Antiplasmodial activity of a series of 1,3,5-triazine-substituted polyamines

Burkhard Klenke1, Michael P. Barrett2, Reto Brun3 and Ian H. Gilbert1,*

1 Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF; 2 Institute of Biomedical and Life Sciences, Division of Infection and Immunity, University of Glasgow, Glasgow G12 8QQ, UK; 3 Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland

Received 30 August 2002; returned 10 January 2003; revised 23 January 2003; accepted 29 April 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Polyamine biosynthesis and function has been shown to be a good drug target in some parasitic protozoa and it is proposed that the pathway might also represent a target in the malaria parasite Plasmodium falciparum. A series of 1,3,5-triazine-substituted polyamine analogues were tested for activity against Plasmodium falciparum in vitro. The series showed activity against the parasites and were generally more active against the chloroquine-resistant line K1 than the chloroquine-susceptible line NF54. Simple unbranched analogues had better activity than analogues carrying branched or cyclic central chains. Addition of multiple triazine units in general led to increased activity of the compounds.

Keywords: Plasmodium falciparum, malaria, parasites, protozoa, polyamine metabolism


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Malaria remains one of the principal diseases of the developing world with an estimated 500 million cases each year and around two million deaths.1 In the absence of effective vaccines, chemotherapy represents the mainstay of malaria control. Resistance to currently used antimalarials, particularly chloroquine and the antifolates, is seriously impeding efforts to control the disease.2 New drugs are urgently needed in the fight against malaria. Polyamine metabolism has been shown to be a good target for chemotherapeutic intervention in diseases caused by other protozoa such as human African trypanosomiasis,3 where the ornithine decarboxylase inhibitor eflornithine has been registered for use against late-stage disease.4

Polyamines are essential for cell proliferation and differentiation. Interference with their biosynthesis or function can block cellular growth and the polyamine pathway has been investigated with regard to anticancer therapy.5 Malaria parasites have a great propensity for rapid proliferation, and interference with polyamine function in these cells is likely to be detrimental. Eflornithine, used in conjunction with bis(benzyl)polyamine analogues, cured rodent malaria models,6 although used alone eflornithine has no curative affect in human malaria. An S-adenosylmethionine decarboxylase inhibitor, methylglyoxalbis(guanyl) hydrazone (MGBG), blocked growth of the erythrocytic stages of Plasmodium falciparum in vitro.7 Moderate antiplasmodial activity was also seen with a series of N-alkylated putrescine derivatives,8 and dicyclohexylamine, an inhibitor of spermidine biosynthesis, also showed antiplasmodial activity.9

We previously reported a series of 1,3,5-triazine-substituted polyamine analogues, which were developed specifically to interfere with polyamine metabolism in African trypanosomes.10 Several of the compounds showed marked trypanocidal activity. We report here the evaluation of the activity of this series against P. falciparum in vitro.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The syntheses of the compounds has been reported previously.10 In vitro activity against erythrocytic stages of P. falciparum was determined using a [3H]hypoxanthine incorporation assay.11,12 One strain suscep- tible to known antimalarial drugs (P. falciparum NF54) and another resistant to chloroquine and pyrimethamine (P. falciparum K1) were used in the assays, and all test compounds were compared for activity with the standard drugs chloroquine (Sigma C6628) and artemisinin (arteannuin, qinghaosu; Sigma 36,159-3). Compounds were dissolved in dimethyl sulfoxide at 10 mg/mL and added to parasite cultures incubated in RPMI 1640 medium without hypoxanthine, supplemented with HEPES (5.94 g/L), NaHCO3 (2.1 g/L), neomycin (100 U/mL) + Albumax (5 g/L) and washed human red cells A+ at 2.5% haematocrit (0.3% parasitaemia). Serial doubling dilutions of each drug were prepared in 96-well microtitre plates and incubated in a humidifying atmosphere at 37°C; 4% CO2, 3% O2, 93% N2.

After 48 h, 50 µL of [3H]hypoxanthine (0.5 µCi) was added to each well of the plate. The plates were incubated for a further 24 h under the same conditions. The plates were then harvested with a Betaplate cell harvester (Wallac, Zurich, Switzerland), and the red blood cells were transferred onto a glass fibre filter and then washed with distilled water. The dried filters were inserted into a plastic foil with 10 mL of scintillation fluid, and counted in a Betaplate liquid scintillation counter (Wallac). IC50 values were calculated from sigmoidal inhibition curves using Microsoft Excel.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The structures of the compounds are shown in Figure 1. Compounds were tested against two strains of P. falciparum. The strains used were NF54, which is susceptible to known antimalarial drugs, and K1, which is resistant to chloroquine and pyrimethamine (Table 1). Compounds were generally more active against the resistant K1 strain than against the susceptible NF54 strain. For simple substituted derivatives, compound 1, activity increased as the length of the central chain increased in the order butyl < nonyl < dodecyl. Branched chain and cyclic central chains had little activity. No major effect was noted on increasing the methylation of the triazine ring for compounds 2a–d and 2e–h. However, against NF54 strain, there was a small increase in activity on methylation, whilst for the K1 strain, there is a small decrease in activity on methylation. No major effect was seen on having one triazine unit compared with two. In general the polyamines substituted with one triazine appeared less active than those substituted with two triazines (comparing 2e–h with 3b–e). The most effective compounds were tetra-substituted methylene-bridged compounds 4, especially where they were methylated. These compounds also showed best activity against Trypanosoma brucei.10 In vitro data did not point to cytotoxicity; however, acute toxicity, manifest in muscular tremor, motor-function disorder and distress that necessitated immediate killing of the animal was observed when compounds 2e, 4b, 4c or 4e were applied to mice at concentrations >10 mg/kg, precluding further in vivo evaluation. Thus in this case, the cellular assay was not a good indicator of toxicity towards the animal. The toxicity may be caused by an effect on a specific signally event or receptor, rather than general cytotoxicity.



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Figure 1. Structures of the 1,3,5-triazine-substituted polyamines used in this study.

 

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Table 1.  Antimalarial activity of compounds against NF54 and K1 strains of P. falciparum
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this report we show that triazine-substituted polyamine analogues have antiplasmodial activity against erythrocytic stages of the parasite in vitro. It was previously shown6 that bis(benzyl) polyamine analogues had activity against malaria parasites in the 0.2–14 µM range, and we demonstrate here that these triazine-substituted compounds are also active in this range. N-alkyl putrescine derivatives have also been shown to be weakly active against Plasmodium, although benzyl derivates were shown to give improved activity in this case as well. Interestingly, chloroquine/pyrimethamine-resistant parasites are more susceptible to these analogues than are susceptible parasites; however, we have not made mechanistic studies that can be used to help interpret this observation. In another study evaluating the effects of spermidine biosynthesis inhibition,9 data were presented showing a 3-fold higher spermidine content in wild-type NF54 parasites compared with a chloroquine-resistant strain. If polyamine deficiency were a common phenotype of chloroquine-resistant lines this could explain the enhanced susceptibility of these cells to polyamine analogues, which might act by displacing polyamines from their normal roles in cells.

However, it is currently not clear how these triazine-substituted polyamine analogues actually exert their action. In rodents, Plasmodium yoelii-infected erythrocytes have been shown to have enhanced polyamine uptake.13 The parasites may induce specific transporters for these metabolites and Plasmodium knowlesi-infected erythrocytes have been shown to have an up-regulated putrescine-specific uptake system.14 It is also possible that the triazine-substituted analogues are preferentially taken into Plasmodium-infected erythrocytes via the new permeation pathway that is induced at the plasma membrane of infected erythrocytes.15 It will be of interest to determine how these analogues enter infected erythrocytes and exert a toxic effect on the parasites. We cannot rule out the possibility that the compounds are oxidized by serum components prior to exerting a toxic effect. Interestingly, it has recently been shown that pentamidine and other diamidine compounds are active against malaria parasites grown in vitro,16 with efficacy in the submicromolar range. It has been proposed that diamidines exert their plasmocidal effect by binding to ferriprotoporphyrin IX and preventing the polymerization of haem that accumulates to toxic levels in these parasites. It is feasible that the triazine-substituted polyamine analogues could act in a similar fashion. Further work is required to elucidate modes of action of these compounds.13


    Acknowledgements
 
The technical assistance of C. Scheurer and J. Easterbrook is acknowledged. We would also like to acknowledge the Wellcome Trust and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) for funding.


    Footnotes
 
* Corresponding author. Tel: +44-29-2087-5800; Fax: +44-29-2087-4149; E-mail: gilbertih{at}cf.ac.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Greenwood, B. & Mutabingwa, T. (2002). Malaria in 2002. Nature 415, 670–2.[CrossRef][ISI][Medline]

2 . Wongsrichanalai, C., Pickard, A. L., Wernsdorfer, W. H. et al. (2002). Epidemiology of drug-resistant malaria. Lancet Infectious Diseases 2, 209–18.[CrossRef][ISI][Medline]

3 . Laurence, J., Marton, L. J. & Pegg, A. E. (1995). Polyamines as targets for therapeutic intervention. Annual Review of Pharmacology and Toxicology 35, 55–91.[CrossRef][ISI][Medline]

4 . Bacchi, C. J., Nathan, H. C., Hutner, S. H. et al. (1980). Polyamine metabolism: a potential therapeutic target in trypanosomes. Science 210, 332–4.[ISI][Medline]

5 . Barrett, S. V. & Barrett, M. P. (2000). Anti-sleeping sickness drugs and cancer chemotherapy. Parasitology Today 16, 7–9.[CrossRef][ISI][Medline]

6 . Bitonti, A. J., Dumont, J. A., Bush, T. L. et al. (1989). Bis(benzyl)polyamine analogs inhibit the growth of chloroquine-resistant human malaria parasites (Plasmodium falciparum) in vitro and in combination with alpha-difluoromethylornithine cure murine malaria. Proceedings of the National Academy of Sciences, USA 86, 651–5.[Abstract]

7 . Wright, P. S., Byers T. L., Cross-Doersen, D. E. et al. (1991). Irreversible inhibition of S-adenosylmethionine decarboxylase in Plasmodium falciparum-infected erythrocytes: growth inhibition in vitro. Biochemical Pharmacology 41, 1713–8.[CrossRef][ISI][Medline]

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9 . Kaiser, A., Gottwald, A., Wiersch, C. et al. (2001). Effect of drugs inhibiting spermidine biosynthesis and metabolism on the in vitro development of Plasmodium falciparum. Parasitology Research 87, 963–72.[ISI][Medline]

10 . Klenke, B., Stewart, M., Barrett, M. P. et al. (2001). Synthesis and biological evaluation of s-triazine substituted polyamines as potential new anti-trypanosomal drugs. Journal of Medicinal Chemistry 44, 3440–52.[CrossRef][ISI][Medline]

11 . Desjardins, R. E., Canfield, C. J., Haynes, D. et al. (1979). Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrobial Agents and Chemotherapy 16, 710–8.[ISI][Medline]

12 . Matile, H. & Pink, J. R. L. (1990). Plasmodium falciparum malaria parasite cultures and their use in immunology. In Immunological Methods (Lefkovits, I. & Pernis, B., Eds), pp. 221–34. Academic Press, San Diego, CA, USA.

13 . Muller, S., Coombs, G. H. & Walter, R. D. (2001). Targeting polyamines of parasitic protozoa in chemotherapy. Trends in Parasitology 17, 242–9.[CrossRef][ISI][Medline]

14 . Singh, S., Puri, S. K., Singh, S. K. et al. (1997). Characterization of simian malarial parasite (Plasmodium knowlesi)-induced putrescine transport in rhesus monkey erythrocytes. A novel putrescine conjugate arrests in vitro growth of simian malarial parasite (Plasmodium knowlesi) and cures multidrug resistant murine malaria (Plasmodium yoelii) infection in vivo. Journal of Biological Chemistry 272, 13506–11.[Abstract/Free Full Text]

15 . Kirk, K., Staines, H. M., Martin, R. E. et al. (1999). Transport properties of the host cell membrane. Novartis Foundation Symposium 226, 55–66; discussion 66–73.

16 . Stead, A. M., Bray, P. G., Edwards, I. G. et al. (2001). Diamidine compounds: selective uptake and targeting in Plasmodium falciparum. Molecular Pharmacology 59, 1298–306.[Abstract/Free Full Text]





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