Philippe Grellier

Antileishmanial and trypanocidal activity of Brazilian Cerrado plants

The side effects and the emerging resistance to the available drugs against leishmaniasis and trypanosomiasis led to the urgent need for new therapeutic agents against these diseases. Thirty one extracts of thirteen medicinal plants from the Brazilian Cerrado were therefore evaluated in vitro for their antiprotozoal activity against promastigotes of Leishmania donovani, and amastigotes of Trypanosoma cruzi. Among the selected plants, Casearia sylvestris var. lingua was the most active against both L. donovani and T. cruzi. Fifteen extracts were active against promastigotes of L. donovani with concentrations inhibiting 50% of parasite growth (IC50) between 0.1-10 microg/ml, particularly those of Annona crassiflora (Annonaceae), Himatanthus obovatus (Apocynaceae), Guarea kunthiana (Meliaceae), Cupania vernalis (Sapindaceae), and Serjania lethalis (Sapindaceae). With regard to amastigotes of T. cruzi, extracts of A. crassiflora, Duguetia furfuracea (Annonaceae), and C. sylvestris var. lingua were active with IC50 values between 0.3-10 microg/ml. Bioassay fractionations of the more active extracts are under progress to identify the active antiparasite compounds.

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.

Novel Antimalarial Compounds Isolated in a Survey of Self-Medicative Behavior of Wild Chimpanzees in Uganda

ABSTRACT Following a veterinary and behavioral survey of chimpanzees from a natural population in Uganda, leaf samples of Trichilia rubescens were collected because of the unusual method of ingestion observed. The methanolic crude extract of T. rubescens leaves exhibited significant antimalarial activity in vitro. Bioassay-directed fractionation provided two new limonoids, trichirubines A and B. A greater understanding of the role of secondary compounds in the primate diet may be helpful in recovering naturally occurring compounds of medicinal significance for human medicine.

New ferrocenic pyrrolo[1,2-a]quinoxaline derivatives: synthesis, and in vitro antimalarial activity.

Following our search for antimalarial compounds, novel series of ferrocenic pyrrolo[1,2-a]quinoxaline derivatives 1-2 were synthesized from various substituted nitroanilines and tested for in vitro activity upon the erythrocytic development of Plasmodiumfalciparum strains with different chloroquine-resistance status. The pyrrolo[1,2-a]quinoxalines 1 were prepared in 6-8 steps through a regioselective palladium-catalyzed monoamination by coupling 4-chloropyrrolo[1,2-a]quinoxalines with 1,3-bis(aminopropyl)piperazine or -methylamine using Xantphos as the ligand. The ferrocenic bispyrrolo[1,2-a]quinoxalines 2 were prepared by reductive amination of previously described bispyrrolo[1,2-a]quinoxalines 9 with ferrocene-carboxaldehyde, by treatment with NaHB(OAc)(3). The best results were observed with ferrocenic pyrrolo[1,2-a]quinoxalines linked by a bis(3-aminopropyl)piperazine. Moreover, it was observed that a methoxy group on the pyrrolo[1,2-a]quinoxaline nucleus and no substitution on the terminal N-ferrocenylmethylamine function enhanced the pharmacological activity. Selected compounds 1b, 1f-h, 1l and 2a were tested for their ability to inhibit beta-haematin formation, the synthetic equivalent of hemozoin, by using the HPIA (heme polymerization inhibitory activity) assay. Of the tested compounds, only 2a showed a beta-haematin formation inhibition, but no inhibition of haem polymerization was observed with the other selected ferrocenic monopyrrolo[1,2-a]quinoxaline derivatives 1b, 1f-h and 1l, as the IC(50) values were superior to 10 equivalents.

Probiotics for the Control of Parasites: An Overview

Probiotics are defined as live organisms, which confer benefits to the host. Their efficiency was demonstrated for the treatment of gastrointestinal disorders, respiratory infections, and allergic symptoms, but their use is mostly limited to bacterial and viral diseases. During the last decade, probiotics as means for the control of parasite infections were reported covering mainly intestinal diseases but also some nongut infections, that are all of human and veterinary importance. In most cases, evidence for a beneficial effect was obtained by studies using animal models. In a few cases, cellular interactions between probiotics and pathogens or relevant host cells were also investigated using in vitro culture systems. However, molecular mechanisms mediating the beneficial effects are as yet poorly understood. These studies indicate that probiotics might indeed provide a strain-specific protection against parasites, probably through multiple mechanisms. But more unravelling studies are needed to justify probiotic utilisation in therapeutics.

Photochemical inactivation with amotosalen and long-wavelength ultraviolet light of Plasmodium and Babesia in platelet and plasma components.

BACKGROUND: Transfusion‐transmitted cases of malaria and babesiosis have been well documented. Current efforts to screen out contaminated blood products result in component wastage due to the lack of specific detection methods while donor deferral does not always guarantee safe blood products. This study evaluated the efficacy of a photochemical treatment (PCT) method with amotosalen and long‐wavelength ultraviolet light (UVA) to inactivate these agents in red blood cells (RBCs) contaminating platelet (PLT) and plasma components.

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.

New ferrocenic pyrrolo[1,2-a]quinoxaline derivatives: Synthesis, and in vitro antimalarial activity – Part II

Following our search for antimalarial compounds, novel series of ferrocenyl-substituted pyrrolo[1,2-a]quinoxalines 1-2 were synthesized from ferrocene-carboxaldehyde and tested for their in vitro activity upon the erythrocytic development of Plasmodium falciparum strains with different chloroquine-resistance status. The ferrocenic pyrrolo[1,2-a]quinoxalines 1-2 were prepared in 6 or 9 steps through a Barton-Zard reaction. Promising pharmacological results against FcB1, K1 and F32 strains were obtained with ferrocenyl pyrrolo[1,2-a]quinoxalines 1j-l linked by a bis-(3-aminopropyl)piperazine linker substituted by a nitrobenzyl moiety.

Design, synthesis and antimalarial activity of novel, quinoline-Based, zinc metallo-aminopeptidase inhibitors

PfA-M1, a neutral zinc aminopeptidase of Plasmodium falciparum, is a new potential target for the discovery of antimalarials. The design and synthesis of a library of 45 quinoline-based inhibitors of PfA-M1 is reported. The best inhibitor displays an IC(50) of 854 nM. The antimalarial activity on a CQ-resistant strain and the specificity towards mammalian aminopeptidase N are also discussed.

Prolyl oligopeptidase of Trypanosoma brucei hydrolyzes native collagen, peptide hormones and is active in the plasma of infected mice.

Proteases play important roles in many biological processes of parasites, including their host interactions. In sleeping sickness, Trypanosoma brucei proteases released into the host bloodstream could hydrolyze host factors, such as hormones, contributing to the development of the diseases symptoms. In this study, we present the identification of the T. brucei prolyl oligopeptidase gene (poptb) and the characterization of its corresponding enzyme, POP Tb. Secondary structure predictions of POP Tb show a structural composition highly similar to other POPs. Recombinant POP Tb produced in E. coli was active and highly sensitive to inhibitors of Trypanosoma cruzi POP Tc80. These inhibitors, which prevent T. cruzi entry into non-phagocytic cells, arrested growth of the T. brucei bloodstream form in a dose-dependent manner. POP Tb hydrolyzes peptide hormones containing Pro or Ala at the P1 position at a slightly alkaline pH, and also cleaves type I collagen in vitro and native collagen present in rat mesentery. Furthermore, POP Tb is released into the bloodstream of T. brucei infected mice where it remains active. These data suggest that POP Tb might contribute to the pathogenesis of sleeping sickness.