The glyoxalase system, inhibition of thioredoxin reductase and use of methylene blue as drug development strategies against the malarial parasite Plasmodium Falciparum
Malaria is a disease caused by protozoan parasites of the genus Plasmodium and is responsible for about half a billion diseases cases and 2-3 million deaths each year. Much of the parasite s success to establish persistent infections is attributed to evasion of the human immune defense system through antigenic variation and increasing development of resistance to all currently available antimalarial drugs except the artemisinins. The difference in structure and mode of action of the artemisinins underlines the fact that new antimalarial drugs with differential modes of action are an urgent priority in order to circumvent plasmodial resistance mechanisms in the absence of effective vaccines or vector control measures.
By means of rational drug design and re-evaluation of an ancient antimalarial drug, three new drug development strategies against the deadliest malarial parasite, Plasmodium falciparum, were developed within the frame of this thesis in order to design possible new mechanism drugs and prevent resistance development to artemisinin.
First, a complete functional glutathione-dependent glyoxalase (Glo) detoxification system comprising a cytosolic GloI (cGloI), a GloI-like protein (GILP) and two GloIIs (cytosolic GloII named cGloII, and tGloII preceded by a targeting sequence) was characterized in direct comparison with the isofunctional human host enzymes. Kinetic and structural similarities of enzymes of both systems were described; however, striking differences especially for the GloIs were also detected which could be exploited for drug development. Various S-(N-hydroxy-N-arylcarbamoyl)glutathiones tested as P. falciparum Glo inhibitors were found to be active in the lower nanomolar range and could be used as lead structures in the development of more selective inhibitors of the P. falciparum glyoxalase system (Akoachere et al., 2005).
Secondly, the characterization of the mode of inhibition of three promising inhibitors of the previously-validated drug target P. falciparum thioredoxin reductase (PfTrxR) is reported in this thesis. The enzyme is a homodimeric flavoenzyme which reduces thioredoxin (Trx) via a C-terminally located CysXXXXCys pair. In this respect PfTrxR differs significantly from its human counterpart which bears a Cys-Sec redox pair at the same position. PfTrxR is essentially involved in antioxidant defence and redox regulation of the parasite and has been validated as a drug target. The inhibitors, 4-nitro-2,1,3-benzothiadiazole (IC50 on PfTrxR = 2 µM), 6,7-nitroquinoxaline (IC50 on PfTrxR = 2 µM), and bis-(2,4-dinitrophenyl)sulfide (IC50 on PfTrxR = 0.5 µM), showed uncompetitive inhibition with respect to both substrates, NADPH and thioredoxin. All three inhibitors were active in the lower micromolar range on the chloroquine resistant P. falciparum strain K1. 4-Nitro-2,1,3-benzothiadiazole was antagonistic with known antimalarials suggesting that the inhibitor uses similar routes of uptake and/or acts on related targets or biochemical pathways (Andricopulo et al., 2005; Andricopulo et al., submitted).
Lastly and most importantly, the renaissance of interest in the ancient antimalarial drug methylene blue (MB) led to the identification of a potential artemisinin-based combination therapy (ACT). A strong synergistic action of MB and artemisinin might be capable of fighting resistant P. falciparum parasites in the field. MB is active against all blood stages of both chloroquine (CQ)-sensitive and CQ-resistant P. falciparum strains with IC50 values in the lower nanomolar range. Ring stages showed the highest susceptibility. As demonstrated by high performance liquid chromatography / tandem mass spectrometry on different cell culture compartments, MB accumulates in malarial parasites. In drug combination assays, MB was found to be antagonistic with CQ and other quinoline antimalarials like piperaquine and amodiaquine; with mefloquine and quinine MB showed additive effects. In contrast, synergistic effects of MB with artemisinin, artesunate, and artemether were observed for all tested parasite strains. Artemisinin/MB concentration combination ratios of 3:1 were found to be advantageous demonstrating that the combination of artemisinin with a smaller amount of MB can be recommended for reaching maximal therapeutic effects. In vitro data reported here indicate that combinations of MB with artemisinin (derivatives) might be a promising option for treating drug resistant malaria. Resistance development under this drug combination is unlikely to occur (Akoachere et al., in press).
Taken together, the results support the feasibility of the rational development of new potential antimalarial drugs. In combination with existing and other promising new malarial-control measures, new antimalarial drugs could greatly contribute to reducing the intolerable global burden of this disease.
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