Mark Loyevsky

Novel aroylhydrazone and thiosemicarbazone iron chelators with anti-malarial activity against chloroquine-resistant and -sensitive parasites

Iron (Fe) is crucial for cellular proliferation, and Fe chelators have shown activity at preventing the growth of the malarial parasite in cell culture and in animal and human studies. We investigated the anti-malarial activity of novel aroylhydrazone and thiosemicarbazone Fe chelators that show high activity at inhibiting the growth of tumour cells in cell culture [Blood 100 (2002) 666]. Experiments with the chelators were performed using the chloroquine-sensitive, 3D7, and chloroquine-resistant, 7G8, strains of Plasmodium falciparum in vitro. The new ligands were significantly more active in both strains than the Fe chelator in widespread clinical use, desferrioxamine (DFO). The most effective chelators examined were 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone and 2-hydroxy-1-naphthylaldehyde-4-phenyl-3-thiosemicarbazone. The anti-malarial activity correlates with anti-proliferative activity against neoplastic cells demonstrated in a previous study. Our studies suggest that this class of lipophilic chelators may have potential as useful agents for the treatment of malaria.

Iron Chelation Therapy for Malaria: A Review

Malaria is one of the major global health problems, and an urgent need for the development of new antimalarial agents faces the scientific community. A considerable number of iron(III) chelators, designed for purposes other than treating malaria, have antimalarial activity in vitro, apparently through the mechanism of withholding iron from vital metabolic pathways of the intra-erythrocytic parasite. Certain iron(II) chelators also have antimalarial activity, but the mechanism of action appears to be the formation of toxic complexes with iron rather than the withholding of iron. Several of the iron(III)-chelating compounds also have antimalarial activity in animal models of plasmodial infection. Iron chelation therapy with desferrioxamine, the only compound of this nature that is widely available for use in humans, has clinical activity in both uncomplicated and severe malaria in humans.

Effects of Root Extracts of Fagara zanthoxyloides on the In Vitro Growth and Stage Distribution of Plasmodium falciparum

ABSTRACT The development of resistance by Plasmodium falciparum to conventional drugs poses a threat to malaria control. There is therefore a need to find new, effective, and affordable remedies for malaria, including those derived from plants. This study demonstrates that crude, reverse-phase high-pressure liquid chromatography (RP-HPLC)-semipurified, and RP-HPLC-purified root extracts of Fagara zanthoxyloides inhibit the growth of P. falciparum in vitro, with 50% inhibitory concentrations (IC50s) of 4.90, 1.00, and 0.13 μg/ml, respectively. Roots of F. zanthoxyloides, known as chewing sticks, are widely used for tooth cleaning in West Africa. Microscopic examination of Giemsa-stained slides showed a virtual absence of schizonts in ring-stage synchronized cultures treated with crude extracts at concentrations of 30 to 60 μg/ml during 36 to 48 h of incubation. These observations suggest that the active constituent in the extract may be cytotoxic for P. falciparum trophozoites, thereby inhibiting their development to the schizont stage. A pure bioreactive fraction was subsequently obtained from the chromatographic separations. When this fraction was mixed with pure fagaronine, the mixture coeluted as a single peak on the analytical RP-HPLC column, suggesting that fagaronine may be the active antimalarial constituent of Fagara root extracts. Additional experiments showed that fagaronine also inhibited P. falciparum growth, with an IC50 of 0.018 μg/ml. The results of this study suggest that the antimalarial activity of fagaronine deserves further investigation.

Dexrazoxane (ICRF-187)

1. Dexrazoxane (ICRF-187) is the only clinically approved drug for use in cancer patients to prevent anthracycline mediated cardiotoxicity. 2. The mode of action appears to be mainly due to the potential of the drug to remove iron from iron/anthracycline complexes and thus reduce free radical formation by these complexes. 3. Dexrazoxane also influences cell biology by its ability to inhibit topoisomerase II and its effects on the regulation of cellular iron homeostasis. 4. Although the cardioprotective effect of dexrazoxane in cancer patients undergoing chemotherapy with anthracyclines is well documented, the potential of this drug to modulate topoisomerase II activity and cellular iron metabolism may hold the key for future applications of dexrazoxane in cancer therapy, immunology, or infectious diseases.

Expression of a recombinant IRP-like Plasmodium falciparum protein that specifically binds putative plasmodial IREs

Plasmodium falciparum iron regulatory-like protein (PfIRPa, accession AJ012289) has homology to a family of iron-responsive element (IRE)-binding proteins (IRPs) found in different species. We have previously demonstrated that erythrocyte P. falciparum PfIRPa binds a mammalian consensus IRE and that the binding activity is regulated by iron status. In the work we now report, we have cloned a C-terminus histidine-tagged PfIRPa and overexpressed it in a bacterial expression system in soluble form capable of binding IREs. To overexpress PfIRPa, we used the T7 promoter-driven vector, pET28a(+), in conjunction with the Rosetta(DE3)pLysS strain of E. coli, which carries extra copies of tRNA genes usually found in organisms such as P. falciparum whose genome is (A+T)-rich. The histidine-tagged recombinant protein (rPfIRPa) in soluble form was partially purified using His-bind resin. We searched the plasmodial database, plasmoDB, to identify sequences capable of forming IRE loops using a specially developed algorithm, and found three plasmodial sequences matching the search criteria. In gel retardation assays, rPfIRPa bound three 32P-labeled putative plasmodial IREs with affinity exceeding the affinity for the mammalian consensus IRE. The binding was concentration-dependent and was not inhibited by heparin, an inhibitor of non-specific binding. Immunodepletion of rPfIRPa resulted in substantial inhibition of the signal intensity in the gel retardation assays and in Western blot-determinations of rPfIRPa protein levels. Endogenous PfIRPa retained all three putative 32P-IREs at the same position on the gel as the recombinant PfIRPa.

Inhibition of in-vitro growth of Plasmodium falciparum by Pseudocedrela kotschyi extract alone and in combination with Fagara zanthoxyloides extract.

Roots of Pseudocedrela kotschyi are commonly used as chewing sticks in West Africa. This study examined the effects of the plant extract on the in-vitro growth of Plasmodium falciparum. Ring-stage synchronised cultures of the malaria parasite were exposed to 30 and 60 microg/ml of P. kotschyi extract for 51 h. Aliquots were taken from the cultures every 3 h for preparation of Giemsa-stained thin films, which were evaluated by light microscopy for degree of parasitaemia and stage distribution of parasite development. The extracts did not show any inhibitory effects on the emergence of trophozoites in treated cultures. However, the results indicate that 80% of inhibition of the parasite transformation into schizont was obtained for both tested concentrations (30 and 60 microg/ml). Experiments with (3)H-hypoxanthine incorporation showed an IC(50) of 16 microg/ml for the Pseudocedrela extract. Pseudocedrela was combined with extract of Fagara zanthoxyloides in various concentrations to determine their interactive effects on the in-vitro cultures. Isobologram analysis of the results indicated a synergistic interaction between the two extracts at low concentrations, while interactions at higher concentrations showed antagonistic effects.

Antimalarial Effect of Iron Chelators

Malaria is a major cause of morbidity and mortality in tropical areas of the world. Despite work on controlling the mosquito vector and on developing an effective vaccine, anti-malarial chemotherapy is at present the fundamental approach to controlloing this infection. Because of widespread development of resistance on the part of the parasite to the presently available anti-malarial drugs, it is important to develop new agents with new mechanisms of action. Iron is an essential nutrient for the malaria parasite, and this chapter reviews the information available on the anti-malarial potential of iron chelating agents.

Assessment of antimalarial effect of ICL670A on in vitro cultures of Plasmodium falciparum.

We tested in vitro the antimalarial properties of ICL670A, a newly developed iron chelator for the long‐term oral treatment of iron overload. Ring‐stage synchronized cultures of Plasmodium falciparum cultured in human erythrocytes were exposed to different concentrations of ICL670A and the conventional iron chelator, desferrioxamine B (DFO), for 48 h. Malarial growth was measured by incorporation of [3H]‐hypoxanthine. ICL670A at 30 µmol/l had marked antimalarial activity that was observable by 6 h after beginning the exposure of ring‐stage parasites to the agent. Over 48 h of culture, malarial growth was significantly lower with ICL670A than with DFO at concentrations of both 30 µmol/l (P = 0·008) and 60 µmol/l (P = 0·001). At 48 h, growth relative to control was 53% with ICL670A and 83% with DFO at concentrations of 30 µmol/l, and 20% with ICL670A and 26% with DFO at concentrations of 60 µmol/l. Standard 50% inhibitory concentrations (IC50s) were similar for ICL670A and DFO. Precomplexation with iron completely abolished the inhibitory effect of ICL670A, indicating that this new agent, like DFO, probably inhibits parasite growth via deprivation of iron from critical targets within the parasite. Further studies to address the question of the antimalarial potential of ICL670A in combination with classic antimalarials would be of interest.

Plasmodium falciparum: Activity of artemisinin against Plasmodium falciparum cultured in sickle trait hemoglobin AS and normal hemoglobin AA red blood cells

The presence of sickle hemoglobin causes accumulation of hemoglobin degradative products that favor oxidative reaction in erythrocytes. Artemisinin derivatives exert antiparasite effects through oxidative reactions within infected erythrocytes. Using [(3)H]-hypoxanthine incorporation, we therefore did an in vitro comparison of IC(50) values for artemisinin in Plasmodium falciparum-infected erythrocytes from sickle cell trait (AS) and normal (AA) individuals. IC(50) values for chloroquine served as control. Without drugs, parasite growth was similar in AA and AS erythrocytes. Gender, age and blood group of donors had no significant effects on parasite growth. IC(50) value for artemisinin was 27+/-14nM in AS (N=22) compared to 24+/-9nM (N=27) in AA erythrocytes (P=0.4). IC(50) values for chloroquine were also similar in AA (22+/-8nM) and AS (20+/-11nM) erythrocytes. These results show no evidence of elevated artemisinin activity on P. falciparum in AS erythrocytes in vitro.