[Snake venom Kunitz/BPTI family: Structure, classification and pharmacological potential]. / LA FAMILE KUNITZ/BPTI DES VENINS DE SERPENTS: STRUCTURE, CIASSIFICATION ET POTENTIEL PHARMACOLOGIQUE.

Snake venoms are rich sources of serine proteinase inhibitors that are members of the KunitzBPTI (bovine pancreatic trypsin inhibitor) family. Generally, these inhibitors are formed by 60 amino acids approximately. Their folding is characterised by a canonical loop that binds in a complementary manner to the active site of serine protease. Some variants from snake venoms show only weak inhibitory activity against proteases while others are neurotoxic. Moreover, proteases inhibitors are involved in various physiological prdcesses, such as blood coagulation, fibrinolysis, and inflammation. Also, these molecules showed an anti-tumoralpotent and anti-metastatic effect. Interestingly, KunitzBPTI peptides can have exquisite binding specificities and possess high potency for their targets making them excellent therapeutic candidates.

[Snake Venom L-Amino Acid Oxidases potential biomedical applications]. / LES L-AMINO ACIDES OXYDASES DES VENINS DE SERPENTS ET LEURS POTENTIELLES APPLICATIONS BIOMEDICALES.

L-amino acid oxidases (LAAOs) are flavoenzymes widely found in several organisms, including venoms snakes, where they contribute to the toxicity of ophidian envenomation. Their toxicity is primarily due to enzymatic activity, but other mechanisms have been proposed recently which require further investigation. LAAOs exert biological and pharmacological effects, including actions on platelet aggregation and the induction of apoptosis, hemorrhage and cytotoxicity. These proteins present a high biotechnological potentialfor the development of antimicrobial, antitumor and antiprotozoan agents. This review summarizes the biochemical properties, structural characteristics and various biological functions of snake venoms' LAAO. Furthermore, the putative mechanisms of action, were well detailed.

MMP inhibitors and cancer treatment trials, limitations and hopes for the future.

Matrix metalloproteinases (MMPs) are a family of enzymes that have been recognized as promising therapeutic and diagnostic targets for the treatment and detection of human cancers. This rises from their unique ability to degrade all components of the extracellular matrix and their overexpression at different stages of tumor progression. The specific involvement of MMPs in the oncogenic processes has speeded up the efforts that have been made for the past 20 years to develop and evaluate MMP inhibitors (MMPIs) as potential anti-cancer agents. However, bringing an MMPI to the point of clinical approval is still a challenge. In this review, we provide an overview of the structure and function of MMPs along with their implication in cancer development. Furthermore, we focus on the literature concerning the development of broad spectrum natural and synthetic MMPIs, with emphasis on their limitations and the disappointing results of most clinical trials. The failure of broad spectrum MMPIs highlighted the need for the development of selective inhibitors that fully discriminate between different members of the MMP family. As a future perspective on the development of potent MMPIs, we also report in this review a novel structure-based strategy developed in our group to design new mini-protein ligands for MMP inhibition by functional motif grafting.

[Selectins as adhesion molecules and potential therapeutic target]. / Les selectines: acteurs de l'adherence cellulaire et potentiel cible therapeutique.

Selectins belong to the family of adhesion molecules that recognize sugars as ligands through their Carbohydrate Recognition Domain (CRD). There are three types of selectin: the L-selectin (CD62L), which is constitutively expressed by most leukocyte populations, the P-selectin (CD62P) is found on activated platelets and endothelial cells, and the E-selectin (CD62E) expressed by activated endothelial cells. These three molecules exhibit high homology in their structures. Selectin-ligand interactions are among the most studied protein-glycan interactions in biology. The selectins and theirs ligands are involved in regulating inflammatory and immunological events that occur at the interface of the bloodstream and vessel walls. Their molecular partners are surface glycoconjugates harboring groups of the sialyl-Lewis antigens. This review presents an inventory of our current knowledge on the structures and functions of selectins and their ligands. We also provide an update on their involvement in pathophysiological processes, especially during inflammation and tumor development.

[Snake venom metalloproteinases: structure, biosynthesis and function(s)]. / Les metalloproteases des venins de serpents: structure, biosynthese et fonction(s).

The biochemical and the pharmacological characterization of snake venoms revealed an important structural and functional polymorphism of proteins which they contain. Among them, snake venom metalloproteases (SVMPs) constitute approximatively 20 to 60% of the whole venom proteins. During the last decades, a significant progress was performed against structure studies and the biosynthesis of the SVMPs. Indeed, several metalloproteases were isolated and characterized against their structural and pharmacological properties. In this review, we report the most important properties concerning the classification, the structure of the various domains of the SVMPs as well as their biosynthesis and their activities as potential therapeutic agents.

[Snake venoms C-type lectins and their receptors on platelets and cancerous cells]. / Les lectines de type C de venins de serpents et leurs récepteurs sur les plaquettes et les cellules cancéreuses.

Snake venoms are a rich natural source of bioactive molecules, such as peptides, proteins and enzymes, more and more used in biomedical research in diagnostic or therapeutic purposes. The protein components of snake venoms belong to diverse families such as serine proteases, phospholipases, disintegrins, metalloproteinases and C-type lectins. Due to their effects on various receptors such as GPIb, GPVI, alpha2beta1..., the C-type lectins were considered, in first time, as modulators of the platelet aggregation. Recently, some of them have been described for their anti-tumoral potential effect due to their capacity to inhibit adhesion, migration, proliferation and invasion of different cancer cell lines. Also, the C-type lectins have a powerful antiangiogenic effect in vivo and in vitro by interacting with integrins of endothelial cells.

[Snake venom disintegrins: classification and therapeutic potential]. / Les désintégrines issues des venins de serpents: classification et potentiel thérapeutique.

Biochemistry and pharmacology of snake venoms reveal structural and functional polymorphisms of proteins they contain. These lead their effects by their enzymatic activities (proteases, phospholipases A2, L-amino acid oxydases...) or by binding to membrane receptors. Disintegrin from snake venoms play a role as antagonists of cell adhesion and migration by binding integrins and blocking their function. Characterization of integrin antagonists from snake venom allows us understanding the function of some integrins providing new information to develop new therapeutic agents. In this review, we report classification and therapeutic implications of disintegrins. In particular the structural and the functional characteristics of lebestatin; a short disintegrin isolated from Tunisian Macrovipera lebetina snake venom.

[Modulation of skeletal muscle contraction by the non-toxic fraction of Buthus occitanus tunetanus venom via the cholinergic receptors]. / Modulation de la contraction du muscle squelettique par la fraction non toxique du venin de Buthus occitanus tunetanus via les récepteurs cholinergiques.

Cholinergic receptors have an essential physiological role in the central nervous system because of their implication in higher functions in the neuromuscular junction within the brain and also in the peripheral nervous system by activating nicotinic (nAChRs) or muscarinic (mAChRs) receptors. Moreover, cholinergic receptors could be recognized by animal toxins isolated from snake venoms or alkaloids having animal or vegetal origin. In this context, we aim to find such molecules in a non toxic venom fraction of Buthus occitanus tunetanus scorpion, M1, which could therefore constitute promising medical tool. We present here a physiological study in skeletal muscle cells that regroups data that have been recently published and some new results reinforcing the last ones. The global effect of M1, was firstly studied on isolated nerve-muscle preparation. In cultured myotubes, we have found that the intracellular calcium increase, induced by M1 was blocked when ryanodine or inositol 1,4,5-triphosphate receptors are inhibited. Moreover, we have shown that M1 application on myotubes, induced a membrane depolarization as seen with acetylcholine. The treatment of myotubes with alpha-bungarotoxin blocked in most parts the depolarization amplitude. Thus, these results confirm the presence of at least one component in M1 active in nAChRs.

[Use of medicinal plants against scorpionic and ophidian venoms]. / Utilisation des plantes médicinales contre l'envenimation scorpionique et ophidienne.

The scorpionic and ophidian envenomations are a serious public health problem in Tunisia especially in Southeastern regions. In these regions Artemisia campestris L is a plant well known which has a very important place in traditional medicine for its effectiveness against alleged venom of scorpions and snakes. In this work, we tested for the first time, the anti-venomous activity of Artemisia campestris L against the scorpion Androctonus australis garzonii and the viper Macrovipera lebetina venoms. Assays were conducted by fixing the dose of extract to3 mg/mouse while doses of venom are variable. The leaves of Artemisia campestris L were extracted by various organic solvents (Ether of oil, ethyl acetate, methanol and ethanol) and each extract was tested for its venom neutralizing capacity. For the ethanolic extract, a significant activity with respect to the venoms of scorpion Androctonus australis garzonii (Aag), was detected. Similarly, a significant neutralizing activity against the venom of a viper Macrovipera lebetina (Ml), was obtained with the dichloromethane extract. These results suggest the presence of two different type of chemical components in this plant: those neutralizing the venom of scorpion are soluble in ethanol whereas those neutralizing the venom of viper are soluble in dichloromethane.

Identification of second lipolysis activating protein from scorpion Buthus occitanus tunetanus.

Besides the previously described LVP1, a second protein, LVP2, inducing a lipolytic response in adipose cells, was purified from scorpion Buthus occitanus tunetanus venom. It represented 2% of crude venom proteins, with pHi = 6 and molecular mass of 16889 Da. The reduction and the alkylation of LVP2 revealed an heterodimeric structure. Isolated alpha and beta chains of LVP2 have a molecular weight (MW) of 8822 Da and 8902, respectively. This protein was not toxic to mice and stimulated lipolysis on freshly dissociated rat adipocytes in a dose-dependent manner with EC50 = 2 +/- 0.75 microg/ml. LVP2 subunits did not display any lipolytic activity. As previously described for venom and LVP1, beta adrenergic receptor (beta AR) antagonists interfere with LVP2 activity. Furthermore, it is shown that LVP2 competes with [3H] CGP 12177 (beta1/beta2 AR antagonist) for binding to adipocyte plasma membrane with an IC50 of about 10(-7)M. Thus, these results bring original information on the existence of proteins that are present in scorpion venoms and can exert a distinct biological activity on adipocyte lipolysis through a beta-type adreno-receptor pathway.

Molecular cloning and nucleotide sequence analysis of encoded anti-insect toxin BotIT2 from the scorpion Buthus occitanus tunetanus venom.

Numerous toxins from scorpion venoms are much more toxic to insects than to other animal classes, and possess high affinity to Na+ channels. Many of them active on insects were purified from the venom of Buthus occitanus tunetanus. Using amino acid sequences of BotIT2 and RACE-PCR amplification (Rapid amplification of cDNA ends) technique, we isolated, identified and sequenced the nucleotide sequence from the venom glands of the scorpion Buthus occitanus tunetanus. The cDNA encodes a precursor of an insect toxin of 60 amino acid residues. The deduced nucleotide sequence toxin was identical to the determined amino acid sequence of BotIT2. BotIT2 is more similar to the excitatory toxins in its mode of action and to the depressant toxins in its primary structure.

[Characterisation of a novel toxin active on potassium apanaine channels, purified from Buthus occitanus tunetanus scorpion venom]. / Caractérisation d'une nouvelle toxine active sur des canaux potassium apamine sensible purifiée à partir du venin de Buthus occitanus tunetanus.

A new peptidyl inhibitor of the small-conductance Ca(2+)-activated K+ channels (SKca) was purified to homogeneity from the venom of the Tunisian scorpion Buthus occitanus tunetanus. The molecular mass determined by SDS-PAGE, shows that it's a short peptide (3300 Da). The primary sequence of this toxin shows that it is a 31-residue polypeptide cross-linked by three disulfide bridges and structurally related to subfamily 5 of short scorpion toxins. This molecule shows similar pharmacological properties with this group of peptides inducing high toxicity in mice after intracerebro-ventricular injection, and competing with iodinated apamin for binding to its receptor site from rat brain synaptosomes (K0.5 = 4 nM).

Round table and synthesis of the meeting.

Our knowledge of the epidemiology of scorpion stings and snakebites remains fragmentary but sufficient, nevertheless, to be able to confirm that envenomations constitute a real public health problem throughout Africa. In order for the health authorities to be able to improve management of this problem, data collection must be enhanced. The objective should be to determine what kinds of intervention are necessary (quantity of antivenom serum and drugs, in particular) and where they should be applied. Specialists must come to a rapid consensus for a simple therapeutic protocol to be used in peripheral health centres where means are often scarce. Training for health personnel is also insufficient. Appropriate courses must be organised for medical doctors and nurses within both their basic and on-going training. These courses must necessarily involve health personnel from rural zones must affected by envenomations. The availability of antivenom serum--the only specific, efficacious drug--must be improved as soon as possible. If quantitative and geographic needs can be determined by epidemiological studies, then distribution must be developed by original means (grouping orders at national level, direct orders) and diversified financial support (purchase on the open market, local authority grants, community participation). The symposium attendees agreed to meet again within two years' time to evaluate progress in the area.

[Effect of Macrovipera lebetina and Cerastes cerastes venoms on adherence to integrins of cancerous cells (IGR39, HT29-D4 and IGROV1)]. / Effet des venins de Macrovipera lebetina et de Cerastes cerastes sur l'adherence aux intégrines des cellules cancéreuses (IGR39, HT29-D4 et IGROV1).

In this work, we provide experimental arguments in favor of the fact that components from Macrovipera lebetina and Cerastes cerastes venoms bind to IGR39 melanoma cells but not to HT29D4 cells that derive from carcinoma adenome. Furthermore, Macrovipera lebetina and Cerastes cerastes venoms inhibit the adherence of IGR39 and HT 29-D4 to various extracellular matrix proteins. Macrovipera lebetina and Cerastes cerastes venoms did not inhibit the non specific adherence of IGR 39 cells to polylysine. In addition, binding of components from Cerastes cerastes venom to IGR39 cells is inhibited by GRGDS peptide and by monoclonal antibidy anti-av, while these two components have no effect on the adherence of IGR39 to Macrovipera lebetina venom.

[In vivo and in vitro protection against lethal activity of Buthus occitanus tunetanus venom with a recombinant protein]. / Induction de protection in vivo et in vitro contre l'activite létale du venin de Buthus occitanus tunetanus avec une protéine recombinante.

We report the use of recombinant scorpion toxin in the form of fusion protein as antigen for mice immunisation. The aim is to produce protective antisera against lethal activity of the venom from Tunisian scorpion Buthus occitanus tunetanus, responsible for several annually reported human cases of scorpion stings. The gene encoding Bot III (the most toxic alpha toxin of Buthus occitanus tunetanus) was fused to the sequence encoding synthetic ZZ domains of staphylococcal protein A. The construct ZZ-Bot III was expressed in the periplasm of E. coli as a fusion protein and purified by affinity chromatography. The recombinant fusion protein was characterized and used as antigen to generate antibodies in mice. The antibodies against the recombinant protein neutralize the toxic venom (10 LD50/ml) and also confer protection for immunized mice against antigenically related mammal toxins.

Use of fusion protein constructs to generate potent immunotherapy and protection against scorpion toxins.

We report the use of recombinant scorpion toxins in the form of fusion proteins as antigens for immunisation in rabbits and mice: the aim was to produce in these animal models protective antisera against the most lethal alpha-type toxins in the venom from the North African scorpion Androctonus australis. The cDNAs encoding AaH I, AaH II and AaH III (the three major alpha-type toxins acting on voltage-sensitive sodium channels) were fused to the sequence encoding the maltose binding protein (MBP). The constructs (MBP-AaH I, MBP-AaH II, MBP-AaH I+II and MBP-AaH III) were expressed in Escherichia coli, and resulting fusion proteins were translocated to the periplasmic space. The recombinant fusion proteins were characterised and used as antigens to generate antibodies in rabbits. These antibodies raised specifically recognised their corresponding radiolabelled-toxin with affinities in the 0.1nM range. In vitro neutralisation assays indicated that 1ml of serum raised against a mixture of fusion proteins was able to neutralise 15 LD(50) of the toxic fraction (AaH-G50) purified from the crude venom by molecular filtration through Sephadex G50. In vivo, the fusion proteins induced a long-term protection in mice against the lethal effects of AaH-G50 or of the native toxins. Ten weeks after the beginning of the immunisation programme, mice were challenged with various toxins or AaH-G50 doses. Mice were fully protected against three LD(50) of AaH-G50. Our work shows that fusion protein constructs can be used as a vaccine providing efficient immune protection against A. australis venom.

Effects of antivenom on Buthus occitanus tunetanus (Bot) scorpion venom pharmacokinetics: towards an optimization of antivenom immunotherapy in a rabbit model.

The pharmacokinetic parameters of Bot venom were determined in a rabbit model using a specific sandwich type ELISA. After intravenous injection, Bot venom seems to follow a three-compartment pharmacokinetic open model. However, after subcutaneous injection, the distribution and elimination kinetics of Bot venom are best characterized by a bi-compartment pharmacokinetic open model. Bot venom is completely absorbed from its SC injection site, since the absolute bioavailability is higher than 95%; the maximum plasma venom concentration is reached between 30 and 60 min after venom injection. Bot venom diffuses rapidly to tissues and is distributed in a high body volume. The total body clearance of Bot venom is relatively high in agreement with a low mean residence time. Antivenom immunotherapy experiments were carried out in the rabbit model, in order to select the most appropriate strategy for the adequate use of this treatment. The effects of the route, the dose and the delay of antivenom injection on Bot venom pharmacokinetic parameters and on the antivenom immunotherapy efficacy were then studied. These studies indicated in particular that: (1) the injection of a minimal neutralizing antivenom dose is required for a complete and permanent neutralization of circulating venom antigens; this dose is named minimal (threshold) efficacious antivenom dose; (2) the intramuscular route is not the most appropriate way for antivenom injection; and (3) a delayed antivenom immunotherapy remains efficacious especially on the neutralization of the remaining circulating venom. In short, these experimental studies show that early intravenous injection of an appropriate antivenom dose (at least the threshold efficacious dose) is the indicated way for a rapid and permanent neutralization of circulating scorpion venom toxins.

Amino acid structure and characterization of a heterodimeric disintegrin from Vipera lebetina venom.

A heterodimeric disintegrin designed as lebein was isolated from crude Vipera lebetina venom using gel filtration, anion and cation exchange chromatographies on FPLC. The amino acid sequence of each subunit determined by Edman degradation contains 64 residues with ten half-cystines and an RGD site at the C-terminal part of the molecule. The molecular mass of native lebein determined by mass spectrometry was found to be 14083.4 Da and those of alpha and beta subunits were 6992.05 and 7117.62, respectively. These value are in good agreement with those calculated from the sequences. This protein strongly inhibits ADP induced platelet aggregation on human platelet rich plasma with IC(50)=160 nM. Sequences of this protein subunits displayed significant sequence similarities with many other monomeric and dimeric disintegrins reported from snake venoms. We identified an amino acid residue (N) in the hairpin loop of both subunits (CNRARGDDMNDYC) which is different from all other reported motifs of disintegrins and this subtle difference may contribute to the distinct affinities and selectivities of this class of proteins.

[Study of the protective capacity of scorpion venom Buthus occitanus tunetanus polymerised to glutaraldehyde in mice strains with different haplotypes]. / Edude de la capacité protectrice du venin du scorpion Buthus occitanus tunetanus (Bot) polymérisé au glutaraldehyde chez des souches de souris d'haplotypes différents.

The immune response obtained against the toxic fraction of the scorpion venom Buthus occitanus tunetanus detoxified by polymerisation with glutaraldehyde, was analysed for low inbred mice having different haplotypes: C57BL/6 (H-2b) et BALB/c (H-2d) and the SWISS outbred mouse. This three strains of mice, immunized with the polymeric form of Bot-G50 are able to induce an immune response with bumoral mediation. The anti-polymers antibodies obtained from immunized mice, cross-react with the native Bot-G50 fraction. Indeed, in vitro protection experiments demonstrated that immune sera were neutralizing (between 150 and 235 micrograms of Bot-G50 ml). The in vivo protection assays showed that immunized mice could resist the challenge by high amount of toxic fraction (between 70 and 80 micrograms of Bot-G50). This protection was found to be long-lived, since immunized SWISS mice could resist the challenge by 4 DL50 of the toxic fraction (80 micrograms) six month after the start of the immunized program.

Lebecetin, a C-lectin protein from the venom of Macrovipera lebetina that inhibits platelet aggregation and adhesion of cancerous cells.

A novel C-lectin protein, lebecetin, was purified and characterized from the venom of Macrovipera lebetina. It is a disulfide-linked heterodimer of 15 and 16 kD. The subunits are homologous to each other and to the other snake venom proteins of the C-type (Ca(2+)-dependent) lectin superfamily. Lebecetin shows a potent inhibitory effect on whole blood and washed platelets induced by different agonists. It inhibits the agglutination of human fixed platelets in the presence of ristocetin. Lebecetin also interferes with the adhesion of IGR39 melanoma and HT29D4 adenocarcinoma cells. These two lines adhere to lebecetin used as matrix. Lebecetin is also able to strongly reduce IGR39 and HT29D4 cell adhesion to fibrinogen and laminin, but not to fibronectin and collagen types I and IV, respectively. Adhesion properties of lebecetin may thus involve integrin receptors.