Glutathione Reductase of the Malarial Parasite Plasmodium falciparum: Crystal Structure and Inhibitor Development

Abstract

The malarial parasite Plasmodium falciparum is known to be sensitive to oxidative stress, and thus the antioxidant enzyme glutathione reductase (GR; NADPH+GSSG+H⁺⇄NADP⁺+2 GSH) has become an attractive drug target for antimalarial drug development. Here, we report the 2.6 Å resolution crystal structure of P. falciparum GR. The homodimeric flavoenzyme is compared to the related human GR with focus on structural aspects relevant for drug design. The most pronounced differences between the two enzymes concern the shape and electrostatics of a large (450 Å³) cavity at the dimer interface. This cavity binds numerous non-competitive inhibitors and is a target for selective drug design. A 34-residue insertion specific for the GRs of malarial parasites shows no density, implying that it is disordered. The precise location of this insertion along the sequence allows us to explain the deleterious effects of a mutant in this region and suggests new functional studies. To complement the structural comparisons, we report the relative susceptibility of human and plasmodial GRs to a series of tricyclic inhibitors as well as to peptides designed to interfere with protein folding and dimerization. Enzyme-kinetic studies on GRs from chloroquine-resistant and chloroquine-sensitive parasite strains were performed and indicate that the structure reported here represents GR of P. falciparum strains in general and thus is a highly relevant target for drug development.

Introduction

Tropical malaria represents an increasing threat to human health and welfare.1., 2. The disease is caused by the multiplication of the protozoan parasite Plasmodium falciparum in human erythrocytes.³ The emerging resistance of Plasmodium to chloroquine and other antimalarials underlines the need for the development of new chemotherapeutic agents with different modes of action.4., 5., 6.

During the erythrocytic stages of its life cycle, the parasite is exposed to oxidative stress produced by activated macrophages of the host and also by toxic heme and other decomposition products of hemoglobin. We are pursuing enhanced oxidative stress as an attractive avenue for drug development, because a number of lines of evidence suggest that this can effectively inhibit parasite growth (reviewed by Schirmer et al.⁷). Indeed the toxic effects of chloroquine and other quinoline antimalarials that inhibit β-hematin formation8., 9., 10., 11., 12. may be partly due to increased levels of O₂-activating heme, and peroxide antimalarials such as artemisinin are believed to be activated by heme resulting in the formation of alkylating free radicals.¹³

The glutathione system plays a key role in the defense of malarial parasites against oxidative stress and in the development of drug resistance. This is supported by the fact that chloroquine-resistant parasites exhibit increased glutathione concentrations.14., 15., 16. The tripeptide GSH detoxifies reactive oxygen species both directly and via glutathione peroxidase and glutathione S-transferase-catalyzed reactions. High levels of reduced glutathione are maintained by de novo synthesis from the constituent amino acids¹⁷ and by the enzyme glutathione reductase (GR), a ubiquitous FAD-containing protein. GR is a homodimer that catalyzes the reduction of glutathione disulfide to GSH using NADPH as the source of reducing equivalents:18., 19., 20.

$$\text{GSSG}\text{+}\text{NADPH}\text{+}\text{H}{}^{\mathrm{+}}\text{⇆}\text{NADP}{}^{\mathrm{+}}\text{+2}\text{GSH}$$

P. falciparum GR (PfGR) has been characterized biochemically and kinetically both in its native authentic form isolated from malarial parasites²¹ and in its recombinant form.22., 23., 24., 25. As systems depending on adequate GR activity, glutaredoxin and glutathione S-transferase have been described in P. falciparum.18., 26. As an intraerythrocytic parasite, P. falciparum depends on an intact host cell milieu. Evidence for this is that a lack of either erythrocyte FAD, the prosthetic group of GR, or of its substrate NADPH, as observed in glucose-6-phosphate dehydrogenase deficiency, leads to significant protection against malaria.27., 28., 29., 30., 31., 32., 33. Red blood cells rendered severely GR-deficient by the drug carmustine were able to fulfil their physiological functions, although for a shortened lifetime, but they did not serve as host cells for P. falciparum.34., 35., 36. Thus both PfGR and hGR are potential targets for the development of antimalarial drugs.23., 35., 37., 38., 39. We have recently shown that PfGR may be the target for methylene blue, an antimalarial which efficiently inhibits PfGR and not hGR at therapeutically used concentrations.²³ Indeed, GR is the only protein that has been identified as a target of methylene blue in the parasite. Furthermore, on the basis of a GR inhibitor a novel strategy for overcoming chloroquine resistance in malarial parasites has recently been developed.37., 40. Quinoline-based alcohols with known antimalarial activity were combined with a GR inhibitor, represented by a menadione derivative, via a metabolically labile ester bond to give double-headed prodrugs. The quinoline moiety served also for directing the prodrug into the parasite. The activity of the compounds was proven in cell culture and in a mouse model.³⁷ Also, the drug combination of methylene blue-chloroquine (BlueCQ) is currently being tested in Nouna, Burkina Faso, as a drug against malaria complicated by methemoglobinemic anemia⁴¹ (B. Coulibaly & R.H.S., unpublished data).

Human GR has been very well studied in terms of enzymology and crystal structure42., 43. (Figure 1). The binding of substrates, substrate analogs and inhibitors including different physiologic nitric oxide-containing compounds44., 45., 46. have been elucidated in detail.20., 42., 47. Steps have already been taken to investigate the inhibitory properties of various drugs on hGR.48., 49., 50. Among these inhibitors are isoalloxazine,50., 51. menadione⁴⁸ and xanthene⁴⁹ derivatives. All of these compounds bind in a cavity that is located at the dimer interface (Figure 1), and inhibit hGR non-competitively, the K_i values being in the lower micromolar range. Indirect evidence suggests that this cavity is also the binding site of methylene blue in PfGR.22., 24., 52. Methylene blue and isoalloxazine derivatives are also considered as constituents of double-headed drugs; the other head is designed to bind at the GSSG site, and the two heads are connected by a linker that fits in the channel connecting the intersubunit cavity with the GSSG site⁴⁶ (Figure 1). In general, phenothiazines and isoalloxazine derivatives are of interest as lead compounds for antimalarial drugs because they show low toxicity and are inexpensive.

There are three major features distinguishing PfGR from hGR that are thought to be of relevance for selective inhibitor design.22., 25. The first is an insertion of 34 residues within the central domain (residues 314–347) of PfGR that is known to be highly antigenic,²² but has an unknown effect on function. The second regards the amino acid residues lining the wall of the cavity at the dimer interface, where only nine out of 21 residues are conserved in PfGR. The third feature is the pair of helices (H11/H11′) at the core of the dimer interface⁴³ (Figure 1). These helices are regarded as a dimerization and folding center of GR53., 54., 55. and it has been shown that synthetic peptides can bind to these helices to interfere with the dimerization of hGR.38., 53., 54.

A valuable stepping stone for the further development of rationally designed drugs against PfGR would be the three-dimensional structure of the enzyme. Here, we describe the three-dimensional structure of P. falciparum glutathione reductase and analyze those features that have been noted as potential targets for the design of selective PfGR inhibitors. In addition we describe novel inhibitor design work related to PfGR as a drug target.