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Targeting Malaria with Polyamines Andrew J. Geall,*,† John A. Baugh,‡ Mark Loyevsky,§ Victor R. Gordeuk,§ Yousef Al-Abed,| and Richard Bucala⊥ Vical Inc., 10390 Pacific Center Court, San Diego, California 92121, The Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland, Center for Sickle Cell Disease/ Department of Microbiology, Howard University College of Medicine, Washington, D.C. 20012, Laboratory of Medicinal Chemistry, North Shore-LIJ Research Institute, Manhasset, New York 10030, and Department of Medicine, and Pathology, Yale University School of Medicine, P.O. Box 208031, New Haven, Connecticut 06520. Received February 21, 2004; Revised Manuscript Received September 13, 2004
During the asexual cycle of Plasmodium falciparum within the host erythrocyte, the parasite induces a stage-dependent elevation in the levels of polyamines by increased metabolism and uptake of extracellular pools. Polyamine amides of N-methylanthranilic acid have been synthesized which have embedded within them putrescine, spermidine, or spermine and from a charge perspective mimic natural polyamines. The interaction of these polyamine conjugates with human erythrocytes infected with malaria is described using fluorescent microscopy. The fluorescent spermine mimic was the only probe to show measurable intracellular accumulation. This was observed in late stage development but not in the ring stages or in uninfected erythrocytes.
INTRODUCTION
Putrescine 1, spermidine 2, and spermine 3 (Chart 1) are naturally occurring polyamines present in all mammalian cell types and are known to be essential for cell growth (1, 2). Changes in the concentration of intracellular polyamine pools are achieved by regulation of a biosynthetic pathway, a catabolic pathway, and an active transport system (1-4). Human erythrocytes contain only trace amounts of polyamines and lack the biosynthetic enzymes for their production (5, 6) but are capable of polyamine transport in vitro (7). During the asexual cycle of Plasmodium falciparum, the parasite induces numerous biochemical, structural, and functional changes in the host erythrocyte. In particular, the parasite induces a stage-dependent elevation in the levels of polyamines by increased metabolism (5, 6) and uptake of extracellular pools (6, 8). Polyamine metabolism by Plasmodium differs from the metabolism in mammalian cells (6). A bifunctional enzyme, ornithine decarboxylase-S-adenosylmethionine decarboxylase, has been characterized (9), and in the plasmodial resource database, PlasmoDB (http://plasmoDB.org), a putative spermidine synthase (gene PF_0301), has been identified (10). The existence of deoxyhypusine synthase and homospermidine synthase has also been established (11); these enzymes are involved in spermidine metabolism in P. falciparum. The biosynthetic steps for the synthesis of putrescine and spermidine have therefore been identified, but the presence of the enzyme spermine synthase and the ability to synthesize spermine is questionable (6), although the * To whom correspondence should be addressed. Tel: 858 646 1260. Fax: 858 646 1150. E-mail:
[email protected]. † Vical Inc. ‡ University College Dublin. § Howard University College of Medicine. | North Shore-LIJ Research Institute. ⊥ Yale University School of Medicine.
Chart 1. Structures of Putrescine 1, Spermidine 2, Spermine 3, and N-Methylanthranilic Acid 4
parasitized erythrocytes contain appreciable amounts of this polyamine (5). During the growth of a malarial parasite, the membrane transport characteristics of the host erythrocyte are modified (6, 8, 12). Increasing evidence suggests that targeting the parasite’s polyamine biosynthetic pathway to inhibit growth, while attractive pharmacologically, may be problematic because of an influx of polyamine from extracellular pools (3, 5, 6, 8, 12). This polyamine transport system remains poorly characterized and it is unclear if the parasite is using the endogenous erythrocytic system or a parasite-derived pathway, although a simian putrescine transporter has been reported in Plasmodium knowlesi-infected erythrocytes (8). The presence of a spermine transport pathway seems likely considering the elevated levels of this polyamine during proliferation (5) and the inability of the parasite to synthesize the molecule biosynthetically. The mammalian polyamine transporter appears to tolerate a degree of structural substrate diversity, but it is quite distinct from systems transporting other cellular components, such as amino acids. Generally, the number of charges, the natural polyamine spacing (propylene or butylene), and a nonbranched structure correlate with high affinity for the transporter (4, 13-16). Fluorescent polyamine conjugates (13, 17) are promising leads for
10.1021/bc0499578 CCC: $27.50 © 2004 American Chemical Society Published on Web 10/07/2004
1162 Bioconjugate Chem., Vol. 15, No. 6, 2004 Chart 2. Structures of the Target Polyamine Amide N-Methylanthranilic Acid Conjugates 5-8
probing and understanding the polyamine transporter for targeted drug delivery. Herein we report the design and controlled synthesis of fluorescent polyamine amides 6-8 (Chart 2) of 2-methylaminobenzoic acid 4 (N-methylanthranilic acid or MANT, Chart 1) that mimic both the charge and regiochemical distribution of natural polyamine substrates 1-3 (Chart 1). We also report the synthesis of fluorescent conjugate 5 (Chart 2), which contains a single primary amine and can be used along with MANT 4 (Chart 1) as nonpolyamine controls. The uptake, via the mammalian polyamine transporter, of conjugates 4-8 into a rapidly proliferating leukemic T-cell line is characterized and shown to be consistent with the literature (13). Transport of conjugates 4-8 into malaria-infected erythrocytes at different stages of maturation is described using fluorescent microscopy. EXPERIMENTAL PROCEDURES
Materials. Analytical grade reagents were purchased from major suppliers. Methods. Details of NMR, fluorescent, and mass spectroscopy are supplied as Supporting Information. Chemical analysis data for compounds 4-8 are supplied as Supporting Information. Cell Culture. Jurkat T cells were cultured at a density of 0.2-0.8 million per milliliter in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) under an atmosphere of 5% CO2-95% air at a temperature of 37 °C. Intracellular accumulation of MANT conjugates was determined in viable Jurkat cells using a FACS Vantage SE flow cytometer. UV excitation coupled with a 424/44 band-pass detection filter allowed visualization of fluorescence on the FL5 channel. Cells were cultured overnight in the presence of the fluorescent probes (50 µM). Prior to FACS analysis, nonspecific membraneassociated compounds were dissociated by incubation for 2 min in 1 mM free spermine (TFA salt) followed by repeated washing in PBS. 3 H-Hypoxanthine Proliferation Assay. The in vitro antimalarial activity of compounds 4-8 was evaluated on the growth of ring-stage-synchronized Plasmodium falciparum (7G8 strain) malaria cultures1 by incorporation of 3H-hypoxanthine. Erythrocyte suspensions with ring-stage-synchronized parasites were distributed into 24-well plates at 2% parasitemia and 2% hematocrit and compounds 4-8 were added at 1, 10, and 100 µm concentrations in triplicate. The plates were incubated in a candle jar at 37 °C for 24 h. 3H-Hypoxanthine was
Geall et al. Chart 3. Structures of the Intermediates in the Synthesis of Polyamine Amide N-Methylanthranilic Acid Conjugates 6
then added to each well at a final activity of 5 µCi/mL. After an additional 24-h incubation period, the cells were harvested onto filters, dried in the oven, transferred to vials with the scintillation fluid, Fluosafe (Fisher Scientific Co., Norcross, GA), and counted on a Beckman LS5000CE scintillation counter. Fluorescent Microscopy. Fluorescent microscopy was used to visualize the intracellular accumulation of the fluorescent probes 4-8 in in vitro Plasmodium falciparum (7G8) cultures1 at 2% hematocrit, 8% parasitemia. Compounds 4-8 at 5 µM concentrations were incubated for 17 h and then separated on a Percoll gradient into mature, ring-infected and -uninfected erythrocytes. Cells were fixed with 4% paraformaldehyde and 0.05% glutaraldehyde and air-dried on polylysine-coated slides. The slides were washed in 1 mM unlabeled spermine in PBS (20 min) and PBS (10 min) to remove nonspecifically bound fluorochrome and mounted using Vectashield (Vector Laboratories, Burlingame, CA). Images were then taken using a 100× oil immersion objective with an Olympus IX70 inverted microscope. A 360/40 band-pass excitation filter was used in conjunction with a multipass emission filter which allowed visualization of the 435 nm emission of MANT. (2-Methylaminobenzoyl)-4-amino-4-aza-butane (5). Putrescine 1 (Chart 1) was selectively N1-acylated with MANT 4 (Chart 1) (1.0 equiv), mediated by DCC (1.5 equiv) and catalytic 1-hydroxybenzotriazole (HOBt) (0.2 equiv, DCM, 25 °C, 72 h), and purified by RP-HPLC over Supelcosil ABZ+Plus (25 cm × 10 mm, 5 µm) (solvent A: 0.1% TFA in MeCN, solvent B: 0.1% aq TFA, gradient elution, 100% A to 65% A, over 20 min, λ ) 340 nm) to afford, after lyophilization, the polytrifluoroacetate salt of polyamine amide 5 (Chart 2, 27%). (2-Methylaminobenzoyl)-1,8-diamino-4-azaoctane (6). Spermidine 2 (Chart 1) was protected as the hexahydropyrimidine 9 (Chart 3), in quantitative yield, as previously described (18). The free primary amino functional group was then orthogonally protected with ethyl trifluoracetate (1.2 equiv, in MeOH, at -78 °C for 1 h and then warmed to room temperature over 1 h) to afford, without purification, the N1-trifluoroacetamide intermediate 10 (Chart 3). N-Acylation, with MANT 4 1 P. falciparum strain 7G8 was used in all experiments. Parasites were maintained in cultures of RPMI-1640 medium supplemented with 25 mM HEPES, 23 mM NaHC03, 10 mM glucose, 10% (v/v) heat-inactivated human plasma (0+ or A+), and washed human erythrocytes (A+) at 2% hematocrit. The growth medium was replaced daily and cultures were gassed with a mixture of 90% N2, 5% CO2, and 5% O2. The morphological characteristics of the parasites and the level of parasitemia were determined by microscopic examination of Giemsa-stained thin blood smears. Alanine lyses erythrocytes infected with the trophozoite stage parasite, leaving intact only erythrocytes with ring-stage parasites. Synchronization to the ring stage was therefore achieved by lysis of cells bearing mature parasites with isoosmotic solution containing 300 mM alanine, 10 mM TrisHCl, pH 7.4.
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(Chart 1) afforded, after purification over silica gel (DCMMeOH 25:1 to 20:1 v/v), the fully protected acylated spermidine intermediate 11 (Chart 3, 81.6%). The TFA protecting group was then cleaved by stirring in concentrated methanolic ammonia (25 °C, 48 h) to afford, after purification over silica gel (DCM-MeOH-concentrated NH4OH 50:10:1 to 40:10:1 v/v/v), the hexahydropyrimidine intermediate 12 (Chart 3, 53%). Treatment with malonic acid (3.6 equiv) and pyridine (3.1 equiv, dry MeOH, reflux, 2 h) (19), afforded, after purification by RP-HPLC, the polytrifluoroacetate salt of polyamine amide 6 (Chart 2, 60%). This deprotection step resulted in a change of retention time by RP-HPLC from 11.5 (compound 12) to 7.6 min (compound 6) ( Supelcosil ABZ+Plus, 5 µm, 15 cm × 4.6 mm, solvent A:0.1% TFA in MeCN, solvent B:0.1% aq. TFA, gradient elution 100% A to 65% A over 20 min, 1.5 mL/min, λ ) 340 nm) loss of the required mass by mass spectroscopy and a 13C NMR spectrum consistent with the required unsymmetrical polyamine amide (20). (2-Methylaminobenzoyl)-1,12-diamino-4,9-diazadodecane (7). N1,N2,N3-Tri-tert-Boc-spermine was synthesized as previously described (21). N-Acylation with MANT 4 afforded, after purification over silica gel (petroleum ether-EtOAc 80:20 to 50:50 v/v), the Bocprotected acylated spermine intermediate (74%). Deprotection with TFA (50:50 TFA-DCM v/v, 25 °C, 2 h) gave, after RP-HPLC purification and lyophilization, the polytrifluoroacetate salt of polyamine amide 7 (Chart 2, 65%). (2-Methylaminobenzoyl)-1,16-diamino-4,8,13-triazahexadecane (8). Incorporation of an intact spermine moiety was achieved using a previously described spermine desymmetrization protocol (22) by reductive alkylation of N1,N2,N3-tri-tert-Boc-spermine with 6-benzyloxycarbonylaminohexanal to afford (N1,N4,N9,N13-tetratert-Boc)-1,19-diamino-4,9,13-triazanonadecane. N-Acylation (88%), poly-Boc deprotection, RP-HPLC purification, and lyophilization afforded the polytrifluoroacetate salt of polyamine amide 8 (Chart 2, 65%). The pKas of polyamines are a function of the interamine distance as well as their substituents (18), so a six-carbon spacer rather than the previously described three-carbon spacer was introduced using aminohexanal. RESULTS
Polyamine amides 5-8 (Chart 2) are homogeneous by RP-HPLC and have been characterized by electrospray MS, 1H, 13C, and HETCOR NMR, and the polyamine substituents were consistent with previously published spectroscopic data of polyamine amides and carbamates (21, 22). The flow cytometry data are shown in Figure 1 and indicate that compounds 6-8 (Chart 2) are all sequestered into viable Jurkat cells. The population shift indicates that approximately 95% of all cells within each population are actively accumulating the fluorescent polyamine analogues. In accord with these data, we have shown that the remaining 5% of the cell population is accounted for by dying cells, as determined by propidium iodide uptake using flow cytometery (data not shown). As approximately 100% of live cells were shown to actively sequester the polyamine analogues, we have used the measure of mean fluorescence intensity (MFI) to give a quantifiable comparison of the degree of uptake of each of the test compounds. These data, Table 1, show that the dicationic putrescine mimic, compound 6, is the most highly sequestered (MFI 372.26), followed by the spermine mimic, compound 8 (MFI 359.69) and then to a lesser degree the spermidine mimic, compound 7 (MFI 204.65). According to flow cytometery, accumulation of
Figure 1. FACS analysis of polyamine amide N-methylanthranilic acid conjugates 5-8 uptake in Jurkat cells. The figure shows fluorescence of MANT conjugate in solid purple with control fluorescence of free MANT-treated cells overlaid in the broken line. Table 1. Mean Fluorescence Intensity of Viable Jurkat Cells compound
mean fluorescence Intensity
4 5 6 7 8
22.51 12.23 372.26 204.65 359.69
the monocationic amine 5 and unconjugated MANT 4 is at background levels. These data are consistent with our results obtained with fluorescence microscopy and fluorimetric analysis of cell lysates (see Supporting Information). Fluorimetry was also used to show that uptake of conjugate 6 was energy-, time-, and concentration-dependent and was competitive with the natural substrates (see Supporting Information). The indication of an active transport system, rather than passive diffusion across a lipid membrane (4), correlates with literature reports of polyamine-MANT conjugates using the polyamine transport pathway (13). The in vitro antimalarial activity of compounds 4-8 was evaluated on the growth of ring-stage-synchronized Plasmodium falciparum (7G8 strain) malaria cultures1 by incorporation of 3H-hypoxanthine. Figure 2 shows that none of the compounds had antimalarial activity, and microscopic examination of Giemsa-stained, thin blood smears showed normal morphological characteristics of
Figure 2. In vitro incorporation of 3H-hypoxanthine into ringstage synchronized Plasmodium falciparum (7G8 strain) for compounds 4-8: (open) 1 µM, (grey) 10 µM, (hashed) 100 µM as compared to control (black).
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Figure 3. Uptake of polyamine MANT conjugate 8 by intraerythrocytic P. falciparum trophozoites as visualized by fluorescent microscopy.
the parasites (data not shown). Thus, these fluorescent polyamine conjugates do not interfere with the normal metabolic function of the malaria parasite within the host erythrocyte and are therefore ideal probes. Fluorescence microscopy was then used to visualize the intracellular accumulation of the fluorescent probes. Compounds 4-7 showed no intracellular accumulation at any of the stages of development of the parasite. However, using fluorescent microscopy, the spermine mimic 8 showed intracellular accumulation in late stage development but not in the ring stages or in uninfected erythrocytes. Figure 3 (right panel) shows the normal phase photograph of trophozoite-infected erythrocytes. The parasites are unstained but the hemozoin within the food vacuole can be seen. The fluorescent image (Figure 3, left panel) shows accumulation of the probe within the cytoplasm of the parasite, which is colocalized with the hemozoin. DISCUSSION
Previously, the uptake of polyamine-N-methylanthranilic acid conjugates into mammalian cells via the polyamine transporter has been characterized (13). The MANT fluorophore shows λmax 250 nm ( 5400) and 333 nm ( 2100) and an emission frequency of 436 nm (excitation 341 nm) and is unlikely to perturb the interactions and subsequent distribution of the polyamine analogue (13). We have synthesized polyamine amide conjugates that have embedded within them putrescine, spermidine, or spermine, and from a charge perspective mimic the natural polyamines. We have characterized their uptake into a human leukemic T-cell line (rapidly proliferating, metabolically active leukemia of lymphoid origin) and shown them to be consistent with literature reports of uptake via the polyamine transporter for MANT conjugates (13). Fluorescence microscopy studies with malaria infected erythrocytes showed only measurable accumulation of the spermine mimic (8). Malaria parasites are capable of synthesizing spermidine and putrescine (5, 6, 8, 9), but it is questionable whether they are capable of synthesizing spermine (6). This fluorescent probe, from a charge perspective, is a mimic of spermine, and we speculate that its accumulation is via the polyamine transporter, although more extensive studies will be required to confirm this. These fluorescent probes will find considerable use in the evaluation of this transport system in malaria for targeted drug therapy. ACKNOWLEDGMENT
We thank Dr. Simon Carrington (University of Bradford, U.K.) for some useful synthetic discussions. Studies were supported by NIH 1RO1AI051306 (R.B.),
RO1AI44857-04 (V.R.G.), UH1-HL03679-05 from National Heart, Lung and Blood Institute, the Office of Research on Minority Health, and by the Howard University General Clinical Research Center Grant M01RR10284. Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Tabor, C. W., and Tabor, H. (1984) Polyamines. Annu. Rev. Biochem. 53, 749-790. (2) Kramer, D. I., Miller, J. T., Bergeron, R. J., Khomutov, R., Knomutov, A., and Porter, C. W. (1993) regulation of polyamine transport by polyamines and polyamine analogs. J. Cell Physiol. 155, 399-407. (3) Marton, L. J., and Pegg, A. E. (1995) Polyamines as targets for therapeutic intervention. Annu. Rev. Pharmacol. Toxicol. 35, 55-91. (4) Seiler, N., Delcros, J. G., and Moulinoux, J. P. (1996) Review: Polyamine transport in mammalian cells. Int. J. Biochem. Cell Biol. 28, 843-861. (5) Assaraf, Y. G., Golenser, J., Spira, D. T., and Bachrach, U. (1984) Polyamine levels and the activity of their biosynthetic enzymes in human erythrocytes infected with the malaria parasite, Plasmodium falciparum. Biochem. J. 222, 815-819. (6) Mu¨ller, S., Coombs, G. H., and Walter, R. D. (2001) Targeting polyamines of parasitic protozoa in chemotherapy. Trends Parasitol. 17, 242-249. (7) Moulinoux, J.-P., Le Calve, M., Quemener, V., and Quash, G. (1984) In vitro studies on the entry of polyamines into normal red blood cells. Biochimie 66, 385-393. (8) Singh, S., Puri, S. K., Singh, S. K., Srivastava, R., Gupta, R C., and Pandey, V. C. (1997) Characterization of simian malarial parasite (Plasmodium falciparum)-induced putrescine transport in rhesus monkey erythroctes. J. Biol. Chem. 272, 13506-13511. (9) Muller, S., Da’dara, A., Luersen, K., Wrenger, C., Das Gupta, R., Madhubala, R., and Walter, R. D. (2000) In the human malaria parasite Plasmodium falciparum, polyamines are synthesized by a bifunctional ornithine decarboxylase, sadenosylmethionine decarboxylase. J. Biol. Chem. 11, 80978102. (10) Le Roch, K. G., Zhou, Y., Blair, P. L., Grainger, M., Moch, J. K., Haynes, J. D., De La Vega, P., Holder, A. A., Batalov, S., Carucci, D. J., and Winzeler, E. A. (2003) Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 301, 1503-1508. (11) Kaiser, A., Gottwald, A., Maier, W. and Seitz, H. M. (2003) Targeting enzymes involved in spermidine metabolism of parasitic protozoa - a possible new strategy for anti-parasitic treatment. Parasitol. Res. 91, 508-516. (12) Ginsburg, H., and Kirk, K. (1998) Membrane Transport in the Malaria-infected Erythrocyte. Malaria: Parasite Biol-
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Communications ogy, Pathogenesis, and Protection (Sherman, I. W., Ed.) pp 219-232, ASM Press, Washington, DC. (13) Cullis, P. M., Green, R. E., Merson-Davies, L., and Travis, N. (1999) Probing the mechanism of transport and compartmentalization of polyamines in mammalian cells. Chem. Biol. 6, 717-729. (14) Porter, C. W., Miller, J., and Bergeron, R. J. (1984) Aliphatic chain-length specificity of the polyamine transportsystem in ascites L1210 leukemia-cells. Cancer Res. 44, 126128. (15) Bergeron, R. J., McManis, J. S., Weimar, W. R., Schreier, K. M., Gao, F., Wu, Q., Oritiz-Ocasio, J., Luchetta, G. R., Porter, C., and Vinson, J. R. T. (1995) The role of charge in polyamine analogue recognition. J. Med. Chem. 38, 22782285. (16) Wang, C., Delcros, J.-G., Cannon, L., Konate, F., Carias, H., Biggerstaff, J., Gardner, R. A., and Phanstiel, O., IV. (2003) Defining the molecular requirements for selective delivery of polyamine conjugates into cells containing active polyamine transporters. J. Med. Chem. 46, 5129-5138. (17) Aziz, S. M., Yatin, M., Worthen, D. R., Lipke, D. W., and Crooks, P. A. (1998) A novel technique for visualizing the intracellular localization and distribution of transported polyamines in cultured pulmonary artery smooth muscle cells. J. Pharm. Biomed. Anal. 17, 307-320.
(18) Ganem, B., and Chantrapromma, K. (1983) Preparation of thermospermine. Methods Enzymol. 94, 416-418. (19) Lurdes, M., Almeida, S., Grehn, L., and Ragnarsson, U. (1988) Selective protection of polyamines: Synthesis of model compounds and spermidine derivatives. J. Chem. Soc., Perkin Trans. 1 1905-1911. (20) -1,8-diamino-4-azaoctane (6). Purified by RP-HPLC (polyTFA salt, tR ) 7 min, Supelcosil ABZ+Plus, 5 µm, 25 cm × 10 mm, solvent A: 0.1% TFA in MeCN, solvent B: 0.1% aq. TFA, gradient elution 100% A to 65% A over 20 min, 6.0 mL/ min, λ ) 340 nm) as a white powder. 13C NMR 67.9 MHz, [2H]6-DMSO, 23.2 (7-CH2); 24.7 (6-CH2); 26.5 (2-CH2); 29.9 (CH3); 36.6 (1-CH2); 38.8 (8-CH2); 45.4 (5-CH2); 46.6 (3-+CH2); 111.2 (1′-C); 114.6 (3′-CH2); 115.4 (5′-CH2); 128.8 (6′-CH2); 133.0 (4′-CH2); 150.5 (2′-C); 170.0 (CCONH). ESI/MS found 279, (M+ + 1), C15H27N4O1 requires M+ + 1 ) 279. (21) Geall, A. J., Taylor, R. J., Earll, M. E., Eaton, M. A. W., and Blagbrough, I. S. (2000) Synthesis of cholesteryl polyamine carbamates: pKa studies and condensation of calf thymus DNA. Bioconjugate Chem. 11, 314-326. (22) Geall, A. J., and Blagbrough, I. S. (2000) Homologation of polyamines in the rapid synthesis of lipospermine conjugates and related lipoplexes. Tetrahedron 56, 2449-2460.
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