Introduction

The emergence of strains resistant to drugs used for prophylaxis and treatment of malaria with presently no vaccine available has contributed to the spread and growth of malaria to various tropical and subtropical regions of the world. Sophisticated defence strategies have evolved in parasitic organisms that enable them to deal with a broad range of foreign and endogenously derived toxic compounds. Most of these structurally diverse, non-polar molecules instead of undergoing modification for their utilisation are detoxified and eliminated by a phase II detoxification enzyme known as glutathione-S-transferase (GST, E.C. 2.5.1.18) which is found in most parasites (Jakoby and Ziegler 1990).

Native GST from Plasmodium yoelii has been purified and characterised (Ahmad and Srivastava 2007a), and GST of P. falciparum (Pf-GST1) has been recombinantly expressed and a substrate as well as inhibitor profiling has also been carried out on the recombinant protein (Harwaldt et al. 2002; Liebau et al. 2002). The enzyme exhibits moderate non-selenium-dependent glutathione peroxidase (GPX) activity and potentially contributes to the protection of the parasite during oxidative stress situations in the parasitised red blood cells. Consequently, it is thereby hypothesised that GST inhibitors would have antimalarial effects per se by preventing parasite resistance against chloroquine (Harwaldt et al. 2002).

Although the utility of this enzyme as a drug target in effectively combating malaria is yet to be established, the critical role played by Pf-GST1 in detoxification makes this enzyme a viable drug target against malaria. The present study investigates the relative susceptibility of GST from rodent malarial parasites P. yoelii to inhibition by various ‘non-substrate ligands’ such as protoporphyrin IX, cibacron blue and menadione (Md), which exhibit non-competitive inhibition towards GST. The data reported here have the potential to form the basis of structure-based design of selective inhibitors, which may serve as antimalarial drug leads.

Materials and methods

Chemicals

Cibacron blue, protoporphyrin IX and menadione sodium bisulfite were purchased from Sigma Chemical Co., USA. All chemicals used in the isolation of malarial parasites, preparation, activity determination and purification of GST were obtained as mentioned in Ahmad and Srivastava (2007a).

P. yoelii nigeriensis infection in Swiss albino mice

In vivo maintenance and isolation of malarial parasites P. yoelii was carried out according to Ahmad and Srivastava (2007a).

Preparation of GST from malarial parasites P. yoelii nigeriensis

GST was prepared from malarial parasites P. yoelii as reported earlier (Ahmad and Srivastava 2007a), and GST activity was determined following the method of Habig et al. (1974). Protein was estimated by the method of Lowry et al. (1951) using BSA as standard.

Purification of GST from P. yoelii

GST was purified from the cytosolic fraction of P. yoelii by the method published earlier (Ahmad and Srivastava 2007a).

Inhibition studies

In order to determine the dose-dependent effect of various inhibitors and their respective inhibitor constants (K i), the enzyme was incubated with varying concentrations of inhibitor for 10 min at room temperature in the presence of 100 mM potassium phosphate buffer and 1.0 mM glutathione (GSH). Reaction was initiated by the addition of 1.0 mM CDNB, and the absorbance at 340 nm was monitored for 5 min at 30-s intervals. The percentage inhibition of the enzyme activity by various inhibitors was calculated by comparing with a control tube. Results were expressed as mean±S.D. based on experiments done in quadruplicates.

Results

Effect of protoporphyrin IX on GST activity from P. yoelii

Protoporphyrin IX caused a concentration-dependent inhibition when studied for its effect in the concentration range of 0.25 to 200 μM (Fig. 1). It inhibited GST to the tune of 31.3% to 85.6% with an apparent K i of around 13 μM.

Fig. 1
figure 1

Inhibition profile of P. yoelii GST by protoporphyrin IX. Results were expressed as mean±S.D. based on experiments done in quadruplicates

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Effect of cibacron blue on GST activity from P. yoelii

The dye cibacron blue also exhibited a concentration-dependent inhibition profile when studied for its effect in the concentration range of 0.5 to 64 μM (Fig. 2). It inhibited GST to the tune of 94.3% at 64 μM concentration with an apparent K i of around 0.4 μM.

Fig. 2
figure 2

Inhibition profile of P. yoelii GST by cibacron blue. Results were expressed as mean±S.D. based on experiments done in quadruplicates

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Effect of menadione on GST activity from P. yoelii

Menadione (2-methyl-1, 4-naphthoquinone, vitamin K), a known inhibitor of mammalian glutamate cysteine ligase and glutathione reductase, has also been used to prevent vitamin K deficiencies, as well as to treat malaria (Ahmad and Srivastava 2007b). Md was found to inhibit P. yoelii GST activity when studied for its effect in the concentration range of 25 to 200 μM (Fig. 3). It inhibited GST to the tune of 3.9% to 43.2% with an apparent K i of around 80 μM.

Fig. 3
figure 3

Inhibition profile of P. yoelii GST by menadione. Results were expressed as mean±S.D. based on experiments done in quadruplicates

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Discussion

In the past decade, there have been great advances in our understanding of the biology of parasites. Recent discoveries regarding the physiology and biochemistry of protozoan and helminth parasites have elucidated many of the prospective targets that are unique to each parasite. Knowledge of the biochemical and molecular differences between the parasite and host and of the rate limiting points in the parasite metabolism are both necessary for the successful selection of prospective targets. Differences between the parasite and host enzymes may include differential sensitivity to inhibitors (Mansour 2002).

Ferriprotoporphyrin IX chloride (hemin), a known inhibitor of mammalian GST, has been demonstrated to exhibit non-competitive inhibition kinetics with respect to substrate GSH (Ahmad and Srivastava 2007a). Protoporphyrin IX and other tetrapyrroles are bound non-covalently (‘liganded’) to GST but not conjugated with reduced glutathione. The IC50 values, i.e. the concentrations of the inhibitors at which there is approximately 50% inhibition in the activity of the enzyme for protoporphyrin IX (12.5 μM), cibacron blue (56.3 μM) and menadione (>200 μM) for P. yoelii GST, were significantly different from those reported for GST from human placenta (Harwaldt et al. 2002).

The present study reports the in vitro effects of three compounds on malarial GST. Several other synthetic compounds belonging to various chemical classes showing in vitro GST inhibitory/modulatory activity have also been identified by us (Ahmad et al. 2007). A few of these compounds have been found to selectively inhibit the parasitic enzyme without affecting the activity of the host (mammalian) enzyme. In vivo experiments using Swiss albino mice are being carried on such compounds for proving the validity of the GST as a target for antimalarial drugs. It remains to be seen which of these compounds inhibit/modulate the activity of P. yoelii GST in vivo as well as the mechanism of inhibition. Inhibition of malarial GST is furthermore expected to act at different vulnerable metabolic sites of the parasite. The lack of functional Plasmodium GST is likely to disturb GSH-dependent detoxification processes, to enhance the levels of cytotoxic peroxides and possibly to increase the concentration of toxic hemin. With the Pf-GST structure and the features of the enzyme at hand, it might be possible to open new avenues for the development of novel antimalarial drugs and of drugs that help in antagonising chloroquine resistance.