Joseph Schrevel

Synthesis, characterization, and in vitro antimalarial and antitumor activity of new ruthenium(II) complexes of chloroquine.

The new Ru(II) chloroquine complexes [Ru(eta(6)-arene)(CQ)Cl2] (CQ = chloroquine; arene = p-cymene 1, benzene 2), [Ru(eta(6)-p-cymene)(CQ)(H2O)2][BF4]2 (3), [Ru(eta(6)-p-cymene)(CQ)(en)][PF6]2 (en = ethylenediamine) (4), and [Ru(eta(6)-p-cymene)(eta(6)-CQDP)][BF4]2 (5, CQDP = chloroquine diphosphate) have been synthesized and characterized by use of a combination of NMR and FTIR spectroscopy with DFT calculations. Each complex is formed as a single coordination isomer: In 1-4, chloroquine binds to ruthenium in the eta(1)-N mode through the quinoline nitrogen atom, whereas in 5 an unprecedented eta(6) bonding through the carbocyclic ring is observed. 1, 2, 3, and 5 are active against CQ-resistant (Dd2, K1, and W2) and CQ-sensitive (FcB1, PFB, F32, and 3D7) malaria parasites (Plasmodium falciparum); importantly, the potency of these complexes against resistant parasites is consistently higher than that of the standard drug chloroquine diphosphate. 1 and 5 also inhibit the growth of colon cancer cells, independently of the p53 status and of liposarcoma tumor cell lines with the latter showing increased sensitivity, especially to 1 (IC50 8 microM); this is significant because this type of tumor does not respond to currently employed chemotherapies.

Properties, stage-dependent expression and localization of Plasmodium falciparum M1 family zinc-aminopeptidase

A Plasmodium falciparum single copy gene predicting a 122 kDa protein belonging to the Ml family of zincmetallopeptidases was previously reported and related to erythrocytic schizont proteins of 96 (p96) and 68 (p68) kDa. By using protease inhibitors during parasite harvest and enzyme preparations, and polyclonal antibodies specific for 2 peptidic domains deduced from the gene, we identified the 120 kDa precursor and demonstrated its processing into p96 and p68. The N-terminal ends of p96 and p68 were mapped between glycine-123 and lysine-163, both proteins thus containing the catalytic domain. The purified enzyme, here named PfA-M1 (p96/p68), displayed strict aminopeptidase activity, optimal at pH 74, with broad substrate spectrum. Its inhibition and reactivation profiles were typical of zinc-metalloaminopeptidases. By Western blotting, PfA-M1 was detected in trophozoites, in addition to schizonts, but not in early rings. PfA-M1 was localized by indirect immunofluorescence confocal microscopy. In trophozoites, the labelling was diffuse in the parasite cytoplasm, with accumulations around the food vacuole. In schizonts, it turned progressively to a vesicle-like pattern, ending as a clear spot in released merozoites. The involvement of PfA-M1 in haemoglobin breakdown and erythrocyte reinvasion is discussed in light of the dual functions recently reported for several P. falciparum proteases.

Molecular, functional and structural properties of the prolyl oligopeptidase of Trypanosoma cruzi (POP Tc80), which is required for parasite entry into mammalian cells

We have demonstrated that the 80 kDa POP Tc80 (prolyl oligopeptidase of Trypanosoma cruzi) is involved in the process of cell invasion, since specific inhibitors block parasite entry into non-phagocytic mammalian host cells. In contrast with other POPs, POP Tc80 is capable of hydrolysing large substrates, such as fibronectin and native collagen. In this study, we present the cloning of the POPTc80 gene, whose deduced amino acid sequence shares considerable identity with other members of the POP family, mainly within its C-terminal portion that forms the catalytic domain. Southern-blot analysis indicated that POPTc80 is present as a single copy in the genome of the parasite. These results are consistent with mapping of POPTc80 to a single chromosome. The active recombinant protein (rPOP Tc80) displayed kinetic properties comparable with those of the native enzyme. Novel inhibitors were assayed with rPOP Tc80, and the most efficient ones presented values of inhibition coefficient Ki < or = 1.52 nM. Infective parasites treated with these specific POP Tc80 inhibitors attached to the surface of mammalian host cells, but were incapable of infecting them. Structural modelling of POP Tc80, based on the crystallized porcine POP, suggested that POP Tc80 is composed of an alpha/beta-hydrolase domain containing the catalytic triad Ser548-Asp631-His667 and a seven-bladed beta-propeller non-catalytic domain. Docking analysis suggests that triple-helical collagen access to the catalytic site of POP Tc80 occurs in the vicinity of the interface between the two domains.

Cytological and immunological responses to Babesia divergens in different hosts: ox, gerbil, man.

A continuous in vitro culture system forBabesia divergens was initiated from a human isolate. It was maintained through 305 subcultures for 3 years using a low concentration of serum and a low haematocrit, with no decrease in the initial virulence. This in vitro system enabled the routine culture of all human and bovineB. divergens isolates thus far tested, with a mean parasitaemia level of 30%–40%. Different cytological aspects observed in the same isolate by optical and electron microscopy were described in parasitized ox, gerbil and human erythrocytes. The sequence ofB. divergens antibody responses was determined in man and ox, enabling the precise identification of majorB. divergens antigens as candidates for vaccines.

A PLASMODIUM FALCIPARUM AMINOPEPTIDASE GENE BELONGING TO THE M1 FAMILY OF ZINC-METALLOPEPTIDASES IS EXPRESSED IN ERYTHROCYTIC STAGES

A new single copy gene has been isolated from Plasmodium falciparum, by immunoscreening a genomic DNA expression library. The gene appears devoid of introns, displays the classical A + T richness and codon usage of P. falciparum genes, and is transcribed into a 4 kb mRNA in erythrocytic stages. The deduced amino acid sequence corresponds to a 1056 residue protein (122 kDa) containing the canonical HExxHx18E signature of zinc-metallopeptidase active sites of the M1 family at position 467-490, a downstream conserved tyrosine residue involved in catalysis in position 551, and the GAMEN conserved motif characteristic of aminopeptidases in the M1 family, at position 431-435. The greatest similarities were found with aminopeptidases N of Escherichia coli and Haemophilius influenza (more than 80% identical residues in the canonical signature of the active site) but significant similarities centred on the active site region exist with all other members of the M1 family such as other prokaryotic aminopeptidases, eukaryotic aminopeptidases A and N and leukotriene A4 hydrolases (40-50% identical residues in the canonical signature of the active site). A polyclonal serum raised to a synthetic peptide deduced from the gene labelled schizont proteins of 96 and 68 kDa purified to homogeneity and both displaying aminopeptidase activity, as well as cytoplasmic structures in schizont stages.

Antimalarial Activity of Cryptolepine and Isocryptolepine, Alkaloids Isolated from Cryptolepis sanguinolenta

Cryptolepis sanguinolenta extracts are currently used by African herbalists to cure malaria but the compounds involved in its antimalarial activity have not been identified. Two alkaloids, cryptolepine and isocryptolepine, have been isolated from the roots of C. sanguinolenta and their antimalarial activity evaluated. Both alkaloids possess intrinsic inhibitory activity against the human malaria parasite, Plasmodium falciparum in vitro, whatever the chloroquine‐resistance status of the strains used. Cryptolepine was slightly more efficient for parasite killing with an IC50 in the range of 0.2 to 0.6 μMSC compared with an IC50 of about 0.8 μMSC for isocryptolepine. The antimalarial activity of cryptolepine was confirmed in vivo on the rodent malarial parasites Plasmodium vinckei petteri and Plasmodium berghei ANKA.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors lovastatin and simvastatin inhibit in vitro development of Plasmodium falciparum and Babesia divergens in human erythrocytes.

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors lovastatin and simvastatin inhibit the in vitro intraerythrocytic development of Plasmodium falciparum and Babesia divergens, with concentrations inhibiting parasite growth by 50% in the ranges of 10 to 20 and 5 to 10 micrograms.ml-1, respectively. For P. falciparum, the 50% inhibitory concentrations were in the same range whatever the chloroquine susceptibility of the strains tested (strain F32/Tanzania [chloroquine susceptible] or FcB.1/Columbia [resistant]). The stage-dependent susceptibility of P. falciparum to simvastatin was studied by subjecting synchronized cultures to 6-h pulses of drug throughout the 48-h erythrocytic life cycle. The most important inhibitory effects were observed between the 12th and 30th hours of the cycle, corresponding to the trophozoite stage. This period precedes the S phase and the nuclear divisions. Parasites in the newly formed ring stage (time zero to the 6th hour of the cycle) and the schizont stage (30th to 48th hour of the cycle) were weakly or not susceptible to simvastatin pulses.

A LARGE MULTIGENE FAMILY EXPRESSED DURING THE ERYTHROCYTIC SCHIZOGONY OF PLASMODIUM FALCIPARUM

We report the identification of a large multigene family of Plasmodium falciparum using a clone isolated with a polyclonal antiserum raised to a Babesia divergens merozoite protein. The recombinant antigen reacted with human sera collected from individuals exposed to malaria. The deduced protein sequence contains a motif homologous to the consensus sequence of merozoite rhoptry proteins encoded by multigene families in several Babesia species. Antibodies raised to the recombinant protein reacted with a 60-kDa merozoite protein both on B. divergens and on P. falciparum immunoblots. The insert hybridized to a large number of fragments on P. falciparum Southern blots and to most chromosomes of the parasite. Specifically, approx. 3-kb RNAs were detected in 4-16-nucleus schizonts. Ten distinct cDNAs were isolated that differed in the size, position and number of restriction sites in the region homologous to the original genomic clone. With about 140 copies per haploid genome, this is the first large multigene family described in malaria parasites. The existence of a multigene family encoding proteins present in the invasive stage of malaria parasites suggests an important role in invasion and denotes a significant potential for generating diversity.

Carboxylic ionophores in malaria chemotherapy: The effects of monensin and nigericin on plasmodium falciparum in vitro and plasmodium vinckei petteri in vivo

Chloroquine is widely used in malaria chemotherapy. Due to its weak base properties, this drug accumulates in the parasite food vacuole where it acts initially by raising the pH of this organelle, thereby reducing the digestion of hemoglobin by the parasite and preventing its growth. Nevertheless, alkalinization of the food vacuole and inhibition of lysosomal protein degradation could also be achieved by means of carboxylic ionophores such as monensin and nigericin. These drugs intercalate into intracellular organelle membranes and exchange protons for K+ or Na+. In the present study, we show that monensin and nigericin exhibit in vitro intrinsic antimalarial activities at nanomolar and picomolar range, respectively, on P.falciparum and thereby appear 25 fold and 30,000 fold more potent than chloroquine. The very low IC50 values exhibited by these two ionophores prompted us to test their antimalarial activities in vivo on Plasmodium vinckei petteri. We found that the ED50 and ED90 values were respectively 1.1mg/kg and 3.5 mg/kg for monensin; 1.8 mg/kg and 4.6 mg/kg for nigericin. In addition, when treated with monensin at 10 mg/kg, 100% of the infected mice were cured. Interestingly, nigericin can be combined with monensin and we show that this combination is synergic. Thus, this finding would allow the use of lower doses of these ionophores and prevent occurrence of drug resistance. Carboxylic ionophores can be viewed as a new strategy in malaria chemotherapy.

Purification and characterization of a new 120 kDa alkaline proteinase of Trypanosoma cruzi.

A new alkaline proteinase activity was identified in cell-free extracts of Trypanosoma cruzi epimastigotes on the basis of its ability to hydrolyze the fluorogenic substrate N-Z-Gly-Gly-Arg-AMC. The optimal activity was at pH 8.0. After a three step-chromatography procedure using two anionic columns (DEAE-Sepharose and Mono Q) and a chromatofocusing column (Mono P), the proteolytic activity was associated with a single 120 kDa protein and was called Tc 120 proteinase. The molecular mass of the proteinase was confirmed by direct visualization of the proteolytic activity using a fluorometric assay on SDS-PAGE. The Tc 120 proteinase which also cleaves N-Z-Arg-AMC, N-Z-Phe-Arg-AMC and N-glutaryl-Gly-Arg-AMC substrates, is a cysteine-type proteinase with an unusual low sensitivity to E-64.