ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1996, p. 1628–1632 0066-4804/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 40, No. 7
Kinetics of Plasmodium falciparum Thymidylate Synthase: Interactions with High-Affinity Metabolites of 5-Fluoroorotate and D1694 MOHAMMAD HEKMAT-NEJAD
AND
PRADIPSINH K. RATHOD*
Institute for Biomolecular Studies, Department of Biology, The Catholic University of America, Washington, D.C. 20064 Received 16 January 1996/Returned for modification 6 March 1996/Accepted 22 April 1996
Consistent with a proposed mechanism for the potent antimalarial activity of 5-fluoroorotate, 5-fluoro-2*deoxyuridylate inhibited Plasmodium falciparum thymidylate synthase with a Ki of 2 nM. Steady-state kinetics revealed no significant differences between malarial and mammalian thymidylate synthases. Thus, additional biochemical parameters must underlie the selective antimalarial activity of 5-fluoroorotate. A polyglutamylated folate analog, D1694-(glu)4, was also a potent inhibitor of malarial thymidylate synthase (Kis 5 1.5 nM). synthase. Additionally, it was suggested that the parasite enzyme binds the nucleotide substrate 29-deoxyuridylate (dUMP) about four times more weakly. The latter observation seemed important because the degree of inhibition of thymidylate synthase by 5-fluoro-29-deoxyuridylate is influenced by competing dUMP (6). However, it was important to reexamine these claims given the high degree of conservation between malarial and mammalian thymidylate synthases (3). Also, the kinetic constants reported by those investigators for mouse thymidylate synthase (26) were significantly different from the published values for well-characterized mammalian thymidylate synthases (8, 9, 23, 27, 32). In addition to questions related to the interactions of malarial thymidylate synthase and 5-fluoro-29-deoxyuridylate, there was also interest in understanding the interactions between this enzyme and folate-based inhibitors of thymidylate synthase. The folate-based thymidylate synthase inhibitor D1694 inhibits mammalian cells in culture (50% inhibitory concentration, 8 nM) (20). This toxicity to mammalian cells can be completely reversed with thymidine (20, 31). This is of special interest because, as noted above, malarial cells do not salvage nucleosides such as thymidine (14, 33, 37). Thus, folate-based thymidylate synthase inhibitors would be expected to be selective antimalarial agents in the presence of thymidine (31). Despite the highly potent and target-selective action of D1694 against mammalian cells, this thymidylate synthase inhibitor does not inhibit malarial parasites in culture until concentrations greater than 10 mM are used (31). One explanation for this finding could be that malarial thymidylate synthase is different from the mammalian enzyme in that it binds D1694 and its metabolites much less tightly than the mammalian enzyme. Alternatively, the lack of potency of D1694 against intact malarial cells may be due to variations in biochemical parameters such as transport and the ease of conversion of D1694 to the polyglutamate form and may not be due to poor binding of the parasite enzyme to the inhibitor. These issues motivated us to study the catalytic properties of P. falciparum thymidylate synthase and to study the binding of this enzyme to potential inhibitors.
The spread of drug-resistant malaria around the world has increased the pressure on investigators to develop new chemotherapeutic strategies (24). Malarial parasites, but not mammalian cells, fail to salvage pyrimidines (14, 33, 37), suggesting that inhibitors of de novo pyrimidine biosynthesis should be considered for malaria chemotherapy (29, 31). Dihydrofolate reductase and thymidylate synthase are essential for de novo pyrimidine biosynthesis. In protozoan parasites, these two enzymes are found on a single polypeptide coded off of a single gene (2, 5, 11, 12). Both dihydrofolate reductase and thymidylate synthase are attractive targets for chemotherapy because even partial inhibition of either of these enzymes leads to nucleotide imbalances and cell death (18, 19, 21, 41). The malarial dihydrofolate reductase domain varies considerably from its mammalian counterpart both in terms of its amino acid sequence (2) and in terms of its ability to bind folate analogs (10). In contrast, the malarial thymidylate synthase domain is highly homologous to its mammalian counterpart (2, 3), thus posing a problem for the selective inactivation of malarial thymidylate synthase. 5-Fluoroorotate was identified as a potent and selective inhibitor of malarial cells in vitro and in vivo (13, 28–30). 5-Fluoroorotate inhibits Plasmodium falciparum with a 50% inhibitory concentration of 6 nM, while it takes a 1,000 times higher concentration to inhibit mammalian cells to the same extent (29). 5-Fluoroorotate, like 5-fluorouracil in mammalian cells, can be metabolized to 5-fluoro-29-deoxyuridylate, a potent inactivator of thymidylate synthase (7, 17, 22, 35). Nanomolar levels of 5-fluoroorotate which kill malarial parasites are also sufficient to inactivate endogenous thymidylate synthase activity (30, 42). During the early hours of thymidylate synthase inactivation, the dihydrofolate reductase activity, on the other half of the bifunctional protein, remains unaffected (30). It is of interest to determine why thymidylate synthase is particularly vulnerable in malarial cells treated with 5-fluoroorotate. Pattanakitsakul et al. (26) have suggested that the thymidylate synthase of malarial parasites may be different from that of mammalian cells. According to those investigators, malarial thymidylate synthase binds 5-fluoro-29-deoxyuridylate 100 times more tightly than the mammalian thymidylate
MATERIALS AND METHODS Materials. Radioactive [6-3H]5-fluoro-29-deoxyuridylate and [5-3H]dUMP were purchased from Moravak Biochemicals, Inc. (Brea, Calif.). Sigma Chemical Co. (St. Louis, Mo.) supplied dUMP, tetrahydrofolate, formaldehyde, folic acid, dihydrofolate, thymidylate, 5-fluoro-29-deoxyuridylate, bovine serum albumin, Luria broth (Miller’s modification), and molecular weight standards for sodium
* Corresponding author. Mailing address: Department of Biology, The Catholic University of America, 620 Michigan Ave., N.E., Washington, DC 20064. Phone: (202) 319-5278. Fax: (202) 319-5721. Electronic mail address:
[email protected]. 1628
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TABLE 1. Purification of P. falciparum thymidylate synthase Enzyme source and purification step
Recombinant P. falciparum enzyme expressed in E. coli Cell lysate Affinity column Native enzyme from infected erythrocytes Cell lysate Affinity column
Total amt of protein (mg)
Total activity (nmol/min)
Sp act (nmol/min/mg)
236 0.125
130 4.4
0.55 35
1 63
100 3.3
98 0.024
2.0 0.6
0.02 25.5
1 1,275
100 30.4
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Isopropylthiogalactose was from Promega (Madison, Wis.). Aldrich Chemicals (Milwaukee, Wis.) provided the activated carbon. Optifluor scintillation fluid was from Packard Instrument Co. (Meriden, Conn.). A formylfolate affinity column was synthesized by the method of Banerjee et al. (1). Centricon 30 filters for ultrafiltration were from Amicon (Beverly, Mass.). The heterologous expression system for P. falciparum dihydrofolate reductase has been described previously (15). Buffers. Lysis buffer consisted of 50 mM N-Tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid (TES; pH 7.5), 75 mM mercaptoethanol, 1 mM EDTA, 10% glycerol, 50 mg of aprotinin per ml, 20 mg of leupeptin per ml, 50 mg of soybean trypsin inhibitor per ml, 1 mM benzamidine, 1 mM 1,10-phenanthroline, and 50 mM phenylmethylsulfonyl fluoride. Buffer A consisted of 50 mM Tris HCl (pH 7.5), 5 mM mercaptoethanol, and 1 mM EDTA. Buffer B consisted of 50 mM Tris HCl (pH 7.5), 5 mM mercaptoethanol, 1 mM EDTA, and 0.45 M NaCl. Preparation of recombinant malarial dihydrofolate reductase-thymidylate synthase. Escherichia coli cells carrying an expression plasmid with the malarial dihydrofolate reductase-thymidylate synthase (15) were grown in Luria broth to an optical density of 0.5 (600 nm) in a total volume of 4 liters. Isopropylthiogalactose was added to a final concentration of 2 mM. After 3 h the cells were harvested by centrifugation at 7,000 3 g for 15 min at 48C. All subsequent steps were performed at 48C unless mentioned otherwise. The pellets were resuspended in 50 ml of lysis buffer. The cells were lysed with a French press at 1,200 lb/in2 (Amino; SLM Instruments, Inc.). The lysate was clarified by centrifugation at 29,000 3 g for 90 min. The affinity purification step was based on the work of Banerjee et al. (1), Sirawaraporn et al. (38), and Zolg et al. (43). In order to purify the enzyme, 40 ml of the cell lysate was applied to a 2-ml formylfolate column at a flow rate of 0.2 ml/min. The flowthrough material was passed through the same affinity column a second time. The column was then washed with buffer B until the UV A280 of the eluate was less than 0.05. The column was then washed with 20 ml of 2 mM dihydrofolate in buffer B, and 2-ml fractions were collected. Malarial dihydrofolate reductase-thymidylate synthase usually eluted between volumes of 4 and 12 ml. To identify the fractions with the highest enzyme activity, 10-ml samples from each fraction were assayed for thymidylate synthase activity by the tritium-release assay described below. Fractions with the highest enzyme activity were pooled and concentrated to a final volume of 1 ml by ultrafiltration. To remove residual dihydrofolate the concentrated sample was dialyzed three times against 1 liter of buffer A. SDS-PAGE of this sample showed a major band with a molecular weight of 71,000 that was about 80% pure (see Fig. 1). The affinity purification gave about 63-fold purification with respect to thymidylate synthase activity (Table 1). Preparation of native P. falciparum dihydrofolate reductase-thymidylate synthase. W2 clones of P. falciparum cells were cultured as described previously (16, 25, 29, 39). P. falciparum cells were isolated from five 60-ml culture batches. Parasites were released from erythrocytes by saponin lysis (30). The cells were collected by centrifugation at 500 3 g for 5 min and treated with 1.5 volumes of 0.2% saponin in phosphate-buffered saline (PBS). After 15 min at room temperature, the cells were diluted with 9 volumes of PBS. Parasites were precipitated by centrifugation at 30,000 3 g for 15 min. After discarding the hemoglobin-rich supernatant, the parasite pellets were washed with 4 ml of cold PBS and were collected by centrifugation. The last step was repeated three times to remove all signs of hemoglobin from the cell pellet. The parasites were stored at 2708C until a total of 300 ml of culture had been processed. The cell pellets were thawed, pooled in 2 ml of lysis buffer, and disrupted by brief sonication (three times for 5 s each time). The lysate was clarified by centrifugation at 12,000 3 g for 30 min. The affinity step allowed for 1,275-fold purification with respect to thymidylate synthase activity (Table 1). Thymidylate synthase activity. (i) 5-Fluoro-2*-deoxyuridylate-binding assay. The 5-fluoro-29-deoxyuridylate-binding assay was based on the previous work of Santi et al. (36) and Hall et al. (15). In a typical assay, 100 ml of sample was mixed with 80 ml of methylenetetrahydrofolate cocktail (0.425 mM tetrahydrofolate, 125 mM TES [pH 7], 62.5 mM MgCl2, 2.5 mM EDTA, 187.5 mM mercaptoethanol, 16.25 mM formaldehyde) and 20 ml of 1 mM [6-3H]5-fluoro-29-deoxyuridylate (23 Ci/mmol). After a 60-min incubation, 100 ml of the reaction mixture was transferred to a nitrocellulose filter (0.45-mm pore size, 2.5 cm; Schleicher & Schuell, Keene, N.H.). The filter was washed with 10 ml of 25 mM potassium phosphate buffer (pH 7.4) at a rate of 2 ml/min. The radioactivity on the filter was
Purification factor
Yield (%)
counted in 7 ml of Optifluor scintillation fluid (Packard). To determine the amount of thymidylate synthase in a sample, the [6-3H]5-fluoro-29-deoxyuridylate-binding assay was performed with at least three different protein concentrations to make certain that the amount of protein used in the assay was not consuming all of the available [6-3H]5-fluoro-29-deoxyuridylate. (ii) Tritium-release assay. The tritium-release assay was based on the previous work of Roberts (34). In a typical assay, 10 ml of enzyme sample was added to a mixture of 20 ml of methylenetetrahydrofolate cocktail (see above) and 10 ml of 1 to 20 mM [5-3H]dUMP. A substrate with a specific activity of 0.4 Ci/mmol was used to assay the recombinant enzyme. The reaction was terminated by adding 20 ml of a stop solution (a mixture of one part 4.34 mM nonradioactive dUMP and three parts 2 N trichloroacetic acid). In order to remove unreacted substrate, 200 ml of 10% charcoal was added to the reaction mixture and the sample was placed on ice. After at least 15 min, the samples were centrifuged at 12,000 3 g for 10 min in a microcentrifuge. The radioactivity in a 100-ml aliquot of the supernatant was determined by liquid scintillation spectroscopy. One unit of enzyme activity is defined as that which generated 1 pmol of tritiated water per min in the presence of saturating concentrations of substrates. Kinetic characterization of malarial thymidylate synthase activity. For determining the effect of various substrate concentrations on enzymatic activity, the reactions were initiated by the addition of enzyme. For each combination of substrates, product formation was measured at a time point that was predetermined to reflect the initial velocity of the reaction. For measuring the inhibitory properties of 5-fluoro-29-deoxyuridylate, the enzyme was preincubated for 10 min with the inhibitor in the presence of methylenetetrahydrofolate. After that, the reaction was initiated with [5-3H]dUMP. For each combination of substrate and inhibitor, product formation was measured at five different time points (0 to 60 min), and velocity was calculated from the linear portion of the productversus-time plot. Inhibition by D1694 and its pentaglutamate derivative were measured in a similar way, but without preincubation.
RESULTS Purification of malarial thymidylate synthase. P. falciparum dihydrofolate reductase-thymidylate synthase has been purified by several groups, but only the dihydrofolate reductase domain has been studied (4, 38, 43). Given the difficulties in generating large numbers of malarial parasites in culture and the notorious instability of the thymidylate synthase domain (4, 11, 38), the recombinant enzyme proved to be a useful source of malarial thymidylate synthase. The kinetic data obtained from the affinity-purified recombinant enzyme were deemed reliable for several reasons. First, a highly specific thymidylate synthase assay was used. The tritium-release assay used in these studies is specific for this reaction, even in crude cell lysates (30). Second, the single formyl-folate affinity chromatography step allowed for the separation of the malarial enzyme from pyrimidine nucleotides and folates in cell lysates. Third, the parasite enzyme was readily separated from bacterial thymidylate synthase in the cell lysates. The recombinant enzyme was judged to be free of bacterial thymidylate synthase because (i) bacterial thymidylate synthase failed to bind to the affinity column and (ii) affinity-purified recombinant malarial dihydrofolate reductase-thymidylate synthase reacted with [3H]5-fluoro-29-deoxyuridylate to generate a labeled species with a molecular weight of 71,000, corresponding to the size of the bifunctional parasite enzyme. [3H]5-fluoro-29-deoxyuridylate did not label a protein with a molecular weight of 31,000 corresponding to the size of the E. coli thymidylate synthase
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FIG. 1. Purification of recombinant P. falciparum thymidylate synthase analyzed by SDS-PAGE. Lane A, concentrated sample after affinity chromatography (10 mg); lane B, high-molecular-weight markers (myosin, 205,000; b-galactosidase, 116,000; phosphorylase b, 97,400; bovine serum albumin, 66,000; ovalbumin, 45,000; and carbonic anhydrase, 29,000).
(data not shown). The affinity-purified recombinant enzyme was about 80% pure, as judged by SDS-PAGE (Fig. 1). While subsequent ion-exchange chromatography could yield a homogeneous enzyme, such preparations were too unstable to generate reliable kinetic values for thymidylate synthase. In contrast, the kinetic data from the affinity-purified recombinant preparation were reproducible from one batch to another. The kinetic data from the recombinant enzyme were validated by independent measurements of native thymidylate synthase isolated directly from a P. falciparum culture (see below). Kinetics of malarial thymidylate synthase. By using the affinity-purified recombinant enzyme, a plot of 1/v (where v is reaction velocity) versus 1/dUMP at various methylenetetrahydrofolate concentrations gave a series of nonparallel lines that intersected away from the vertical axis (Fig. 2). Secondary plots indicated that P. falciparum thymidylate synthase had a Km of 2 mM for dUMP and a Km of 39 mM for methylenetetrahydro-
ANTIMICROB. AGENTS CHEMOTHER.
folate. Similar values were established for the native enzyme isolated from parasites in culture (Table 2). Binding of 5-fluoro-2*-deoxyuridylate. To measure the affinity of the malarial thymidylate synthase for 5-fluoro-29-deoxyuridylate, the interactions between malarial thymidylate synthase and 5-fluoro-29-deoxyuridylate were studied. In the presence of 0.9 mM methylenetetrahydrofolate, 5-fluoro-29deoxyuridylate inhibited malarial thymidylate synthase activity with a Ki of 2 nM (Fig. 3). kcat of malarial thymidylate synthase. To determine the number of active sites per unit volume of the recombinant enzyme preparation, interactions between malarial thymidylate synthase and 5-fluoro-29-deoxyuridylate were studied by the nitrocellulose assay. On the basis of the Vmax value, calculated from the intercepts of Fig. 2 and the number of 5-fluoro29-deoxyuridylate-binding sites, it was possible to assign a kcat value of 118/s for the recombinant P. falciparum thymidylate synthase. An almost identical value of kcat was established for the native enzyme from cells in culture (Table 2). Binding to D1694 and its pentaglutamate form. To measure the affinity of the malarial thymidylate synthase for D1694, the compound was incubated with various concentrations of methylenetetrahydrofolate. Double-reciprocal plots revealed that D1694 had a Ki (slope) of 2.8 mM and a Ki (intercept) of 28 mM. These data suggested that D1694 is a slightly poorer inhibitor of malarial thymidylate synthase than of the mammalian enzyme (Table 2), but not enough to completely explain the 10,000-fold poorer inhibition of malarial parasites by D1694 (31). Since previous studies with mammalian thymidylate synthase suggested that the pentaglutamate form of this compound binds much more strongly to the enzyme than the monoglutamate form (20, 40), the inhibitory property of the pentaglutamate against malarial thymidylate synthase was also examined. The polyglutamylated form, D1694-(glu)4, inhibited malarial thymidylate synthase about 1,000 times more strongly than the monoglutamate form. The pentaglutamate derivative had a Ki (slope) of 1.6 nM and a Ki (intercept) of 44 nM (Fig. 4). There was virtually no difference between the malarial and mammalian thymidylate synthases in binding to the polyglutamylated D1694 (Table 2). DISCUSSION
FIG. 2. Response of P. falciparum thymidylate synthase activity to changing substrate concentrations: Lineweaver-Burk plots of 1/v versus 1/dUMP. A total of 2.1 U of recombinant P. falciparum thymidylate synthase was incubated with various concentrations of 29-deoxyuridylate (0.9, 1.8, 2.6, 4.4, 8.9, and 17.8 mM) and methylenetetrahydrofolate (10 mM [■], 20 mM [å], 40 mM [ç], and 160 mM [F]). After 30 min, the release of tritiated water was determined as described in the text. Each datum point is the average of three determinations.
Malarial parasites are susceptible to 5-fluoroorotate at a concentration 1,000 times lower than that at which mammalian cells are susceptible (29, 42). It is also known that nanomolar levels of 5-fluoroorotate that are sufficient to inhibit the proliferation of malarial parasites are also sufficient to inactivate malarial thymidylate synthase (30). It was proposed that the potent toxicity of 5-fluoroorotate against malarial parasites was mediated through the tight binding of 5-fluoro-29-deoxyuridylate to malarial thymidylate synthase (29, 30). In the present study, it was directly demonstrated that malarial thymidylate synthase has a high affinity for 5-fluoro-29-deoxyuridylate. While this may explain the potency of 5-fluoroorotate, the molecular basis for the selectivity of 5-fluoroorotate lies elsewhere (see below). When the kinetic properties of the malarial thymidylate synthase were compared with those of the host enzyme, no differences could be detected with respect to saturation by substrates, kcat, or the affinity for the nucleotidebased inhibitor 5-fluoro-29-deoxynucleotide or the affinity for the folate-based inhibitor D1694(glu)4 (Table 2). While it is conceivable that some subtle aspects of the active site of malarial thymidylate synthase may someday be identified and exploited for selective binding, all present indications are that the high level of amino acid sequence homology seen between
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TABLE 2. Comparison of kinetic constants of thymidylate synthases from P. falciparum and mammalian cells Km (mM) Enzyme source
P. falciparum Recombinantb Nativeb Mammalian
kcat (per FdUMPa binding site) (min21)
Ki (nM)
29-Deoxyuridylate
Methylenetetrahydrofolate
2 1.3
39 30
118 120
2
1.8c, 3.4d
31c, 8d, 27e
150d
1.7c, 5f
FdUMP
D1694 monoglutamate
D1694 pentaglutamate
2.8
1.5
0.06e
1.0e
a
FdUMP, for 5-fluoro-29-deoxyuridylate. Kinetics of malarial thymidylate synthase (present studies). Kinetics of human thymidylate synthase by Dolnick and Cheng (9). d Kinetics of human thymidylate synthase by Davisson et al. (8). e Kinetics of mouse thymidylate synthase by Ward et al. (40). f Kinetics of Ehrlich ascites carcinoma thymidylate synthase by Reyes and Heidelberger (32). b c
the parasite and host enzymes (2) translates into a highly homologous active site. Early suggestions indicating large differences between mammalian and malarial thymidylate synthases (26) were not supported by the present study. The conservation of the thymidylate synthase active site in the malarial parasite, and most likely in other pathogens, need not rule out the design of selective chemotherapeutic agents against this otherwise attractive target. Efforts to develop selective chemotherapy directed at malarial thymidylate synthase may circumvent the conservation of active sites by exploiting the inability of erythrocytic forms of malarial parasites to utilize exogenous thymidine (14, 31, 33, 37) and by exploiting host-pathogen differences in metabolic steps that are involved in the transport and activation of prodrugs into the toxic products that bind thymidylate synthase (29, 30). With respect to 5-fluoro-29-deoxyuridylate, the patterns of pyrimidine metabolism in malarial parasites clearly indicated that fluoropyrimidines such as 5-fluorouracil and 5-fluoro-29deoxyuridine that are well suited to the generation of 5-fluoro-
FIG. 3. Inhibition of P. falciparum thymidylate synthase by 5-fluoro-29-deoxyuridylate. A total of 0.71 U of P. falciparum thymidylate synthase was preincubated with 960 mM methylenetetrahydrofolate and various concentrations of 5-fluoro-29-deoxyuridylate (0 nM [E], 0.5 nM [ç], 1 nM [å], and 5 nM [■]). The reaction was initiated with various concentrations of 29-deoxyuridylate (1, 2, 3.3, 5, 6.7, 10, and 20 mM). The release of tritiated water was determined at five different time points ranging from 10 to 60 min. The rate of the reaction was determined from the linear portion of the product-versus-time plot. Each datum point is the average of two determinations.
29-deoxyuridylate in mammalian cells were not well suited to killing malarial parasites (29). P. falciparum cells have very low or nondetectable levels of the enzymes required to activate uracil, uridine, and 29-deoxyuridine (33). However, these cells have high levels of orotate phosphoribosyltransferase and are known to efficiently incorporate exogenous orotate into parasite nucleic acids (14, 30, 33). Therefore, it was correctly anticipated that 5-fluoroorotate would be a more potent antimalarial agent than 5-fluorouracil, 5-fluorouridine, or 5-fluoro-29deoxyuridine (29). The lessons learned from 5-fluoro-29-deoxyuridylate and 5-fluoroorotate may be applicable to the case of folate-based inhibitors of thymidylate synthase directed at malarial parasites. The present studies indicate that the pentaglutamate form of D1694 inhibits malarial thymidylate synthase at least as well as 5-fluoro-29-deoxyuridylate does. The 10,000 times lower toxicity of D1694 toward malarial cells in culture compared with that toward mammalian cells must reflect the fact that, like 5-fluoro-29-deoxyuridine, D1694 was developed and optimized for inhibiting the proliferation of mammalian cells (20).
FIG. 4. Inhibition of P. falciparum thymidylate synthase by the folate analog D1694-(glu)4. A total of 2.6 U of P. falciparum thymidylate synthase was preincubated with 50 mM dUMP, various concentrations of methylenetetrahydrofolate (MTHF; 25, 33, 50, 66, 100, and 200 mM), and various concentrations of D1694-(glu)4 (0 nM [å], 2.5 nM [{], 5 nM [}], 10 nM [E], and 20 nM [F]). After 20 min, the release of tritiated water was determined as described in the text.
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Its poor activity against malarial cells must be due to poor transport or poor polyglutamylation of D1694 in malarial parasites. Identification of the structural basis for this major difference in host-parasite folate metabolism could lead to compounds that are transported and processed successfully while still maintaining a high affinity for the active site of malarial thymidylate synthase; such insights would also allow for the development of new strategies for the selective inhibition of the malarial dihydrofolate reductase. ACKNOWLEDGMENTS P.K.R. and M.H.-N. thank John Hyde (University of Manchester, Manchester, United Kingdom) for kindly providing us with the P. falciparum dihydrofolate reductase-thymidylate synthase heterologous expression system; the host strain AB1899 (with plasmid pACYC lac i-2 to express the lac repressor) was provided to J. Hyde by Glaxo Group Research Ltd., Stevenage, United Kingdom. We also thank F. T. Boyle (Zeneca Pharmaceuticals, Cheshire, United Kingdom) for providing us with D1694 and its pentaglutamate derivative. This work was supported by a Public Health Service grant from the National Institute of Allergy and Infectious Diseases (AI 26912). P.K.R. is also a recipient of a Research Career Development Award from the National Institute of Allergy and Infectious Diseases (AI 01112). REFERENCES 1. Banerjee, C. K., L. L. Bennett, Jr., R. W. Brockman, B. P. Sani, and C. Temple, Jr. 1982. A convenient procedure for purification of thymidylate synthase from L1210 cells. Anal. Biochem. 121:275–280. 2. Bzik, D. J., W. Li, T. Horii, and J. Insulburg. 1987. Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductasethymidylate synthetase gene. Proc. Natl. Acad. Sci. USA 84:8360–8364. 3. Carreras, C. W., and D. V. Santi. 1995. The catalytic mechanism and structure of thymidylate synthase. Annu. Rev. Biochem. 64:721–762. 4. Chen, G., and J. W. Zolg. 1987. Purification of the bifunctional thymidylate synthase-dihydrofolate reductase complex from the human malaria parasite Plasmodium falciparum. Mol. Pharmacol. 32:723–730. 5. Coderre, J. A., S. M. Beverly, T. R. Schimke, and D. V. Santi. 1983. Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica. Proc. Natl. Acad. Sci. USA 80:2132–2136. 6. Danenberg, P. V., and K. D. Danenberg. 1978. Effect of 5,10-methylenetetrahydrofolate on the dissociation of 59-fluoro-29-deoxyuridylate from thymidylate synthase: evidence for an ordered mechanism. Biochemistry 17:4018– 4024. 7. Danenberg, P. V., and A. Lockshin. 1981. Fluorinated pyrimidines as tightbinding inhibitors of thymidylate synthetase. Pharmacol. Ther. 13:69–90. 8. Davisson, V. J., W. Sirawaraporn, and D. V. Santi. 1989. Expression of human thymidylate synthase in Escherichia coli. J. Biol. Chem. 264:9145–9148. 9. Dolnick, B. J., and Y.-C. Cheng. 1977. Human thymidylate synthetase derived from blast cells of patients with acute myelocytic leukemia. Purification and characterization. J. Biol. Chem. 252:7697–7703. 10. Ferone, R., J. J. Burchall, and G. H. Hitchings. 1969. Plasmodium berghei dihydrofolate reductase. Isolation, properties, and inhibition by antifolates. Mol. Pharmacol. 5:49–59. 11. Ferone, R., and S. Roland. 1980. Dihydrofolate reductase: thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata. Proc. Natl. Acad. Sci. USA 77:5802–5806. 12. Garrett, C. E., J. A. Coderre, T. D. Meek, E. P. Garvey, D. Claman, S. M. Beverly, and D. V. Santi. 1984. A bifunctional thymidylate synthetase-dihydrofolate reductase in protozoa. Mol. Biochem. Parasitol. 11:257–265. 13. Gomez, Z., and P. K. Rathod. 1990. Antimalarial activity of a 5-fluoroorotate and uridine combination in mice. Antimicrob. Agents Chemother. 34:1371– 1375. 14. Gutteridge, W. E., and P. I. Trigg. 1970. Incorporation of radioactive precursors into DNA and RNA of Plasmodium knowlesi in vitro. J. Protozool. 17:89–96. 15. Hall, S. J., P. F. G. Sims, and J. Hyde. 1991. Functional expression of the dihydrofolate reductase and thymidylate synthase activities of the human malaria parasite Plasmodium falciparum in Escherichia coli. Mol. Biochem. Parasitol. 45:317–330. 16. Haynes, J. D., C. L. Diggs, F. A. Hines, and R. E. Desjardines. 1976. Culture of human malaria parasites, Plasmodium falciparum. Nature (London) 263: 767–769. 17. Heidelberger, C., P. V. Danenberg, and R. G. Moran. 1983. Fluorinated pyrimidines and their nucleosides. Adv. Enzymol. 54:57–119. 18. Houghton, P. J., G. S. Germain, B. J. Hazelton, J. W. Pennington, and J. A.
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