Old and new targets for innovative antimalarial compounds: the different strategies of the Italian Malaria Network.

Clinical treatment-failures to affordable drugs encouraged new investigation for discovery and development of new prophylactic and therapeutic interventions against malaria. The Drug Discovery Cluster (DDcl) of the Italian Malaria Network gathers several highly integrated and complementary laboratories from different Italian Institutions to identify, synthesise, screen in vitro and in vivo new antimalarial molecules directed against the intraerythrocytic stage of P. falciparum parasites and/or with transmission blocking activity to select lead compounds for further development. Complementary research activities, both in vitro and in the clinics, aim at investigating the pathogenetic mechanisms of severe malaria anaemia and the different manifestations of the disease in malaria-HIV co-infected patients to identify new therapies and improve survival.

A fine balance between oxidised and reduced haem controls the survival of intraerythrocytic plasmodia.

The polymerisation of haemoglobin-derived ferri-protoporphyrin IX (Fe(III)PPIX) to haemozoin (malaria pigment) is a crucial process for intraerythrocytic plasmodia to prevent haem toxicity. It is also the target of in-use antimalarial drugs and newer compounds. This reaction and the inhibition thereof can be reproduced and studied in vitro.

Ultrastructural characteristics, biological activity and pharmacological relevance of synthetic malaria pigment (beta-haematin).

Malaria pigment (haemozoin, HZ) is the detoxification product of haemoglobin-derived haem of intraerythrocytic malaria parasites. At schizont rupture, haemozoin accumulates inside host phagocytic cells. The chemical structure and the spectroscopic characteristics of haemozoin are identical to those of beta-haematin (BH), a synthetic pigment obtained from Ferriprotoporphyrin IX (Fe (III) PPIX) in acidic conditions. The process of BH formation is the target of quinoline antimalarials. Here, we summarise the results of our studies on the ultrastructural characteristics, biological and pharmacological relevance of synthetic vs. native haemozoin. 1) By electron microscopy, native HZ and synthetic BH appear as dark brown crystals, morphologically indistinguishable and are internalised by phagocytes at the same extent. 2) Both HZ and BH modulate the production of cytokines (TNF and NO) and increase the susceptibility to lipid peroxidation of mouse or human phagocytes. The antioxidant status of the phagocytes regulates the susceptibility to BH/HZ-mediated effects. 3) The process of BH formation from Fe(III)PPIX, hence haem detoxification, can be inhibited by electrochemically-reduced, Fe(II)PPIX molecules. Maintaining iron in the reduced state can thus be considered a new pharmacological target. This was confirmed by the observation that thiol-reducing agents (NAC, cystein) were able to inhibit BH formation and were toxic to parasites in vitro.

Plasmodium falciparum parasitized red blood cells modulate the production of endothelin-1 by human endothelial cells.

AIM: Plasmodium falciparum (P. falciparum) malaria is the most important parasitic infection of humans, responsible for about 2,000,000 deaths every year. Cytoadherence of P. falciparum parasitized erythrocytes (pRBC) to vascular endothelium contributes to the pathogenesis of severe malaria causing microcirculatory obstruction and subsequent tissue hypoxia. Several cytokines and vasoactive mediators are involved in this process. The aim of this paper was to investigate the production of endothelin-1 (ET-1), a potent vasoconstrictor agent, by endothelial cells from large vessels (human umbilical vein endothelial cells, HUVEC) or the microvasculature (human microvascular endothelial cells, HMEC-1), co-cultured with different strains of P. falciparum pRBC under normoxic or hypoxic conditions. METHODS: HMEC-1, immortalized by SV 40 large Tontigen, were maintained in MCDB 131 medium supplement ed with 10% fetal calf serum, 10 ng/ml of epidermal growth factor, 1 microg/ml of hydrocortisone, 2 mM glutamine, 100 U/ml of penicillin, 100 microg/ml of streptomycin and 20 mM Hepes buffer. The levels of ET-1 in the supernatants were measured by immunoenzymatic assay. RESULTS: The results indicated that IL1-beta and hypoxia were able to induce ET-1 production by both HUVEC and HMEC-1. However, the co-incubation of HUVEC or HMEC-1 with pRBC induced a dose-dependent decrease of both constitutive and IL1- or hypoxia-induced ET-1 production. The inhibition was independent from the parasite strain used and from the origin of endothelial cells. CONCLUSION: These results show that pRBC by modulating both constitutive and stimulated ET-1 release from endothelial cells can induce modifications of the vascular tone in different anatomical districts. This could be of relevance in the pathogenesis of severe malaria.

Standardization of the physicochemical parameters to assess in vitro the beta-hematin inhibitory activity of antimalarial drugs.

Intraerythrocytic plasmodia form hemozoin as a detoxification product of hemoglobin-derived heme. An identical substance, beta-hematin (BH), can be obtained in vitro from hematin at acidic pH. Quinoline-antimalarials inhibit BH formation. Standardization of test conditions is essential for studying the interaction of compounds with this process and screening potential inhibitors. A spectrophotometric microassay of heme polymerization inhibitory activity (HPIA) (Basilico et al., Journal of Antimicrobial Chemotherapy 42, 55-60, 1998) previously reported was used to investigate the effect of pH and salt concentration on BH formation. The yield of BH formation decreased with pH. Moreover, under conditions used in the above HPIA assay (18 h, 37 degrees C, pH = 2.7), several salts including chloride and phosphate inhibited the process. Aminoquinoline drugs formulated as salts (chloroquine-phosphate, primaquine-diphosphate), but not chloroquine-base, also inhibited the reaction. Interference by salts was highest at low pH and decreased at higher pH (pH 4). Here, we describe different assay conditions that eliminate these problems (BHIA, beta-hematin inhibitory activity). By replacing hematin with hemin as the porphyrin and NaOH solution with DMSO as solvent, the formation of BH was independent of pH up to pH 5.1. No interference by salts was observed over the pH range 2.7-5.1. Dose-dependent inhibition of BH formation was obtained with chloroquine-base, chloroquine-phosphate, and chloroquine-sulfate at pH 5.1. Primaquine was not inhibitory. The final product, characterized by solubility in DMSO, consists of pure BH by FT-IR spectroscopy. The BHIA assay (hemin in DMSO, acetate buffer pH 5 +/- 0.1, 18 h at 37 degrees C) is designed to screen for those molecules forming pi-pi interactions with hematin and thus inhibiting beta-hematin formation.