The phytochemical EA is a known inhibitor of GSTs and previously has been shown to possess antiplasmodial activity (2
). To study the potential of EA-derived compounds as antimalarial leads, we assessed the effects of EA, FEA, and CEA on plasmodial and mammalian cell systems, redox-related enzymes, and transcripts.
GST represents a novel GST isoform, and its H-site differs significantly from those of its human counterparts (13
). In contrast to all other GSTs, P. falciparum
GST contains only five residues following helix α8, which is too short to form a wall (Mu or Pi class) or an α-helix (alpha class). This leads to a more solvent-accessible H-site in P. falciparum
GST, which suggests that the substrate spectrum of P. falciparum
GST is broader, includes amphiphilic compounds, and is accessible to amphiphilic inhibitors that are not able to enter the H-site of the human isoforms. Based on these considerations, one or two additional hydroxyl groups were inserted into EA, resulting in FEA and CEA, respectively (Fig. ). CEA was found to be more effective on P. falciparum
GST than on hGST. The inhibition of the plasmodial enzyme increased significantly with increasing hydrophilicity, indicating that the structural properties of the active site of P. falciparum
GST can be exploited for rational drug design using these planar polyaromatic ring systems (Table ).
It had been shown previously that EA inhibits the growth of CQ-sensitive and -resistant malarial parasites in vitro, with an IC50
in the range of 200 to 500 nM (2
). In order to compare the inhibitory effect of EA to those of FEA and CEA, we tested all compounds on four P. falciparum
strains with various degrees of CQ resistance. The previously reported antiplasmodial effect of EA could be reproduced, yielding IC50
s between ~350 and ~1,300 nM (Table ).
The introduction of one additional hydroxyl group into EA (yielding FEA) increased antiplasmodial activity by around one order of magnitude. The addition of a second hydroxyl group, yielding CEA, led to a further reduction of the IC50 by around 50%. Thus, the two derivatives had much more potent antiplasmodial activity than EA (Table ). These data support the notion that FEA and CEA are promising antimalarial leads that are particularly effective against CQ-resistant P. falciparum strains, as indicated by IC50s of 95 and 86 nM, respectively, on the resistant strain K1 (Table ). Under the same conditions, CQ was active at an IC50 of 163 nM.
A comparison of the effects of the inhibitors on recombinant P. falciparum
GST and P. falciparum
in cell culture suggests that all three compounds are likely to have additional targets in the parasite. The activity of a specific inhibitor with only one target usually decreases by one order of magnitude from enzyme to cell culture. For our compounds, however, the effects on cell culture were about two orders of magnitude stronger (IC50
around 100 nM) than the inhibition of P. falciparum
around 10 μM). Studying other Plasmodium
enzymes indeed demonstrated that P. falciparum
GR and P. falciparum
TrxR were effectively inhibited by the substances, with IC50
s in the same range as that determined for P. falciparum
GST (Table ). As shown previously, EA furthermore inhibits the formation of beta-hematin, and plasmepsin II has been suggested as a target for EA (8
). Since multiple drug targets reduce the risk of resistance development, this mode of action is not a principle hindrance for further lead optimization.
The malaria pigment, also called hemozoin, is composed of an ordered arrangement of reciprocal heme dimers (4
). Interestingly, in vitro heme aggregation assays revealed that EA, as well as the two structural derivatives, may exert some of their antiplasmodial activity by forming soluble complexes with free heme in the parasite's digestive vacuole, thereby blocking the formation of hemozoin (Fig. ). In the time-dependent assay (Fig. ), CEA showed the strongest effects. Similar results and IC50
s in the lower-micromolar range were obtained by Ignatushchenko et al. (19
) for xanthones. Xanthones are, like EA and derivatives, planar aromatic systems, and the authors suggest that the ability to inhibit heme aggregation represents the mode of the antiplasmodial action of these compounds.
Drug combination assays revealed no synergistic action for the polyphenolic compounds with either of the antimalarials tested. Rather, EA, FEA, and CEA acted antagonistically in vitro. Whether CEA, FEA, or their potential derivatives could be successful in combination therapy with known antimalarials in vivo remains to be studied.
The potential of CEA and FEA as antimalarial lead compounds is substantiated by their low cytotoxicity in human cells. At low-nanomolar CEA concentrations, which would be required for the inhibition of P. falciparum
growth in vitro, the number of human A549 cells undergoing cell death is marginal. These data are further supported by the fact that EA and related compounds have even been recommended as cancer-chemopreventive agents in humans (33
The impact of this finding also is reflected in transcript levels and enzyme activities of A549 cells after treatment with EA and its analogues (Table ). Two different concentrations of each compound were applied on the basis of preliminary inhibitory tests at which less than 10% cell death in the culture occurred after 24 h of incubation. The cellular effects caused by EA, FEA, and CEA were negligible considering the much lower inhibitor concentrations required for antiplasmodial activity. The inhibition (about 60%) of human TrxR activity by CEA should be mentioned (Table ); it occurred at an inhibitor concentration near the IC50 on the isolated enzyme (Table ).
All aspects delineated above feature CEA as the most potent of the compounds tested. In vivo studies in a P. berghei
mouse model indicated that CEA is able to improve the survival of infected animals. The slow decrease of parasitemia matches the in vitro data on stage specificity, which demonstrate that CEA is a slowly acting compound. CQ, which is known to be a fast-acting antimalarial, was assessed in parallel. An exposure time of ≥1 h at a high compound concentration range was sufficient to achieve substantial growth inhibition of all parasite stages. Our stage dependency data are in good agreement with other studies using similar techniques and reporting that trophozoite and schizont stages are considerably more sensitive to CQ than ring stages (25
). CEA, in contrast, was found to be strongly stage specific for trophozoites, for which a long exposure time seemed to be more critical than a high compound concentration (Fig. ). Thus, CEA is a promising candidate for use in combination therapy with the fast-acting drugs that now are applied in order to prevent and circumvent drug resistance (35
). The fact that CEA showed the highest activity against parasites in the trophozoite stage, when hemoglobin catabolism is maximal, is consistent with the observation that CEA inhibits heme aggregation.
As rapidly growing and multiplying cells, malarial parasites rely on functional and efficient antioxidant systems for survival (3
). Transcriptional studies revealed that CEA-treated parasites possess impaired redox capacities, since the mRNA levels of several antioxidant enzymes, including GST, GR, and TrxR, were remarkably downregulated (Fig. ). It is unlikely that this downregulation of redox-active genes is simply a cell death-related event: (i) the genes were downregulated compared to the levels of two independent reference genes (LDH and 18S rRNA), and (ii) other genes (for HSP, GS, and Sel1) showed stable transcription or even upregulation. Furthermore, to rule out that the observed downregulation of redox genes is simply an effect of any toxic agent, we determined the effects of inducers of oxidative stress (20 μM paraquat) or nitrosative stress (10 μM sodium nitroprusside) on TrxR, TPx1, and Sel1 transcript levels in P. falciparum
3D7. After an incubation time of 15 h, all three genes were clearly upregulated using the same reference gene (18S rRNA) and, thus, followed a trend opposite that observed for CEA (data not shown).
In summary, the two compounds structurally related to EA show improved antiplasmodial activity. Combined with their low cytotoxicity, they represent promising antimalarial lead compounds (international patent application no. PCT/EP2006/060707) that might be particularly effective against CQ-resistant P. falciparum.