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Antimicrobial Agents and Chemotherapy, November 2005, p. 4649-4657, Vol. 49, No. 11
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.11.4649-4657.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A Class of Pantothenic Acid Analogs Inhibits Plasmodium falciparum Pantothenate Kinase and Represses the Proliferation of Malaria Parasites{dagger}

Christina Spry,1 Christina L. L. Chai,3,4 Kiaran Kirk,1 and Kevin J. Saliba1,2*

School of Biochemistry and Molecular Biology,1 Medical School,2 Department of Chemistry, The Australian National University, Canberra ACT 0200, Australia,3 Institute of Chemical and Engineering Sciences, 1 Pesek Road, Jurong Island, Singapore 6278334

Received 12 May 2005/ Returned for modification 14 June 2005/ Accepted 22 August 2005


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ABSTRACT
 
The growth and proliferation of the human malaria parasite Plasmodium falciparum are dependent on the parasite's ability to obtain essential nutrients. One nutrient for which the parasite has an absolute requirement is the water-soluble vitamin pantothenic acid (vitamin B5). In this study, a series of pantothenic acid analogs which retain the 2,4-dihydroxy-3,3-dimethylbutyramide core of pantothenic acid but deviate in structure from one another and from pantothenic acid in the nature of the substituent attached to the amide nitrogen were synthesized using an efficient single-step synthetic route. Eight of 10 analogs tested inhibited the proliferation of intraerythrocytic P. falciparum parasites in vitro, doing so with 50% inhibitory concentrations between 15 and 200 µM. The compounds were generally selective, inhibiting the proliferation of a human cell line (the Jurkat cell line) only at concentrations severalfold higher than those required for inhibition of parasite growth. It was demonstrated that compounds in this series inhibited the phosphorylation of pantothenic acid by pantothenate kinase, the first step in the parasite's biosynthesis of the essential enzyme cofactor coenzyme A, doing so competitively, with Ki values in the nanomolar range.


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INTRODUCTION
 
Growth of the human malaria parasite, Plasmodium falciparum, during the intraerythrocytic stage of its life cycle is dependent on the parasite's ability to obtain essential nutrients from the external medium. One nutrient for which the parasite has an absolute requirement is pantothenic acid (vitamin B5; see Table 1 for its structure) (5, 13), the precursor of the essential enzyme cofactor coenzyme A. In the past 60 years, there have been numerous reports on the synthesis of pantothenic acid analogs with inhibitory activity against a range of pantothenic acid-requiring microorganisms (3, 10, 17, 19). For the majority of analogs, the inhibitory activity was antagonized by pantothenic acid, consistent with their inhibitory effect being due to their interference with pantothenic acid utilization (10, 17). A selection of these compounds was tested against avian malaria parasites in vivo and demonstrated potent antiplasmodial activity (2). Subsequently, after devising a method for maintaining P. falciparum parasites in cultures in vitro for short lengths of time, Trager showed that pantothenic acid analogs also inhibited proliferation of the human malaria parasite (21, 22).


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  		TABLE 1. Structures and antiproliferative activities of pantothenic acid analogues against P. falciparum and human Jurkat cells in vitro {zac0110554050t1a}

Since the initial discovery of the antiplasmodial activity of certain pantothenic acid analogs, there have been several developments in understanding the human malaria parasite's pantothenic acid requirement and the mechanisms by which the parasite transports and metabolizes this essential vitamin. It was shown that pantothenate (the ionized form of pantothenic acid) is taken up by the P. falciparum-infected erythrocyte via the "new permeation pathways" expressed on the erythrocyte membrane (14), transported into the parasite by a low-affinity H+-pantothenate symporter on the parasite plasma membrane, and then, once inside the parasite, phosphorylated by a high-affinity pantothenate kinase as the first step in the conversion of pantothenate to coenzyme A (15).

The progress made in this area, combined with the pressing need for new antimalarial agents that are effective against increasingly drug-resistant malaria parasites, has renewed interest in pantothenic acid utilization as an antimalarial drug target. Recently Saliba et al. (13) reported that pantothenol, the provitamin of pantothenic acid, which was previously recognized for its antibacterial properties (17), inhibited the proliferation of P. falciparum in vitro and significantly reduced the parasitemia of mice infected with Plasmodium vinckei vinckei. This report was followed by the finding that a pantothenic acid analog called CJ-15,801, isolated from a fungus (Seimatosporium sp. strain CL28611), also possessed antiplasmodial activity against P. falciparum (16). Both compounds were shown to inhibit pantothenate kinase activity in the parasite.

In this study, we report the synthesis of a series of pantothenic acid analogs which retain the pantothenic acid 2,4-dihydroxy-3,3-dimethylbutyryl, or "pantoyl," moiety but which differ from pantothenic acid and pantothenol in the structure of the substituent attached to the amide nitrogen (N substituent). An examination of the antiplasmodial activities of these compounds allowed the structural requirements for parasite inhibition to be probed and provided insight into structure-activity relationships. We demonstrate that these structural analogs, like pantothenol and CJ-15,801, inhibit the enzyme pantothenate kinase. In addition, we show that for at least three of the compounds in this series, the inhibition is competitive in nature, and that the analogs bind the target enzyme with nanomolar affinity.


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MATERIALS AND METHODS
 
Reagents. Pantothenol and ethyl pantothenol (compound 2) were obtained from Daiichi Pharmaceutical Co. Ltd., Japan. [14C]pantothenate was purchased from New England Nuclear (51.5 mCi/mmol) and American Radiolabeled Chemicals, Inc. (55 mCi/mmol). [3H]hypoxanthine (14.7 Ci/mmol) and [14C]choline (55 mCi/mmol) were purchased from Amersham Biosciences.

Chemical synthesis. Pantothenic acid analogs 1 and 3 to 10 were prepared by a procedure adapted from the work of Snell and Shive (17). Details of chemical procedures and analytical data for the compounds are reported in the supplemental material.

Cell culture. All in vitro experiments involving malaria parasites were performed using the 3D7 strain of P. falciparum. The parasites were maintained in synchronous continuous cultures as described elsewhere (1).

To investigate selectivity, in vitro experiments were performed using Jurkat cells, a human leukemic T-lymphocyte cell line. The Jurkat cells were maintained in RPMI 1640 supplemented with 25 mM HEPES, 11 mM glucose, 24 µg/ml gentamicin, and 10% (vol/vol) fetal calf serum at 37°C under an atmosphere of 5% CO2.

In vitro growth assays. The in vitro antiplasmodial activity of the compounds was measured using a standard [3H]hypoxanthine incorporation assay (4) as described by Saliba et al. (13). Briefly, P. falciparum-infected erythrocytes were incubated in 96-well microtiter plates in low-hypoxanthine culture medium (RPMI 1640 supplemented with 25 mM HEPES, 11 mM glucose, 2.4 µM hypoxanthine, 24 µg/ml gentamicin, and 0.6% [wt/vol] Albumax II) containing twofold dilutions of the test compounds. For compounds dissolved in dimethyl sulfoxide (compound 10), the concentration of dimethyl sulfoxide introduced into cultures never exceeded 0.1% (vol/vol). Plates were incubated for 72 h before [3H]hypoxanthine (0.4 µCi) was added to the wells. After a further 24 h of incubation, the cells were harvested onto glass fiber filters, and the [3H]hypoxanthine incorporated into nucleic acids was measured using scintillation counting. The 50% inhibitory concentration (IC50) of each drug was calculated by fitting the data to a sigmoidal curve using nonlinear least-square regression (SigmaPlot) and averaging the IC50 estimates from several independent experiments. IC50 values were analyzed statistically by performing unpaired t tests on the log-transformed data and adjusting the P values using the Bonferroni correction for multiple comparisons (11).

The effect of the pantothenic acid analogs on the proliferation of Jurkat cells was measured using a variation of the [3H]hypoxanthine incorporation assay described above. Jurkat cells, seeded at a density of 5,000 cells/ml, were incubated in Jurkat cell culture medium containing twofold dilutions of the test compounds. Cells incubated with culture medium alone served to estimate 100% cell growth. Assays included doxycycline, an antimalarial shown previously to inhibit the proliferation of Jurkat cells (9), as a positive control (IC50 = 9.1 ± 1.3 µM [mean ± standard error of the mean {SEM}]; n = 3).

Both the parasite and Jurkat cell proliferation assays were performed in the presence of 1 and 20 µM pantothenate to test the effect of the pantothenate concentration on the antiproliferative activity of the analogs of interest. The normal concentration of pantothenate in human whole blood is 1.57 to 2.66 µM (24). To compare statistically the activities of each of the compounds at 1 and 20 µM, unpaired t tests were performed on the log-transformed data.

[14C]pantothenate accumulation. To investigate the mechanism of action of the analogs, pantothenate accumulation by malaria parasites "isolated" from their host erythrocytes was measured in the presence and absence of each compound. Mature trophozoite-stage parasites were isolated from their host erythrocytes by the treatment of parasitized erythrocyte suspensions with saponin (0.05% [wt/vol]), as described previously (14). This has the effect of permeabilizing the erythrocyte and parasitophorous vacuole membranes while leaving the parasite plasma membrane intact. The accumulation of [14C]pantothenate was measured using a variation of the protocol described by Saliba et al. (14). Briefly, to initiate the accumulation reaction, isolated parasites, washed (five times) and resuspended in saline (125 mM NaCl, 5 mM KCl, 20 mM glucose, 25 mM HEPES, 1 mM MgCl2, pH 7.1), were added to a saline solution at 37°C containing [14C]pantothenate and one of the analogs to be tested or an equivalent concentration of water or dimethyl sulfoxide. The final concentration of [14C]pantothenate in the reaction was 0.1 µCi/ml (2 µM), the inhibitor (when present) was at a concentration of 1 mM, and the final cell concentration was typically 2 x 107 to 6 x 107 cells/ml. Aliquots (300 µl) were removed from the suspensions in duplicate at appropriate time intervals and centrifuged through 300 µl of oil (5:4 mixture of dibutyl phthalate:dioctyl phthalate) at 15,800 x g for 2 min to terminate the reaction by sedimenting the cells below the oil. The cells were processed for scintillation counting as described previously (14). The amount of [14C]pantothenate trapped in the extracellular space within the cell pellet was estimated as described previously (13). [14C]pantothenate distribution ratios (i.e., the concentration of [14C]pantothenate inside the cell relative to that in the extracellular solution) were calculated by assuming that the intracellular water volume of the parasite is 28 fl (14).

The effect of the analogs on [14C]choline accumulation by P. falciparum parasites was measured using the same procedure as that described above.

The effect of the analogs on [14C]pantothenate accumulation by Jurkat cells was investigated using essentially the same method. Before commencement of the accumulation experiment, the Jurkat cells were washed (three times) and resuspended in an appropriate volume of saline. The final concentration of [14C]pantothenate in the reaction was 0.1 µCi/ml (2 µM), the inhibitor (when present) was at a concentration of 1 mM, and the final cell concentration was typically 1 x 106 to 3 x 106 cells/ml. Distribution ratios were calculated by assuming the intracellular water volume of Jurkat cells to be 632 fl/cell (7).

The amount of [14C]pantothenate or [14C]choline accumulated in the presence of each inhibitor was statistically compared to that accumulated in the absence of inhibitors, using a paired t test. P values were adjusted using the Bonferroni correction for multiple comparisons.

[14C]pantothenate phosphorylation. To evaluate the effect of the analogs on P. falciparum pantothenate kinase, the phosphorylation of [14C]pantothenate in parasite lysates was measured in the presence and absence of the analogs. Lysates were prepared from isolated trophozoite-stage parasites as described elsewhere (14). [14C]pantothenate phosphorylation was assayed using the Somogyi reagent [a combination of ZnSO4 and Ba(OH)2 which precipitates phosphorylated compounds from solution (18)] as described by Saliba et al. (14). Briefly, each lysate was added to a solution (at 37°C) containing 50 mM Tris, 5 mM ATP, 5 mM MgCl2, [14C]pantothenate (at a final concentration of 0.01 µCi/ml, or 0.2 µM), and one of the inhibitors (at a final concentration of 1 mM) or an equivalent volume of water. Typically, a lysate prepared from 4 x 107 to 9 x 107 parasites was present in each ml of reaction solution. At predetermined intervals, 200-µl aliquots were transferred to microcentrifuge tubes containing 500 µl 5.6% (wt/vol) perchloric acid to terminate the reaction. Samples were processed for scintillation counting as described elsewhere (14).

The effect of the compounds on Jurkat pantothenate kinase was determined in the same manner. For the preparation of cell lysates, cells were washed three times with saline before being resuspended in a 10 mM phosphate buffer (pH 7.4) containing a protease inhibitor cocktail (1 Complete Mini tablet/10 ml; Roche). The suspension was subsequently freeze-thawed (three times) in liquid nitrogen and triturated (15 times) through a 27-gauge needle. The lysate was centrifuged three times at 2,060 x g for 5 min, with the supernatant being transferred each time to a new microcentrifuge tube. Typically, a lysate prepared from 1 x 106 to 5 x 106 cells was present in each ml of reaction solution.

The amount of phosphorylation measured in the presence of each inhibitor was compared to that measured when inhibitors were absent, using a paired t test. P values were adjusted using the Bonferroni correction for multiple comparisons.

Kinetic analysis of the mode of inhibition. The initial velocity of the pantothenate phosphorylation reaction in the presence of compound 1 was measured over a range of concentrations of the compound (0.02 to 200 µM). Phosphorylation was measured as described above at a single pantothenate concentration (0.2 µM). The data were fitted by nonlinear least-square regression to a sigmoidal curve using SigmaPlot software, and the IC50 was thereby determined. Subsequently, for compound 1 and an additional two analogs (compounds 3 and 6), the initial velocity of the phosphorylation reaction was measured at various pantothenate concentrations (0.1 to 1.6 µM) in the presence of the analogs, each at concentrations of 0, 0.5, and 1 µM. The reciprocals of the pantothenate concentration and the initial reaction velocity were plotted, and inhibition constants (Ki) were calculated from the plot using the Lineweaver-Burk equation for competitive inhibition, as follows: 1/v0 = {[(1+[I]/Ki)Km]/Vmax} x (1/[S]) + 1/Vmax, where v0 is the initial pantothenate phosphorylation rate, Km is the pantothenate concentration at which the phosphorylation reaction rate is half-maximal, [S] is the concentration of pantothenate, [I] is the concentration of inhibitor, and Vmax is the maximal velocity of the phosphorylation reaction.


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RESULTS
 
Chemistry. Using pantothenol as a prototype, a series of pantothenic acid analogs was synthesized. As shown in Table 1, the analogs all retained the pantoyl moiety but varied in the nature of the substituent attached to the amide nitrogen. Compound 1 was synthesized to determine the effect of replacing the terminal hydroxyl group of pantothenol with a methyl ether. With the ethyl ether analog (compound 2) being commercially available, the effect of varying the length of the alkyl ether could also be investigated. The thiomethyl ether analog (compound 3) permitted an examination of the importance of the oxygen heteroatom. Compounds 4 and 5 allowed the importance of the terminal functionality of pantothenol and the effect of varying the carbon chain length to be examined. Compound 6, a double bond analog of compound 5, was synthesized to determine the effect of introducing an electron-rich double bond into the N substituent. The importance of the position of the terminal hydroxyl group in pantothenol could be probed with compound 7, and the effect of additional hydroxyl substituents was probed with compound 8. Compounds 9 and 10 also retained a pantoyl moiety but differed more considerably from pantothenol in the nature of their N substituents. Compound 9, in which the N substituent is simply an amino group forming part of a hydrazide functional group, was synthesized because it is a known inhibitor of pantothenic acid-requiring bacteria (10). Compound 10 was synthesized to probe the steric and electronic effects of the terminal nitrogen atom of compound 9.

The analogs were prepared by the condensation of R-pantolactone with the corresponding amine or hydrazine, using the general synthetic procedure reported by Snell and Shive (17). The preparation of compounds 1, 4 to 7, and 9 has been described previously; to our knowledge, compounds 3, 8, and 10 are novel. The reaction yields (after purification) were generally between 74 and 92%, reflecting the efficiency of the single-step condensation reaction. With the exception of compounds 8 and 10, the analogs were obtained as viscous liquids. As previously described by Snell and Shive (17), low-boiling-point starting materials were removed from the reaction products in vacuo; however, to improve the purity of the preparations, the compounds were purified further by flash chromatography (compounds 1 and 3 to 9) or recrystallization (compound 10). The purified compounds were demonstrated to be of sufficient purity for use in biological assays (>95%) by 13C nuclear magnetic resonance (13C NMR) and combustion analysis (see the supplemental material). With the exception of compound 8, all analogs were prepared as single stereoisomers. Since compound 8 was synthesized from racemic 3-amino-1,2-propanediol, the product was a mixture of two diastereomers. This mixture was assayed first before determining the need to investigate the activity of either isomer further; the low antiplasmodial activity of the mixture, however, made further investigation unnecessary (see below).

In the 1H NMR spectra of the pantothenic acid analogs, resonances corresponding to the six methyl protons, the two methylene protons, and the single methine proton of the common pantoyl moiety appeared between 0.80 and 1.02 ppm, 3.23 and 3.51 ppm, and 3.79 and 4.05 ppm, respectively. In the 13C NMR spectra, resonances corresponding to the two methyl carbons, the methylene carbon, the quarternary carbon, the methine carbon, and the carbonyl carbon of the common pantoyl moiety were observed between 20.00 and 21.57 ppm, 70.09 and 71.28 ppm, 39.23 and 40.58 ppm, 76.81 and 77.50 ppm, and 173.19 and 176.53 ppm, respectively. The presence of these characteristic resonances in the NMR spectra of the synthesized compounds, in combination with resonances unique to each compound, helped to confirm the identity of the analogs.

In vitro antiplasmodial activity. The in vitro antiplasmodial activity of the pantothenic acid analogs was measured with the 3D7 strain of P. falciparum. For each compound, the concentration required for a 50% inhibition of parasite proliferation (IC50) is reported in Table 1. At an extracellular pantothenate concentration of 1 µM (close to the normal whole-blood concentration [24]), all of the compounds, with the exception of compounds 7 and 8, inhibited P. falciparum proliferation, with IC50 values below 200 µM. An IC50 value of 129 ± 3 µM (mean ± SEM) was measured for pantothenol, which was slightly higher than that measured in a different strain (FAF6) of P. falciparum (60 ± 4 µM) (13). The potency of compounds 1 to 6 was comparable to that of pantothenol, demonstrating that the terminal hydroxyl group of the N substituent is not critical. Its removal or replacement with an alkyl ether, thioalkyl ether, or methyl group did not result in a significant attenuation of activity (P > 0.21; unpaired t test with Bonferroni correction). When a hydroxyl group was introduced two carbon units from the amide nitrogen, as in compounds 7 and 8, a loss of activity of >10-fold was observed (P < 0.04; unpaired t test with Bonferroni correction), suggesting that this substituent may interfere with the mechanism of inhibition. In this assay, compound 9 was the most active of the analogs, inhibiting parasite growth approximately 10-fold more effectively than pantothenol (P = 0.02; unpaired t test with Bonferroni correction). When a hydrogen atom on the terminal nitrogen of the hydrazide was replaced with a phenyl group (compound 10), a substitution that changes the electronic and steric properties of the nitrogen atom, potency was significantly reduced (P = 0.001; unpaired t test with Bonferroni correction). This suggests that the terminal nitrogen atom of compound 9 has an important role in the mechanism by which the compound inhibits parasite growth.

When the extracellular pantothenate concentration was increased from 1 to 20 µM, the concentration-response curves for compounds 1 to 7 shifted to the right (see Fig. 1 for the concentration-response curve shift for compound 1). This shift is reflected in the significant increase in IC50 values reported for compounds 1 to 7 (P < 0.03; unpaired t test) (Table 1). The increase in the concentration of compounds required to inhibit parasite proliferation in the presence of a 20-fold higher concentration of pantothenate is consistent with the analogs exerting their effect on parasite proliferation by competitively inhibiting the parasite's utilization of pantothenate. The effect of the 20-fold increase in pantothenate on antiplasmodial activity varied between compounds, with IC50 values increasing by as little as 3-fold or as much as 18-fold. Increasing the concentration of pantothenic acid 20-fold did not antagonize the antiplasmodial activity of compounds 9 and 10 (P > 0.52; unpaired t test), suggesting that a higher concentration of pantothenate is required to antagonize the activity of these inhibitors, that they inhibit parasite proliferation by a noncompetitive inhibition of pantothenic acid utilization, or that they inhibit parasite proliferation by a mechanism that is unrelated to pantothenic acid utilization.



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  		FIG. 1. Inhibition of in vitro proliferation of P. falciparum and Jurkat cells by compound 1. The concentration-response curves show the effects of increasing concentrations of compound 1 on [3H]hypoxanthine incorporation by parasites cultured (for 96 h) in medium containing 1 µM pantothenate (filled circles), parasites cultured (for 96 h) in medium containing 20 µM pantothenate (open squares), and Jurkat cells cultured (for 96 h) in medium containing 1 µM pantothenate (filled triangles). The data are averages from three or more independent experiments, with each performed in triplicate. Error bars represent SEM, and where not shown, fall within the symbols. For clarity, negative error bars only are shown for the 1 µM pantothenate parasite plot, positive error bars only are shown for the 20 µM pantothenate parasite plot, and for the Jurkat cell plot error bars (most of which fell within the symbols) have been omitted.

In vitro cytotoxicity against Jurkat cells. Compounds 1 to 7 and 9 displayed relatively low toxicities when tested against the human Jurkat cell line, inhibiting proliferation only at concentrations much higher than those required to inhibit parasite proliferation (Table 1; Fig. 1). The selectivity indexes of these compounds, i.e., the ratios of the IC50 values against Jurkat cells to the IC50 values against P. falciparum, ranged from >4 to >38. Compound 10 was the only analog with antiplasmodial activity that did not show selectivity: the IC50 measured against the Jurkat cell line did not differ significantly from the IC50 measured against P. falciparum (P = 0.42; unpaired t test).

The IC50 values (against Jurkat cells) of the three compounds that inhibited Jurkat cell proliferation by 40% or more at concentrations below 2 mM (compounds 6, 9, and 10) did not increase when the concentration of pantothenate was increased 20-fold (data not shown). This suggests that either a higher concentration of pantothenate is required to attenuate the activity or that the antiproliferative activity of these compounds against the Jurkat cell line is not a result of competitive inhibition of pantothenate utilization.

Inhibition of pantothenate accumulation. The effect of the pantothenic acid analogs on the parasite's utilization of pantothenate was investigated directly by measuring the uptake of [14C]pantothenate into isolated P. falciparum parasites with and without the compounds being present. Initially, one of the active compounds (compound 4) was selected, and its effect on pantothenate uptake was measured over a 30-min time course. Similar to results reported previously for pantothenol (13), compound 4 inhibited the accumulation of [14C]pantothenate in the parasite (Fig. 2A). Under control conditions, [14C]pantothenate accumulated in the parasite throughout the time course, reaching a distribution ratio (the ratio of the concentration of [14C]pantothenate inside the cell to that in the extracellular solution) of approximately 30 after 30 min. However, when compound 4 (1 mM) was present, a distribution ratio of >1 was reached within 5 min, but thereafter [14C]pantothenate accumulated very slowly, reaching a distribution ratio of only 3 after 30 min.



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  		FIG. 2. Effect of pantothenic acid analogs on the accumulation of [14C]pantothenate by saponin-isolated P. falciparum trophozoites and Jurkat cells. (A) Time courses for the accumulation of [14C]pantothenate by isolated parasites in the presence (open squares) or absence (filled circles) of compound 4 (1 mM). Accumulation is expressed as the [14C]pantothenate distribution ratio, i.e., the ratio of the concentration of [14C]pantothenate inside the cell to that in the extracellular solution. (B) Accumulation of [14C]pantothenate (open bars) and [14C]choline (striped bars) by isolated parasites during a 10-min incubation with [14C]pantothenate and pantothenol or compounds 1 to 7, 9, and 10 (1 mM). The amount of [14C]pantothenate or [14C]choline accumulated is shown as a percentage of that accumulated by parasites in the presence of the appropriate solvent controls (closed bar). The reduction in [14C]pantothenate accumulation was significant for all compounds (P < 0.005; paired t test with Bonferroni correction). None of the compounds caused a significant reduction in [14C]choline accumulation (P > 0.70; paired t test with Bonferroni correction). (C) Time course for the accumulation of [14C]pantothenate by Jurkat cells in the absence of inhibitors. Accumulation is expressed as the [14C] distribution ratio. (D) Accumulation of [14C]pantothenate by Jurkat cells during a 2-min incubation with [14C]pantothenate and pantothenol or compounds 1 to 7, 9, and 10 (open bars). The amount of [14C]pantothenate accumulated is shown as a percentage of that accumulated by cells in the presence of the appropriate solvent controls (closed bar). Accumulation was not significantly reduced in the presence of the compounds (P > 0.17; paired t test with Bonferroni correction). The data are averages from two independent experiments, with each carried out in duplicate. Error bars represent the range/2, and where not shown, fall within the symbols.

The effect of the other analogs was determined by measuring the amount of [14C]pantothenate accumulated by parasites during a 10-min incubation with each analog, a time period in which pantothenate was shown to accumulate linearly with time in the absence of inhibitors (Fig. 2A). All of the compounds tested, when present at a concentration of 1 mM, significantly reduced the amount of [14C]pantothenate accumulated by the parasites (P < 0.005; paired t test with Bonferroni correction) (Fig. 2B). In the presence of pantothenol and compounds 1 to 7 and 10, the amount of [14C]pantothenate accumulated in the 10-min period was reduced >75%. Compound 9 was less effective (P < 0.004; unpaired t test with Bonferroni correction), reducing accumulation by only 43%. Due to its low antiplasmodial activity, the effect of compound 8 on the parasite’s accumulation of [14C]pantothenate was not investigated.

To determine whether the effect on pantothenate accumulation was due to a direct effect on the parasite's pantothenate utilization or was a result of a generally deleterious effect on the parasite, the accumulation of [14C]choline, a phospholipid precursor unrelated to pantothenate, which like pantothenate is accumulated by the parasite under normal conditions (8), was measured in a series of parallel experiments. None of the analogs that inhibited pantothenate accumulation significantly inhibited the accumulation of [14C]choline (P > 0.70; paired t test with Bonferroni correction) (Fig. 2B).

The mechanism for the selectivity of the pantothenic acid analogs was investigated by measuring the effect of the compounds of interest on the uptake of [14C]pantothenate by Jurkat cells. [14C]pantothenate accumulation was measured over a 40-min time course. The accumulation of [14C]pantothenate by Jurkat cells was initially linear with time, slowed after about 5 min, and then leveled off at a distribution ratio of approximately 40 after 20 min (Fig. 2C). None of the analogs had a significant effect on the amount of [14C]pantothenate accumulated by Jurkat cells in the initial 2-minute linear portion of the uptake time course, when added at a concentration of 1 mM (P > 0.17; paired t test with Bonferroni correction) (Fig. 2D). The contrasting effects of the analogs on the pantothenate accumulation of P. falciparum and human Jurkat cells (Fig. 2B and D) are consistent with the selective inhibition of parasite proliferation being due to the ability of the compounds to inhibit selectively the accumulation of pantothenate in the parasite.

Inhibition of pantothenate phosphorylation. The accumulation of pantothenate by isolated P. falciparum trophozoites represents the combined effects of the transport of the vitamin into the parasite (via a H+-pantothenate symport mechanism) and its subsequent phosphorylation (and thus trapping) within the parasite (15). The results shown in Fig. 2A and B are consistent with the view that in the presence of the analogs, pantothenate entered the parasite (reaching a distribution ratio of >1) (Fig. 2A) but was not effectively trapped, i.e., phosphorylation occurred at a rate substantially lower than that in cells under control conditions (reducing accumulation). Saliba et al. (13) demonstrated previously that the inhibitory effect of pantothenol on the parasite's ability to accumulate pantothenate resulted from the inhibition of pantothenate phosphorylation by pantothenate kinase. To test directly whether the same was true of the compounds of interest here, the effect of the analogs on P. falciparum pantothenate kinase activity was determined by measuring the amount of [14C]pantothenate phosphorylated by parasite lysates during a 10-min incubation (during which pantothenate phosphorylation increased linearly with time under control conditions) (Fig. 3A) in the presence and absence of the compounds (1 mM). All of the compounds tested reduced the amount of [14C]pantothenate phosphorylated by parasite lysates (P < 0.02; paired t test with Bonferroni correction) (Fig. 3B). Compounds 1 to 7 and 10 reduced the amount of pantothenate phosphorylated by >85%. Compound 9 was less effective (P < 0.05; unpaired t test with Bonferroni correction), inhibiting phosphorylation by 73%, consistent with the observation that it is a less effective inhibitor of pantothenate accumulation by intact parasites (Fig. 2B). As in the accumulation assay, compound 8 was not tested due to its low antiplasmodial activity.



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  		FIG. 3. Effect of pantothenic acid analogs on the phosphorylation of [14C]pantothenate by trophozoite-stage P. falciparum and Jurkat cell lysates. (A) Time course for the phosphorylation of [14C]pantothenate by parasite lysates in the absence of inhibitors. Phosphorylation is expressed as the percentage of [14C]pantothenate that was phosphorylated. (B) Phosphorylation of [14C]pantothenate by parasite lysates during a 10-min incubation with [14C]pantothenate and pantothenol or compounds 1 to 7, 9, and 10 (open bars). Phosphorylation is expressed as a percentage of the amount of [14C]pantothenate phosphorylated in the presence of appropriate solvent controls (closed bar). The reduction in phosphorylation caused by all compounds was statistically significant (P < 0.02; paired t test with Bonferroni correction). (C) Time course for the phosphorylation of [14C]pantothenate by Jurkat cell lysates in the absence of inhibitors. Phosphorylation is expressed as the percentage of [14C]pantothenate that was phosphorylated. (D) Phosphorylation of [14C]pantothenate by Jurkat lysates during a 20-min incubation with [14C]pantothenate and pantothenol or compounds 1 to 7, 9, and 10 (open bars). Phosphorylation is expressed as a percentage of the amount of [14C]pantothenate phosphorylated in the presence of appropriate solvent controls (closed bar). The reduction in phosphorylation caused by all compounds was statistically significant (P < 0.05; paired t test with Bonferroni correction). The data are averages from two independent experiments, with each carried out in duplicate. Error bars represent the range/2, and where not shown, fall within the symbols.

The selectivity of the analogs was investigated further by measuring their effect on the phosphorylation of [14C]pantothenate by Jurkat cell lysates. Phosphorylation was measured for 20 min, during which the phosphorylation of [14C]pantothenate by Jurkat cell lysates increased linearly with time in the absence of inhibitors (Fig. 3C). As shown in Fig. 3D, [14C]pantothenate phosphorylation by Jurkat cell lysates was inhibited by all of the analogs tested (P < 0.05; paired t test with Bonferroni correction). Hence, the pantothenic acid analogs in this series, when present at a concentration of 1 mM, inhibited the phosphorylation of pantothenate by both P. falciparum and Jurkat cell lysates.

Kinetic analysis of the mode of inhibition. The effect on pantothenate kinase of three of the more potent inhibitors of parasite proliferation (compounds 1, 3, and 6) was investigated further. When the rate of pantothenate phosphorylation in parasite lysates was measured at various concentrations of analog 1, the pantothenate phosphorylation rate was shown to decrease with increasing concentrations of the inhibitor (Fig. 4A), with an IC50 of 0.3 µM being measured. The mode of analog binding to pantothenate kinase was determined for compounds 1, 3, and 6 by measuring the initial phosphorylation rate over a range of pantothenate concentrations, both in the absence of inhibitor and in the presence of 0.5 and 1 µM inhibitor (two concentrations that fell within the steep part of the concentration-response curve of compound 1) (Fig. 4A). For compound 1, the three double reciprocal plots of (pantothenate concentration)–1 versus (pantothenate phosphorylation rate)–1 intersected approximately at the (pantothenate phosphorylation rate)–1 axis, consistent with the compound inhibiting pantothenate kinase in a competitive manner (Fig. 4B). Similar data were obtained for compounds 3 and 6 (Fig. 4C and D). Using the Lineweaver-Burk equation for competitive inhibition, Ki values of 0.46 ± 0.04, 0.37 ± 0.05, and 0.51 ± 0.21 µM (mean ± SEM) were calculated for compounds 1, 3, and 6, respectively. Analogs 1, 3, and 6 therefore bind the enzyme with comparable affinities to that of the natural substrate, pantothenate, which has a Km of 0.28 ± 0.03 µM (15).



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  		FIG. 4. Effects of compounds 1, 3, and 6 on phosphorylation of [14C]pantothenate by pantothenate kinase in trophozoite-stage P. falciparum lysates. (A) Concentration-response curve showing the effect of increasing concentrations of compound 1 on the phosphorylation of [14C]pantothenate. The data are averages from a single experiment carried out in duplicate. (B to D) Lineweaver-Burk plots [(pantothenate concentration)–1 versus (pantothenate phosphorylation rate)–1] for inhibition of pantothenate kinase activity at various concentrations of pantothenate and in the absence (filled circles) and presence of 0.5 µM (open squares) and 1 µM (filled triangles) compound 1 (B), compound 3 (C), and compound 6 (D). The data are averages from two independent experiments. Error bars represent the range/2. Where not shown, error bars fall within the symbols. For clarity, negative error bars only are shown for the 0.5 µM plots and positive error bars only are shown for the 1 µM plots.


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DISCUSSION
 
Following on from the initial discovery that the survival of the avian malaria parasite Plasmodium lophurae was favored by the addition of pantothenate to an appropriate culture medium (20), it was demonstrated that the growth and replication of P. falciparum, the most virulent human malaria parasite, are dependent on an extracellular source of the vitamin pantothenic acid (5, 13). The antiplasmodial activity of pantothenic acid analogs in both early in vitro culture experiments and in vivo avian malaria models has been reported (2, 21, 22). Nonetheless, since the advent of a method for the continuous culture of P. falciparum in vitro (23), there have been only two reports of pantothenic acid analogs acting as inhibitors of P. falciparum (13, 16). In this paper, we report the synthesis and characterization of a series of pantothenic acid analogs that inhibit P. falciparum's utilization of pantothenate and its proliferation.

The lead compound, pantothenol, inhibits the proliferation of P. falciparum (3D7 strain) with a modest potency (IC50, 129 µM). Eight of the 10 pantothenic acid analogs reported in this study (compounds 1 to 6, 9, and 10) were also shown to be moderately potent inhibitors of P. falciparum proliferation (IC50, 15 to 200 µM). Of the eight active compounds, compound 9 was the only one that was significantly more potent than pantothenol. The relative potency of compound 9 suggests that further examination of the activity of derivatives of this compound may be worthwhile. In addition, the finding that compound 9 (an antibacterial agent) inhibited P. falciparum proliferation highlights the possibility that more antibacterial pantothenic acid analogs (such as those in references 3 and 6) could also be effective antiplasmodial agents. Even though only compound 9 displayed improved activity relative to that of pantothenol, the structural variation between the analogs that retained antiplasmodial activity suggests that there is plenty of scope for the further synthesis of analogs for testing against the malaria parasite.

The two other compounds synthesized in this study (compounds 7 and 8) were >10-fold less potent than pantothenol. This result is consistent with the hydroxyl substituent on the second carbon from the amide nitrogen, which is absent from pantothenol but common to both analogs, interfering with the interaction between these inhibitors and their molecular target. The reduced activities of compounds 7 and 8 could also be attributed to a diminished ability to access the parasite target, resulting from factors such as decreased influx into the infected erythrocyte and thereafter the parasite or increased efflux, metabolism, or nonspecific protein binding, all of which would reduce the effective intracellular concentration of the compounds.

The antiplasmodial activity of compounds 1 to 7 was attenuated by pantothenate, consistent with the inhibition of parasite proliferation resulting from the competitive inhibition of pantothenate utilization. These seven compounds were shown to inhibit P. falciparum's accumulation of [14C]pantothenate via inhibition of pantothenate kinase, the enzyme responsible for phosphorylating, and hence trapping, pantothenate within the parasite. The data in Fig. 4 show that for at least three of the analogs (compounds 1, 3, and 6), the inhibition was competitive in nature. Kinetic analysis revealed that pantothenate kinase binds to the analogs with a high affinity (Ki, 370 to 510 nM), comparable with the enzyme's affinity for pantothenate (Km, 280 nM) (15).

Although compounds 9 and 10 also inhibited the accumulation and phosphorylation of pantothenate by P. falciparum, there was no increase in IC50 values when the pantothenate concentration was increased 20-fold. These data are consistent with compounds 9 and 10 inhibiting parasite proliferation by interfering with pantothenate utilization via an alternative, noncompetitive mechanism. The reactive terminal nitrogen atom of compound 9 could undergo an irreversible interaction with the enzyme pantothenate kinase; the comparably lower potency of compound 10 may be accounted for by a decrease in the nucleophilicity of this terminal nitrogen atom, a result of electron withdrawal by the aromatic ring through resonance, and by steric effects resulting from the replacement of a hydrogen atom on the terminal nitrogen with an aromatic ring. An alternative explanation is that higher-affinity binding of these compounds to their target may result in a pantothenate concentration of >20 µM being required to increase the IC50. The inhibition of parasite proliferation could also result from the inhibition of one or more alternative parasite targets at a concentration lower than that required for the inhibition of pantothenate utilization. The latter explanation is also consistent with compound 9, the least effective inhibitor of pantothenate accumulation and phosphorylation, being the most effective inhibitor of parasite proliferation.

To our knowledge, this is the first report to describe the effects of a series of pantothenic acid analogs on the proliferation and pantothenate utilization of a mammalian cell line. With the exception of compound 10, the antiplasmodial analogs of this series behaved as selective inhibitors of parasite proliferation, inhibiting proliferation of the Jurkat cell line only at concentrations much higher than those required to inhibit parasite growth. Although this result is clearly promising, the mechanism for selectivity remains unclear. The activities (against Jurkat cells) of the three analogs that inhibited Jurkat cell proliferation by 40% or more at the concentrations tested (compounds 6, 9, and 10) were not attenuated by a 20-fold increase in the pantothenate concentration (data not shown). For compound 6 (but not compounds 9 and 10), this was surprising, as a 20-fold increase in pantothenate had resulted in a significant increase in its IC50 against the parasite. This result is consistent with the analog (and perhaps others in this series) inhibiting P. falciparum proliferation by a mechanism that differs from the mechanism by which it inhibits the growth of Jurkat cells.

Although the pantothenic acid analogs inhibited pantothenate kinase activity in both parasite and Jurkat cell lysates, the accumulation of pantothenate by intact cells was inhibited only in the parasites. There are a number of possible explanations for this. One possibility is that under physiological conditions the analogs are unable to gain access to the Jurkat cell cytosol and hence the enzyme. An alternative explanation is that phosphorylation does not contribute significantly to the observed accumulation of pantothenate in mammalian cells and that accumulation occurs instead primarily via an electrogenic membrane transport process (12) that is unaffected by the analogs.

In a preliminary experiment, five of the analogs were tested for in vivo antiplasmodial activity in P. vinckei vinckei-infected mice. Unfortunately, signs of toxicity in some of the mice treated with compounds 3 to 6 required the in vivo experiment to be abandoned. Pantothenol, when administered orally to P. vinckei vinckei-infected mice, reduces parasite proliferation (13). It would therefore be of interest to determine whether pantothenic acid analogs similar to those in this series (but which do not display toxicity in vivo) have the potential to be orally active antimalarial agents.

In this paper, we have described a series of pantothenic acid analogs which inhibit pantothenate kinase and repress P. falciparum proliferation in vitro. The antiplasmodial activity and the high affinity with which the analogs in this series bind the target enzyme raise the possibility that further investigation into the antiplasmodial activity of similar pantothenic acid analogs could yield potent inhibitors of P. falciparum.


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ACKNOWLEDGMENTS
 
This work was supported by a grant from the Australian National Health and Medical Research Council (224245).

We are grateful to the Canberra Branch of the Australian Red Cross Blood Service for the provision of blood, to Daiichi Pharmaceutical Co. Ltd., Japan, for the provision of pantothenol and ethyl pantothenol (compound 2), to John Allen, Gordon Lockhart, and Anitha Jeyasingham of the ANU Mass Spectrometry Unit for their assistance with mass spectrometry, to Sasha Melnitchenko and Viki Withers of the ANU Analytical Unit for their assistance with microanalysis, and to Isabelle Ferru for her assistance with the in vivo experiment.


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FOOTNOTES
 
* Corresponding author. Mailing address: School of Biochemistry and Molecular Biology, The Australian National University, Canberra ACT 0200, Australia. Phone: 61 2 6125 7549. Fax: 61 2 6125 0313. E-mail: kevin.saliba{at}anu.edu.au. Back

{dagger} Supplemental material for this article may be found at http://aac.asm.org/. Back


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Antimicrobial Agents and Chemotherapy, November 2005, p. 4649-4657, Vol. 49, No. 11
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.11.4649-4657.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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