(DFG, Be 1540/4-4). Cooperation with Prof. Heiner Schirmer, Biochemistry Center, Heidelberg, University
The malarial parasite Plasmodium falciparum multiplies in human hepatocytes and erythrocytes as well as in the midgut of the transmitting mosquito Anopheles. For the survival and growth of the parasite in these very different environments, glutathione homeostasis is of particular importance. The tripeptide glutathione (γ-Glu-Cys-Gly) serves to detoxify electrophilic pathophysiological compounds such as hemin and methylglyoxal and to quench reactive oxygen and nitrogen species. There is furthermore evidence that the detoxification of antimalarial drugs and thus mechanisms of resistance are linked to the glutathione system. Within the scope of this project, we plan to continue our work on the glutathione-dependent redox milieu and glutathione-dependent proteins of the malarial parasite, employing methods of protein biochemistry and enzyme kinetics as well as transcriptome and proteome analyses. As in the preceding project, we will focus on glutathione reductase, glyoxalase I, glyoxalase II, and the parasite-specific plasmoredoxin. These proteins are also of interest as drug targets. On the basis of three-dimensional structures which were elucidated during the last funding period or will be elucidated, further enzyme inhibitors, particularly subversive substrates, will be developed and tested. The clinical dose-finding studies for methylene blue, a subversive substrate of parasite disulfide reductases, will be supported by in vitro studies.
Oxidative stress in malarial parasites. Malarial parasites are continuously exposed to oxidative and nitrosative stress. Such stress might be exogenously produced by the immune response of the host as well as endogenously by the high metabolic rate of Plasmodium and hemoglobin degradation within the parasite. Oxidants derived from the host's diet are another potential source of exogenous oxidative stress. Over the last few years Plasmodium has been shown to possess a whole range of antioxidant defense mechanisms, namely a complete glutathione system comprising NADPH, highly active glutathione reductase (GR), glutathione, and different glutaredoxin-like proteins as well as a functional glutathione-dependent glyoxalase system and a glutathione S-transferase with peroxidase activity. Additionally, a complete thioredoxin system comprising NADPH, thioredoxin reductase (TrxR), different thioredoxin-like proteins, and thioredoxin-dependent peroxidases (TPx) has been characterized, and two functional superoxide dismutases as well as two lipoamide dehydrogenase-like proteins are present. The thioredoxin systems of the malaria vector Anopheles gambiae and the related insect Drosophila melanogaster were also characterized.
As indicated by erythrocyte glucose 6-phosphate dehydrogenase (G6PD) deficiency, limited availability of reducing equivalents in the form of NADPH confers protection from malaria. The mechanism of protection from severe malaria by G6PD deficiency is probably not growth inhibition of the parasite. Rather it involves the oxidation of hemoglobin to membrane-associated hemochromes, which finally leads to IgG-based early detection and degradation of parasitized G6PD-deficient erythrocytes.
Two major antioxidant enzymes, catalase and glutathione peroxidase, do not occur in the parasite. This constellation offers great potential for the development of chemotherapeutic agents that act by perturbing the redox equilibrium of the parasite. Additionally, the redox metabolism is involved in the pathology (virulence) and it has been demonstrated to play a major role in the action of and resistance to clinically used antimalarial drugs. Transcriptome analysis has been applied to the antioxidant protein network of Plasmodium falciparum under various conditions.
The glutathione system. As in many other organisms, the cysteine-containing tripeptide glutathione is the most abundant low molecular weight antioxidant in malarial parasites. At physiological ionic strength 25°C and pH 7.0, the standard redox potential of the GSSG/2GSH system is -240 mV. For conditions probably prevailing in the trophozoite cytosol (37°C and pH 7.2) a value of –270 mV has been estimated. Since the GSH concentration (1-5 mm) is much higher than that of NADPH (<100 µm) and Trx(SH)2 (<50 µm), the overall redox environment in the trophozoite results largely from the contribution of the glutathione redox couple. This implies that glutathione is the major redox buffer in the parasites. A high GSH/GSSG ratio is established by the flavoenzyme glutathione reductase, and GR is abundant in trophozoites, having an activity of up to 10 U/ml cytosol. Under conditions when GR activity is insufficient, GSSG can also be reduced by other processes such as protein thiol glutathionylation, by reduction with Trx(SH)2 (see below), and probably by dihydrolipoate-dependent reactions catalyzed by glutaredoxin. The system of GSSG export, compensated by highly efficient synthesis of intraparasitic GSH from the constituent amino acids, has a net role as an efficient GSSG reduction system.
Glutathione reductase. High levels of GSH in malarial parasites are NADPH-dependently maintained by the homodimeric FAD-containing enzyme GR. P. falciparum GR has been characterized biochemically and kinetically. A number of reports indicate that a lack of FAD, the prosthetic group of GR, or of the cosubstrate NADPH, as observed in glucose-6-phosphate dehydrogenase deficiency, lead to substantial protection from severe malaria. These clinical and epidemiological observations suggest that both human GR and PfGR are potential targets for the development of antimalarial drugs. PfGR is – at therapeutically used concentrations – the only verified enzymic target of the antimalarial drug methylene blue. As a novel class of potential antimalarial agents, double-headed prodrugs – containing a PfGR inhibitor linked via a hydrolase-labile ester bond to a 4-anilinoquinoline – were synthesized and successfully tested in vitro and in vivo. Like other members of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes, each subunit of PfGR contains a disulfide (Cys39 and Cys44) and a flavin that are in redox contact.
The catalytic mechanism of PfGR was studied in detail by Böhme et al. in 2000. The data indicate that EH4, the four-electron reduced form of the enzyme, is likely to be catalytically inactive and labile. The oxidized, stable, and intensely studied form of PfGR, Eox, is but a minor form in vivo whereas intermediate forms of the enzyme – such as EH2 x NADPH, S58-glutathionyl-PfGR – are likely to be accessible to specific inhibitors. EH4, an over-reduced standby form of the enzyme, and the glutathionylated enzyme probably occur exclusively in the parasite.
Human GR has been very well studied in terms of enzymology and crystal structure. The binding of substrates, substrate analogs, and inhibitors has also been investigated. Steps have already been taken to study the inhibitory properties of various drugs on human GR. These inhibitors are safranine, isoalloxazines, menadione, and xanthene derivatives. They non-competitively inhibit human GR by binding in the cavity at the dimer interface. It has been proposed that this cavity is also the binding site of methylene blue in PfGR. Therefore a valuable stepping-stone for the development of rational drugs designed against PfGR was to have the three-dimensional structure of the enzyme (see below).
Glyoxalases. The glyoxalase system consists of glyoxalase I (GloI), glyoxalase II (GloII), and the coenzyme glutathione. It is a cyclic metabolic pathway that removes toxic 2-oxoaldehydes such as methylglyoxal by converting them to the corresponding non-toxic 2-hydroxycarboxylic acids such as D-lactate. Glyoxalase I catalyzes the formation of S-2-hydroxyacylglutathione (a thioester of GSH); glyoxalase II then hydrolyzes the ester, thus producing GSH and a free 2-hydroxycarboxylic acid. With the absence of a typical citric acid cycle and the capacity of electron transport activity in the respiratory chain appearing to be much lower than in mammals, Plasmodium is bound to maintain high glycolytic activity. This is indicated by the 100-fold increase in glucose consumption in P. falciparum-infected erythrocytes in comparison to non-infected cells, which exposes the parasite to higher fluxes of methylglyoxal produced by the non-enzymatic conversion of triose phosphates. On parasitation of red blood cells, the formation of D-lactate from methylglyoxal was found to be increased by a factor of 30, and there were higher GloI and GloII activities than in uninfected erythrocytes. The antimalarial activity of Glo-inhibitors was demonstrated by the growth inhibition caused by S-p-bromobenzylglutathione diethyl ester with IC50 values in the lower micromolar range.
Glutathione S-transferase. Glutathione transferases (GST) exist in most organisms as numerous isoforms. They serve in the intracellular detoxification of noxious chemicals by linking them to glutathione. GSTs can furthermore detoxify lipid peroxidation products and serve as carrier proteins, so-called ligandins, of certain organic molecules, which leads to the inactivation and immobilization of these compounds. In chloroquine-resistant parasites GST activity is directly and positively related to drug pressure. The P. falciparum GST gene is located on chromosome 14 and codes for the only GST isoenzyme in the parasite. PfGST is a homodimer of 26 kDa subunit size. A number of PfGST inhibitors – including ferriprotoporphyrin IX – have been identified. The enzyme represents ~1% of the cellular protein and might therefore serve as an efficient in vivo buffer for parasitotoxic hemin, which binds uncompetitively to GST (Ki = 6.5 µm = [GST x GSH] [hemin]/[GST x GSH x hemin]) and is toxic to the parasite.