Jacques Bouvier

A new class of anthelmintics effective against drug-resistant nematodes

Anthelmintic resistance in human and animal pathogenic helminths has been spreading in prevalence and severity to a point where multidrug resistance against the three major classes of anthelmintics—the benzimidazoles, imidazothiazoles and macrocyclic lactones—has become a global phenomenon in gastrointestinal nematodes of farm animals. Hence, there is an urgent need for an anthelmintic with a new mode of action. Here we report the discovery of the amino-acetonitrile derivatives (AADs) as a new chemical class of synthetic anthelmintics and describe the development of drug candidates that are efficacious against various species of livestock-pathogenic nematodes. These drug candidates seem to have a novel mode of action involving a unique, nematode-specific clade of acetylcholine receptor subunits. The AADs are well tolerated and of low toxicity to mammals, and overcome existing resistances to the currently available anthelmintics.

A unique signature identifies a family of zinc-dependent metallopeptidases

The primary sequence motif HExxH has been found in many zinc‐dependent endopeptidases. We show that a larger signature comprising this sequence is common to most of the known zinc‐dependent endopeptidases, and that the presence of the signature can be indicative of membership in the family. A search of the protein sequence databases for entries containing the signature retrieved several unexpected potential zinc endopeptidases.

Evidence that the major surface proteins of three Leishmania species are structurally related

Promastigotes of Leishmania major LRC-L137, L. donovani LEM 75, and L. tropica LRC-L32 were surface radioiodinated. The proteins of the parasites were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and labeled molecules were revealed by fluorography. A single major iodinated protein of Mr 63 000 (p63) was identified in each of the three species. These proteins were partially purified by phase separation in Triton X-114 solution, demonstrating that the p63 of each of the three species is the most abundant integral membrane protein in the promastigote. Peptide maps were obtained by partial proteolysis with N-chlorosuccinimide or Staphylococcus V8 protease followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The maps of L. major and L. donovani were identical, but only partially homologous to the maps of L. tropica p63. Finally, immunological crossreactivity among the three p63s was demonstrated with the serum of a mouse immunized with purified L. major p63, and the serum of a dog naturally infected with L. donovani. The data show that the major surface proteins found on promastigotes of three Old World Leishmania species are structurally related.

Amplification and sequencing of genomic DNA fragments encoding cysteine proteases from protozoan parasites.

Cysteine protease gene fragments from three protozoan parasites Trypanosoma cruzi, Trypanosoma brucei, and Entamoeba histolytica were amplified by the polymerase chain reaction (PCR) from genomic DNA using degenerate oligonucleotide primers. The primers used for the amplification were designed based upon amino acid sequences flanking the active site cysteine and asparagine residues that are conserved in the eukaryotic cysteine proteases analyzed to date. The amplified DNA fragments, representing approximately 70% of the coding regions of the cysteine protease genes, were subcloned and sequenced. Sequence analysis and alignment showed significant sequence similarity to other members of the eukaryotic cysteine protease family (45% identical to chicken cathepsin L) and conservation of the cysteine, histidine, and asparagine residues which form the catalytic triad. These gene fragments provide molecular probes for further analysis of the structure and function of these important metabolic enzymes.

Identification of the promastigote surface protease in seven species of Leishmania

Twelve different strains of Leishmania, including L. major, L. donovani, L. infantum, L. tropica, L. mexicana, L. amazonensis, L. braziliensis, and L. enriettii were examined for the presence of an ectoenzyme structurally and functionally related to the promastigote surface protease found in L. major LEM 513. All strains examined possess a protease that is labelled by surface iodination of living promastigotes. The electrophoretic migrations of the labelled proteases are similar in all species showing distinct ectoprotease activity. In addition, proteases that cross-react immunologically with the polypeptide moiety of the surface protease of L. major LEM 513 were found in 10 strains. These proteases were in all cases labelled by surface radioiodination. Two of the strains, L. amazonensis and L. braziliensis, do not show a strict correlation between protease activity, surface iodination, and immunological cross-reactivity with the promastigote surface protease of L. major LEM 513, although both strains possess distinct neutral proteases with electrophoretic behavior similar to that of the enzyme of L. major. The amount of proteolytic activity detected at the surface of living cells depends on the strain tested, and correlates qualitatively with the amount of promastigote surface protease detected on zymograms. We conclude that the proteolytic activity found at the surface of Leishmania promastigotes is a common feature of the species infective for humans and that the promastigote surface protease described in this article is structurally and functionally conserved in Old and New World Leishmania.

Characterization of the promastigote surface protease of Leishmania as a membrane-bound zinc endopeptidase

The effects of a variety of inhibitors suggested that the promastigote surface protease (PSP) of Leishmania might be a zinc metalloprotease. To investigate this possibility, we conducted atomic emission and absorption spectroscopic analyses, which show that PSP contains 1 atom of zinc per 63-kDa monomer. Further studies showed that the enzyme can be biosynthetically labeled with 65ZnCl2. The comparison of the amino acid sequence of Leishmania major PSP with nine other zinc metalloproteinases revealed significant similarity in the area of their zinc-binding sites. These data show clearly that the promastigote surface protease of Leishmania is a zinc metalloproteinase. Secondary structure analysis by circular dichroism spectroscopy indicates that PSP contains over 40% beta-strand and less than 20% alpha-helical structure. The molecular masses of amphiphilic PSP (152 kDa) and of hydrophilic PSP (142 kDa), determined by quantitative electron scattering, suggest that the purified enzyme occurs in solution, and presumably at the cell surface, as a non-covalent homodimer.

Leishmania major: Differential regulation of the surface metalloprotease in amastigote and promastigote stages

During its life cycle, the protozoan parasite Leishmania major alternates from an intracellular amastigote form in the mammalian host to a flagellated promastigote form in the insect vector. The expression of the surface metalloprotease (PSP) during differentiation in vitro was investigated by Western and Northern blots, by immunoprecipitation of cells metabolically labeled with [35S]methionine or labeled at the surface with radioactive iodine, and by quantification of the proteolytic activity in substrate-containing polyacrylamide gels. We report that the surface metalloprotease is down-regulated at both the mRNA and the protein level in amastigotes, where it represents less than 1% of the equivalent proteolytic activity detected in promastigotes. A significant amount of mRNA is detected 4 hr after the onset of differentiation. The expression of the protease begins at that time and reaches steady state 8 hr later. The synthesis of PSP precedes the complete morphological differentiation to the promastigote stage and the appearance of the lipophosphoglycan, another major promastigote surface component. In contrast to PSP, a family of mercaptoethanol-activated proteases present in the amastigote exists only at a reduced level in the promastigote. The confinement of the surface metalloprotease to the insect stage of the parasite suggests that it has no physiological function in the parasitism maintenance of mammalian host macrophages.

Discovery of amino-acetonitrile derivatives, a new class of synthetic anthelmintic compounds

A new series of amino-acetonitrile derivatives (AAD) have been discovered that exhibit high anthelmintic activity against parasitic nematode species such as Haemonchus contortus and Trichostrongylus colubriformis. Significantly, these compounds also demonstrate activity against nematode strains resistant to the currently available broad-spectrum anthelmintics. The discovery, synthesis, structure-activity relationship and biological results are presented.

Novel peptide inhibitors of Leishmania gp63 based on the cleavage site of MARCKS (myristoylated alanine-rich C kinase substrate)-related protein

The zinc metalloprotease gp63 (leishmanolysin; promastigote surface protease) is expressed at high density at the surface of Leishmania promastigotes. Efficient non-toxic inhibitors of gp63 do not exist, and its precise role in parasite physiology remains unknown. MARCKS (myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in various cells, including macrophages. We reported previously that MRP is an excellent substrate for gp63. A major cleavage site was identified within the MRP effector domain (ED), a highly basic 24-amino-acid sequence, and the synthetic ED peptide (MRP(ED)) was shown to inhibit MRP hydrolysis. In the present study, MRP cleavage was used as an assay to measure the capacity of various MRP or MARCKS ED peptides to block gp63 activity. On a molar basis, MRP(ED) inhibited gp63 to a greater extent than two previously described gp63 inhibitors, o -phenanthroline and benzyloxycarbonyl-Tyr-Leu-NHOH. MARCKS(ED) analogues containing modifications in the gp63 consensus cleavage site showed significant differences in inhibitory capacity. As phosphorylation of ED serine residues prevented gp63-mediated MRP degradation, we synthesized a pseudophosphorylated peptide in which serine residues were substituted by aspartate (3DMRP(ED)). 3DMRP(ED) was a highly effective inhibitor of both soluble and parasite-associated gp63. Finally, MRP ED peptides were synthesized together with an N-terminal HIV-1 Tat transduction domain (TD) to obtain cell-permeant peptide constructs. Such peptides retained gp63 inhibitory activity and efficiently entered both macrophages and parasites in a Tat TD-dependent manner. These studies may provide the basis for developing potent cell-permeant inhibitors of gp63.

A new in vitro test to evaluate the resistance level against acaricides of the cattle tick, Rhipicephalus (Boophilus) microplus

In this article we present a new bioassay to assess the resistance status of ticks to acaricides. The Larval Tarsal Test (LTT) is a sensitive, highly time-effective in vitro test. It allows the investigation of a large number of compounds and doses on the cattle tick Rhipicephalus (Boophilus) microplus in a short period of time. The ability of the LTT to assess the lethal concentration at 50% mortality (LC(50)) and resistance ratios (RRs) of a susceptible and a resistant R. microplus strain was compared with the FAO-recommended Larval Packet Test (LPT). Representative compounds of the carbamate, organophosphate (OP), synthetic pyrethroid (SP), formamidine (FOR), macrocyclic lactone and pyrazole classes were used for this comparison. The resistance status against OP, SP and FOR of the resistant R. microplus strain was confirmed in vivo. The LTT resulted in resistance ratios comparable to those obtained with the LPT. However, the lethal concentrations were up to 150-fold lower in the LTT than in the LPT. The advantage of the LTT is to simplify the methodology by avoiding the handling of larvae and using multi-well plates. The LTT is therefore a suitable test for the assessment of the level of resistance of R. microplus and is very promising to evaluate the resistance profile of field strains. Additionally, the LTT is also suitable to test other ixodid species.