2-thiouracil (ICN), 5-bromouracil, and 5-bromouridine (Avocado Research Chemicals, Heysham, UK), 2-thiouridine and 3⬘-deoxyuridine (Trilink Biotechnologies, San Diego, CA), 4-thiouridine, 4-thiouracil, 3-deazauracil, 1-methyluracil, and 2,4-dithiouracil (Aldrich). Dibutylphthalate was from BDH. Radiolabelled nucleosides and bases ([5-3H]cytidine (21.5 Ci/mmol), [5-3H]cytosine (22.8 Ci/mmol), [methyl3 H]thymidine (60.3 Ci/mmol), [methyl-3H]thymine (61 Ci/ mmol), and [6-3H]5-Xuorouracil (20 Ci/mmol)) were obtained from Moravek Biochemicals Inc. (Brea, CA) or from Amersham Pharmacia Biotech UK Ltd ([5,6-3H] uridine, 37 Ci/mmol). Cell culture reagents were from Gibco. Fetal bovine serum was purchased from BioSera (Ringmer, East Sussex, UK).

119

2.2. Trypanosome culture Procyclic forms of T. b. brucei strain 427 were grown at 25 °C as described by Brun and Schonenberger (1979) in SDM-79 medium supplemented with 10% (v/v) fetal bovine serum. Bloodstream forms of T. b. brucei strain 427 were grown at 37 °C in a 5% CO2 atmosphere in HMI-9 medium (Invitrogen) supplemented with 10% fetal bovine serum. Cells (procyclic or bloodstream forms) at the midlogarithmic stage of growth were harvested and washed twice in assay buVer (33 mM Hepes, 98 mM NaCl, 4.6 mM KCl, 0.3 mM CaCl2, 0.07 mM MgSO4, 5.8 mM NaH2PO4, and 14 mM glucose, pH 7.3). Cells were resuspended in this buVer at approximately 108 cells/ml prior to use in transport experiments. Cell counts were performed using a haemocytometer. 2.3. Transport assays Transport assays with procyclic or bloodstream form trypanosomes were performed at room temperature (»22 °C), essentially as described previously for uracil uptake (De Koning and Jarvis, 1998). Procyclic cells were harvested by centrifugation washed into assay buVer (108 cells/ml). Bloodstream forms for transport assays were grown in adult female Wistar rats, collected by exsanguination and isolated as described by anion exchange chromatography prior to washing into the same assay buVer as procyclics (Wallace et al., 2002). One hundred microliters of an inhibitor/radiolabel mixture was added to 1.5 ml microcentrifuge tubes, which contained 300 l of oil mixture [di-n-butylphthalate (BDH) and mineral oil (Sigma); 7:1 v/v]. One hundred microliters of cells were added to each microcentrifuge tube and incubated for a predetermined time, after which 1 ml of ice-cold stop-solution (4000 M of unlabelled permeant in assay buVer) was added to preclude further mediated uptake. The cells were then separated from the extracellular label by centrifugation (12,000g, 30 s). The microcentrifuge tubes were Xash frozen in liquid nitrogen. The bottom of the tubes, containing the pellets, were cut oV using tube cutters and collected in scintillation vials to which 250 l of SDS 2% was added. The pellets were left for 20 min at room temperature, after which 3 ml of scintillation Xuid [Optiphase HiSafe III (Perkin-Elmer)] was added to each vial and radiation was determined in a liquid scintillation counter. To correct for non-speciWc association of radiolabel with the cell pellets, parallel triplicate determinations of uptake in the presence of saturating concentrations of unlabelled permeants were included in each experiment. These values were subtracted to yield saturable, mediated uptake. 2.4. Drug sensitivity assays A number of pyrimidine antimetabolites were tested for trypanocidal activity against bloodstream forms in culture, using the standard Alamar blue protocol, exactly as

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described previously (Wallace et al., 2002). BrieXy, cells were cultured in 96-well plates in HMI-9 medium (Invitrogen), in the presence or absence of serial dilutions of test compound. After 48 h of culture Alamar Blue reagent (BioSource, Camarillo, CA) was added to 10% (v/v) and the plates incubated for a further 24 h at 37 °C and Xuorescence determined in a Perkin-Elmer LS55B Xuorimeter. 2.5. Data analysis All experiments were performed in triplicate and repeated at least twice. Errors given in the text and Wgures are standard errors. Graphs shown are of representative experiments performed in triplicate. Full dose–response curves with a minimum of three points over a suitable range, were Wtted using the Prism software package (Graphpad). The 50% inhibition value, IC50, was established from the sigmoidal inhibition proWles of the test compounds. This value was then used in the Cheng–PrusoV equation to determine the inhibition constant: Ki D IC50/ [1 + (L/Km)] where L is the permeant concentration and Km the Michaelis–Menten constant of the permeant. 3. Results 3.1. Cytosine and cytidine uptake Uptake of [3H]cytosine at 0.1 M was linear during 150 s, with a rate of 0.006 § 0.0004 pmol(107 cells) ¡1 s¡1 (r2 D 0.97) but barely detectable in the presence of 1000 M of unlabelled cytosine (0.00016 § 0.00004 pmol(107 cells)¡1 s¡1), which may represent the rate of cytosine diVusion (Fig. 1A). Unusually, the linear regression of cytosine uptake intercepted positively with the ordinate when uptake of zero seconds in the presence of 1000 M of unlabelled cytosine was taken as zero transport. As the zero second point of 0.1 M [3H]cytosine is part of the linearity over 150 s, it follows that the discrepancy is due to externally bound radiolabel rather than uptake, which could be displaced by high levels of unlabelled cytosine. Subsequent experiments with [3H]cytosine were performed using 90 s incubations, well within the linear phase of uptake.

A

Inhibition of [3H]cytosine uptake (0.03 M) by unlabelled cytosine over a range of 0.01–1000 M showed that cytosine uptake was saturable and conformed to Michaelis–Menten kinetics (Fig. 1B), indicating the presence of a saturable transporter with a Km value of 0.048 § 0.009 M and a Vmax of 0.0050 § 0.0029 pmol (10 7 cells)¡1 s¡1 (n D 4). Hill coeYcients were consistent with the presence of a single high-aYnity transporter for cytosine, designated C1. The eVects of cytidine and other pyrimidines were tested against the uptake of 0.03 M [3H]cytosine in order to determine the speciWcity of the cytosine transporter. Cytidine and uracil inhibited cytosine uptake with respective Ki values of 0.42 § 0.16 and 0.36 § 0.06 M (n D 3) (Fig. 1B). Thymine inhibited almost 50% of cytosine uptake at 1000 M, whereas it was virtually ineVective at 100 M (n D 3). The high aYnity of this transporter for cytosine, cytidine, and uracil are consistent with a single, high-aYnity transporter distinct from the U1 uracil transporter of T. b. brucei procyclics, which displays a high selectivity for uracil and low aYnity for cytosine and cytidine (De Koning and Jarvis, 1998). In Wve out of six separate experiments we could detect no signiWcant uptake of 1 or 10 M [3H]cytidine over 300–600 s by T. b. brucei procyclics (slope not signiWcantly diVerent from zero; P > 0.05, F test) and not aVected by the presence of 1000 M of unlabelled cytidine or cytosine (not shown). We therefore conclude that little or no cytidine uptake occurs in T. b. brucei procyclics, and inhibition of cytosine uptake by cytidine is likely to represent inhibition of cytosine transport by C1. 3.2. Thymine and thymidine uptake Uptake of [3H]thymine and [ 3H]thymidine at up to 10 M of label by T. b. brucei procyclic cells was linear for at least 5 min with a rate of 0.0010 § 0.0005 pmol(107 cells) ¡1 s¡1 (n D 2) for thymine and 0.0006 pmol(107 cells)¡1 s¡1 (n D 1) for thymidine. However, uptake was not signiWcantly reduced in the presence of 1000 M of unlabelled thymine or thymidine (P > 0.05; comparison of correlation coeYcients).

B

150

Cytosine Uptake (% of control)

(pmol(107 cells)-1)

Cytosine Uptake

1.6

1.2

0.8

0.4

0.0 0

30

60

90 120

150 180 210 240

Time (s)

100

50

0 -9

-8

-7

-6

-5

-4

-3

log[Inhibitor] (M)

Fig. 1. Cytosine uptake in T. b. brucei procyclic cells. (A) Uptake of 0.1 M [3H]cytosine in the presence (䉱) or absence (䊏) of 1000 M of unlabelled cytosine over 240 s. Linear regression was carried out over the interval 0–150 s. (B) Inhibition of 0.03 M of [3H]cytosine uptake over 90 s, by increasing concentrations of unlabelled cytosine (䉲), cytidine (䊏), uracil (䊊), and thymine (䊐).

S. Gudin et al. / Experimental Parasitology 114 (2006) 118–125

3.3. Uridine uptake

121

A

Uridine Uptake

Uptake of [3H]uridine at 1 M by T. b. brucei procyclic was linear over 240 s and uptake was inhibited almost entirely in the presence of 2500 M of unlabelled uridine but incompletely by 1000 M thymidine (Fig. 2A). Subsequent experiments with [3H]uridine were performed using 120 s incubations. Inhibition of [3H]uridine uptake by unlabelled up to 2500 M uridine or 100 M uracil showed that [3H]uridine uptake was saturable, and inhibited in a biphasic manner by these pyrimidines (Fig. 2B). In inhibition experiments, thymidine and cytidine inhibited only 50–70% of uridine uptake (Fig. 2C). These data are consistent with uridine uptake by at least two distinct transporters, one with high aYnity for uridine and the second with much lower aYnity, and only one of them sensitive to inhibition by thymidine and cytidine. To characterize the kinetic parameters of both transporters, inhibition of [3H]uridine uptake by unlabelled uridine in the presence of 100 M of thymidine was performed (Fig. 2D). We observed that the thymidine-insensitive transporter has relatively low aYnity for uridine, with a Km value of 34 § 5 M and a Vmax of 0.87 § 0.27 pmol(107 cells)¡1 s¡1 (n D 3), determined in the presence of 100 M of thymidine. This carrier displayed a Ki value of 0.76 § 0.12 M for uracil (n D 4) (Fig. 3) and was almost certainly identical to the previously identiWed U1 transporter, which has a reported Ki value of 45 § 15 M for uridine and a Km value for uracil of 0.45 § 0.09 M (De Koning and Jarvis, 1998).

(pmol(107 cells)-1s-1)

0.012 0.010 0.008 0.006 0.004 0.002 0.000 0

-8

-7

-6

-5

-4

-3

log[inhibitor] (M) Fig. 3. Inhibition of 1 M [3H]uridine uptake (120 s) by increasing concentrations of unlabelled uracil in the presence of 100 M of unlabelled thymidine by T. b. brucei procyclic. IC50 value for this experiment was 0.97 M.

The higher-aYnity uridine transporter, designated U2, displayed an apparent Km of 4.1 M (n D 2) and a Vmax of 0.019 § 0.0086 pmol(107 cells)¡1 s¡1 (n D 3) and is highly sensitive to inhibition by thymidine and cytidine, with Ki values of 0.38 § 0.07 M (n D 6) and 0.041 § 0.023 M (n D 3), though neither would be expected to be a permeant (see Sections 3.1 and 3.2), as well as to uracil. 3.4. Sensitivity of bloodstream forms to pyrimidine antimetabolites A number of pyrimidine analogues were tested for activity against bloodstream forms of T. b. brucei, using the standard Alamar blue protocol. Each compound was tested three separate times, using doubling dilutions starting at B

2.0

Uridine Uptake

1.5

1.0

0.5

(pmol(107 cells)-1s-1)

Uridine Uptake

(pmol(107 cells)-1)

0.05

0.0

0.04 0.03 0.02 0.01 0.00

0

60

120

180

240

0

-8

Time (s) 0.03

Uridine Uptake

D

0.02

0.01

(pmol(107 cells)-1s-1)

Uridine Uptake

(pmol(107 cells)-1s-1)

C

-7

-6

-5

-4

-3

log[inhibitor] (M) 0.15

0.10

0.05

0.00

0.00 0

-8

-7

-6

-5

log[inhibitor] (M)

-4

-3

0

-8

-7

-6

-5

-4

-3

log[inhibitor] (M)

Fig. 2. Uridine uptake by T. b. brucei procyclics. (A) Uptake of 1 M [3H]uridine [䊏, 0.0050 § 0.0004 pmol(107 cells)¡1 s¡1], or 1 M in the presence of 1000 M of unlabelled thymidine [䉱, 0.0029 § 0.0006 pmol(107 cells)¡1 s¡1] or 1 M in the presence of 2500 M of unlabelled uridine [䊉, 0.0005 § 0.00007 pmol(107 cells)¡1 s¡1]. (B–D) Inhibition of 1 M of [3H]uridine uptake over 120 s by increasing concentration of unlabelled. (B) Uridine (䊏) and uracil (䉱) or (C) thymidine (䊏) and cytidine (䉱) or (D) thymidine (䊏) or uridine in the presence of 100 M of unlabelled thymidine (䉱).

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100 M. The veterinary trypanocide diminazene aceturate was included in each assay as a positive control, whereas incubations without test compounds served as negative controls. The following compounds did not have a clear eVect, deWned as >50% inhibition at 100 M, on trypanosome growth: uracil, 5-bromouracil, 5-chlorouracil, 2-thiouracil, 4-thiouracil, 2,4-dithiouracil, 3-deazauracil, 1-methyluracil, uridine, 5-Xuorouridine, 5-bromouridine, 2-thiouridine, 4-thiouridine, 3⬘-deoxyuridine. However, 5Xuorouracil consistently inhibited growth of bloodstream trypanosomes, with an IC50 value of 7.1 § 2.0 M, compared to 0.065 § 0.010 for diminazene aceturate (Fig. 4).

Fluorescence

250 200 150 100 50 0 0

-8

-7

-6

-5

-4

log[test compound] (M)

0.04

Purine uptake in T. b. brucei is a highly eYcient process with submicromolar or low micromolar Km values, and Vmax ranging from 0.3–4.5 pmol(107 cells) ¡1 s¡1 for the various purine transporters (Carter and Fairlamb, 1993; De Koning and Jarvis, 1997a,b; Natto et al., 2005). With B Uracil Uptake (pmol(107 cells)-1s-1)

Uracil Uptake (pmol(107 cells)-1s-1)

Because of the trypanocidal activity of 5-Xuorouracil, uptake of uracil and 5-Xuorouracil in bloodstream forms was brieXy addressed. Uptake of [3H]uracil in bloodstream forms appeared similar to that in procyclics, with a highaYnity, high-capacity transporter that was inhibited eYciently by 5-Xuorouracil, with a Ki value of 0.31 § 0.08 M (n D 3), but low aYnity for 5-chlorouracil (Fig. 5A). The Km of the uracil transporter in bloodstream forms was determined as 0.57 § 0.08 M and the Vmax as 0.36 § 0.06 pmol(107 cells)¡1 s¡1 (Fig. 5B; n D 3). Transport of [3H]5-Xuorouracil was measured over 120 s at various radiolabel concentrations and appeared saturable, reaching a maximum uptake rate at around 1 M (Fig. 5C). Inputting these values into the Michaelis– Menten equation yielded a Km value of 0.55 M (not shown) and a separate experiment, measuring [3H]5-Xuorouracil transport over 10 s in the presence of 0.03–250 M of unlabelled 5-Xuorouracil, produced a Km value of 0.61 M (Fig. 5D). Vmax values were 0.11 and 0.078 pmol(107 cells)¡1 s¡1, respectively, for the two experiments. Judging from the Vmax/Km ratios for the two radiolabels, T. b. brucei bloodstream forms thus transport uracil with »4-fold higher eYciency than 5-Xuorouracil. 4. Discussion

Fig. 4. Sensitivity of T. b. brucei bloodstream forms to pyrimidines and trypanocides. Cells were incubated with the indicated concentrations of diminazene aceturate (䊉), 5-Xuorouracil (䊐) or 5-Xuorouridine (䉱), and the Xuorophore Alamar Blue, generating a Xuorescence signal proportional to cell number. IC50 values were calculated using non-linear regression. The data shown are of a representative experiment performed in duplicate.

A

3.5. Uptake of 5-Xuorouracil in bloodstream T. b. brucei

0.03

0.02

0.01

0.00

0.3

0.2

0.1

0.0 0

-8

-7

-6

-5

-4

-3

0

1

2

log[Inhibitor] (M) 6 5 4 3 2 1 0 0

30

60

Time (s)

90

120

D

0.12

5-Fluorouracil Uptake (pmol(107 cells)-1s-1)

5-Fluorouracil Uptake (pmol(107 cells)-1)

C

3

4

[Uracil] (μM)

0.09 0.06 0.03 0.00 0

1

2

3

4

[5-Fluorouracil] (μM)

Fig. 5. Pyrimidine uptake in T. b. brucei bloodstream forms. (A) Uptake of 0.055 M [3H]uracil over 10 s was inhibited by various concentrations of uracil (䊏), 5-Xuorouracil (䊊) or 5-chlorouracil (䉱). (B) Conversion of uracil inhibition data from frame A to Michaelis–Menten plot. (C) Uptake of various concentrations of [3H]5-Xuorouracil over 120 s: (䊐), 0.01 M; (䊏), 0.1 M; (䉱), 1 M; (䊊), 5 M; (䉲), 0.1 M label + 1 mM of unlabelled 5-Xuorouracil. (D) Michaelis–Menten plot of [3H]5-Xuorouracil over 10 s.

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the exception of uracil uptake by the previously described TbU1 transporter (De Koning and Jarvis, 1998), the eYciency of pyrimidine transport, expressed as Vmax/Km, seems to be much less than purine uptake in T. b. brucei procyclics (Table 1). The cytosine transporter, designated C1, does have an unusually high aYnity, allowing it to scavenge extremely low concentrations of cytosine, but a very low capacity under assay conditions with in vitro cultured procyclics. The apparent low capacity of C1 might reXect low levels of expression as a result of cell culture conditions or alternatively inherent properties of the transporter itself. Unfortunately, it is not feasible to re-do these experiments with procyclic cells freshly isolated from tsetse Xy midguts, but expression levels of C1 might well be increased under some conditions. For example, purine transport activities in procyclic trypanosomes can be upregulated by purine starvation (De Koning et al., 2000). Indeed, the large variation in cytosine uptake rates, but crucially not in Km or Ki values, in our experiments does seem to indicate that expression levels of this transporter vary considerably (Table 1), and this phenomenon warrants further investigation. The aYnity of this transporter for uracil but not for thymine indicates that the methyl group in the position 5 of the ring prevents binding, most probably as a result of steric hindrance. Uridine is taken up by at least two transporters in procyclics, designated U1 and U2, which display moderately high aYnity for this nucleoside. However, the transport eYciency of uridine through U2, in particular, is very low, due to the apparent low Vmax. As with C1, it is not possible to assess whether this low level of uptake is due to very low expression levels of the transporter as a result of cell culture conditions. We can conclude, however, that U1 is primarily a uracil carrier, as its eYciency of transport for the nucleobase is >40-fold higher than for the corresponding nucleoside (Table 1). The inhibition experiments with [3H]uridine consistently indicate a two-transporter system, but the aYnities of the two transporters for uracil are too close to resolve with suYcient conWdence. We conclude that U2 displays high aYnity for uracil and may well be able to transport it, though its contribution to the overall rate of uracil uptake in procyclics is likely to be minor (De Koning and Jarvis, 1998). U2 also displays high aYnity for cytidine and thymidine, but, we were unable to detect any mediated

123

uptake of either nucleoside and in both cases it is probable this reXects inhibition rather than any signiWcant rate of transport. C1 is inhibited by cytidine and uracil with high aYnity but no cytidine uptake is detectable in cultured procyclics. It must be pointed out, however, that transport of [3H]cytosine (of the highest speciWc activity available) was only just suYcient to obtain reliable Km values. If cytidine is a minor permeant for this transporter it would be undetectable by currently available techniques. As for uracil, it is very eYciently transported by U1 and in the absence of a speciWc U1 inhibitor, a minor Xux through C1 or U2 would also be impossible to single out. The only way these issues can be clariWed by the expression of individual transporter genes in a suitable system lacking all pyrimidine transport capacity, but no protozoan, or metazoan, pyrimidine transporters have been cloned to date. Thymine inhibits C1 with a Ki of around 1000 M, but no mediated uptake of thymine seems to occur in procyclics. A rate of uptake could be established for both thymine and thymidine, when using high radiolabel concentrations. However, uptake for these pyrimidines was non-saturable and we thus conclude that the observed uptake of thymine and thymidine is the result of passive diVusion. This conclusion is in agreement with the observation that C1 displays no signiWcant aYnity for thymine (thymidine was not tested) and that U1 displays no aYnity for thymine or thymidine (De Koning and Jarvis, 1998). We therefore postulate that C1 is a high-aYnity cytosine transporter and that cytidine and uracil are likely to be inhibitors rather than permeants for this carrier. Like U1, C1 is thus a highly speciWc transporter. But, whereas U1 speciWcity is the result of extreme size exclusion of the binding pocket, preventing the binding of other substrates (De Koning and Jarvis, 1998; Papageorgiou et al., 2005), C1 appears to be able to bind other pyrimidines but may be unable to translocate them eVectively across the membrane. Whereas purine transporters in protozoa exhibit a broad speciWcity for a wide range of natural and synthetic purine bases and/or nucleosides (De Koning et al., 2005), pyrimidine transporters thus appear to be much more exclusive and thymidine, thymine and cytidine appear not to be salvaged by procyclic T. b. brucei. Supply of these pyrimidines appears to be dependent on synthesis from UMP

Table 1 Kinetic parameters of purine and pyrimidine transport in procyclic T. b. brucei Transporter

Permeant

H1 H4 H4 P1 P1 U1 U1 U2 C1

Hypoxanthine Hypoxanthine Adenine Inosine Adenosine Uracil Uridine Uridine Cytosine

a

Km (M) 9.3 § 2.0 0.55 § 0.07 2.1 § 0.6a 0.36 § 0.04 0.26 § 0.02 0.46 § 0.09 33 § 4.9 4.1 § 2.1 0.048 § 0.009

Vmax (pmol(107 cells)¡1 s¡1)

Vmax/Km

References

4.5 § 0.8 0.27 § 0.08 0.41 § 0.14a 0.40 § 0.02 0.63 § 0.18 0.65 § 0.08 0.87 § 0.27 0.019 § 0.009 0.0049 § 0.0029

0.48 0.49 0.2a 1.1 2.4 1.4 0.03 0.005 0.10

De Koning and Jarvis (1997a) Natto et al. (2005) Burchmore et al. (2003) De Koning et al. (1998) De Koning et al. (1998) De Koning and Jarvis (1998) This manuscript This manuscript This manuscript

Expression of the encoding TbNBT1 gene in Saccharomyces cerevisiae.

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(Randolph et al., 1995), which the cell obtains through de novo biosynthesis or through phosphoribosylation of uracil with uracil phosphoribosyltransferase (UPRT) (Hammond and Gutteridge, 1982). Salvaged uridine would Wrst need to be converted to uracil by uridine phosphorylase, as the kinetoplastida lack uridine kinase activity (Hammond and Gutteridge, 1982; Randolph et al., 1995). Thus, the eYcient uptake of uracil and a secondary capacity to salvage uridine appears to conWrm the central role UMP in trypanosomatid pyrimidine metabolism. The capacity for pyrimidine salvage, when the parasite is capable of de novo synthesis, could be vital in the absence of the starting materials of the synthesis (glutamate and carbamoyl phosphate). The broad speciWcity and high eYciency of the T. b. brucei purine transporters make them excellent conduits for potential antimetabolites (De Koning et al., 2005; Wallace et al., 2004). In contrast, the very narrow selectivity and low eYciency of pyrimidine salvage in T. b. brucei procyclics appears to severely limit the prospect of any pyrimidinebased chemotherapeutic strategies. This was reXected in the almost complete lack of activity of known pyrimidine antimetabolites against bloodstream trypanosomes. However, 5-Xuorouracil, the only pyrimidine analogue with high aYnity for U1, the only signiWcant pyrimidine transporter, had a clear eVect. A very similar pattern was established recently for L. major (Papageorgiou et al., 2005). Our preliminary observations did show that uracil transport in bloodstream forms closely resembles that of procyclics, though a full study is beyond the scope of the present paper. We have demonstrated, however, that [3H]5-Xuorouracil is salvaged moderately eYciently by bloodstream forms and its in vivo activity may warrant further investigation. Acknowledgments S.G. was supported by a stipend from the Conseil regional de Lorraine and the Magistere of Microbiology and Enzymology, Université Henri Poincaré of Nancy (France), N.B.Q. is supported by a Getfund Scholarship Award from the Ghana Education Trust Fund and M.I.A. is supported by a scholarship from the Libyan Government. We thank Professor C.M.R. Turner for helpful discussions. References Bhattacharya, B.K., Otter, B.A., Berens, R.L., Klein, R.S., 1990. Studies on the synthesis of furo[3,2-d]pyrimidine C-nucleosides: new inosine analogues with antiprotozoan activity. Nucleosides Nucleotides 9, 1021–1043. Bressi, J.C., Verlinde, C.L., Aronov, A.M., Shaw, M.L., Shin, S.S., Nguyen, L.N., Suresh, S., Buckner, F.S., Van Voorhis, W.C., Kuntz, I.D., Hol, W.G., Gelb, M.H., 2001. Adenosine analogues as selective inhibitors of glyceraldehyde-3-phosphate dehydrogenase of Trypanosomatidae via structure-based drug design. Journal of Medicinal Chemistry 44, 2080–2093. Brun, R., Schonenberger, M., 1979. Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-deWned medium. Acta Tropica 36, 289–292.

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