[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.

Cerastocytin, a new thrombin-like platelet activator from the venom of the Tunisian viper Cerastes cerastes.

Cerastocytin, a thrombin-like enzyme from the venom of the desert viper, Cerastes cerastes, has been purified to homogeneity by fast performance liquid chromatography (FPLC) on Mono-Q and Mono-S columns. It is a basic protein (isoelectric point higher than 9) made of a single polypeptide chain of 38 kDa. Its N-terminal polypeptide sequence shows strong similarities with other thrombin-like enzymes from snake venoms. Nanomolar concentrations of cerastocytin induce aggregation of blood platelets. This activity is inhibited by chlorpromazine, theophylline and mepacrine, as in the case of platelet aggregation stimulated by low doses of thrombin. Cerastocytin also possesses an amidolytic activity measured with the thrombin chromogenic substrate S-2238. The platelet aggregating activity and the amidolytic activity of cerastocytin were inhibited by PMSF, TPCK, TLCK and soybean trypsin inhibitors, suggesting that cerastocytin is a serine proteinase. On the other hand, both amidolytic activity and platelet aggregating activity of cerastocytin were unaffected by hirudin or by antithrombin III in the presence of heparin. High concentrations of cerastocytin (1-10 microM) also cleaved prothrombin and Factor X.

Amino acid sequence of VlF: identification in the C-terminal domain of residues common to non-hemorrhagic metalloproteinases from snake venoms.

The complete amino acid sequence of a non-hemorrhagic fibrino(geno)lytic enzyme (VlF) isolated from Vipera lebetina venom has been determined. VlF was subjected to separate enzymatic and chemical digestions. Resulting fragments were purified by RP-HPLC and subjected for sequencing by automated Edman degradation. The amino terminus of VlF was determined by mass spectrometry. VlF was shown to be composed of 202 residues having a relative molecular mass of 22,826 Da and containing a zinc-binding site and a catalytically active residue. It displayed significant sequence similarities with many other mature metalloproteinases reported from snake venoms. Sequence comparison of hemorrhagic and non-hemorrhagic mature metalloproteinases revealed the presence at the C-terminal part of the enzymes of two residues common to only hemorrhagic metalloproteinases and two others shared by only non-hemorrhagic ones.

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.

Immunochemistry of scorpion alpha-toxins: purification and characterization of two functionally independent IgG populations raised against toxin II of Androctonus australis Hector.

We report the isolation and characterization of two IgG populations specific to two synthetic peptides corresponding to two antigenic sites of toxin II of the North African scorpion Androctonus australis Hector. Firstly, thanks to the use of: (1) antigenic homology studies between toxin II of A. australis Hector and toxin III of Buthus occitanus tunetanus, (2) chemical modification of toxin II of A. australis Hector, and (3) prediction of the localization of the four major antigenic sites of scorpion alpha-toxins by the method developed by Hopp and Woods [Proc. natn. Acad. Sci. U.S.A. 78, 3824-3828 (1981)], we have established that the region around the disulfide bridge between cysteines 12 and 63 as well as the stretch of residues 50-59 probably each enclosed an antigenic site. Secondly, the synthetic replicates of these regions linked to Sepharose allowed us to isolate, by immunoaffinity chromatography, two IgG populations from the whole anti-toxin II of A. australis Hector IgGs. Finally, each of these two IgG populations was shown to be specific to one antigenic site as evidenced by the multideterminant effect on the slopes of binding curves developed by Berzofsky et al. [Biochemistry 15, 2113-2121 (1976)]. Furthermore, these two IgG populations were found to be functionally independent and this could be related to the fact that the two regions carrying the two antigenic sites are not close to each other in space and that there is neither steric hindrance nor cooperative effects between them. The association constant of these site-specific IgG populations was calculated and found to be equal to 1.18-5.14 X 10(9) l/mole for IgG anti-site 1 and 1.16-5.62 X 10(9) l/mole for IgG anti-site 2 respectively by Sips [J. chem. Phys. 16, 490-495 (1948)], Scatchard [Am. N.Y. Acad. Sci. 51, 660-772 (1949)] and Steward and Petty [Immunology 23, 881-887 (1972)] representations. The index of heterogeneity of 0.9 for anti-P1 and anti-P2 indicates the purification of essentially homogeneous affinity IgG populations.

The antigenic structure of a scorpion toxin.

Scorpion toxins constitute a family of homologous proteins that exert potent pharmacological effects on ion channels. These proteins are immunogenic and constitute a good model for investigation of the molecular basis of antigenicity. In the first part of this article we summarize the results we have obtained in recent years concerning the location of the main antigenic regions of a model toxin, toxin II of the North African scorpion Androctonus australis Hector. Then, thanks to the recently available atomic coordinates of this toxin, we analyzed the relationships between the structural features of the protein and the location of the antigenic regions: we found that antigenic regions are located at exposed parts of the molecular surface, i.e. in reverse turns and the alpha-helix. These surface parts also correspond to segments of the polypeptide chain which are most accessible to a large spherical probe modelizing an antibody molecule. Finally, we obtained a general idea of what could be the main discontinuous antigenic determinants by looking for the neighboring relationships between the most exposed residues of the protein.

Immunochemistry of scorpion alpha-neurotoxins. Determination of the antigenic site number and isolation of a highly enriched antibody specific to a single antigenic site of toxin II of Androctonus australis Hector.

Scorpion neurotoxins active on mammals, i.e. alpha- and beta-toxins, have been divided into several groups according to structural and also immunological criteria. A study of the alpha-toxins has been performed in order to determine the number of antigenic sites; a minimum of four Fab fragments were found to bind simultaneously to toxins I and II of Androctonus australis Hector, and toxin I of Buthus occitanus tunetanus. Taking advantage of the loss of one common antigenic site between toxin II of Androctonus australis Hector and toxin III of Buthus occitanus tunetanus, a highly purified antibody population towards a single site of toxin II of Androctonus australis Hector was isolated and characterized. This result is discussed in terms of sequence homologies between the two proteins as the amino acid sequence of toxin III of Buthus occitanus tunetanus has been determined using standard procedures: the two proteins differ at three positions, two of which (positions 10 and 64) are in the vicinity of the disulfide bridge Cys 12-Cys 63, the third (position 51) is the only one to be conservative.

Immunochemistry of scorpion alpha-toxins: study with synthetic peptides of the antigenicity of four regions of toxin II of Androctonus australis Hector.

Sequences 19-29 and 28-39 of toxin II of the North African scorpion Androctonus australis Hector have been synthesized. These two peptides correspond to the highest peaks in the hydrophilicity profile of toxin II and were thus believed to account for a significant proportion of toxin antigenicity. Affinity chromatography of solid-phase-bonded peptides allowed us to purify two sub-populations from the total IgGs raised against the native toxin. They both still bound to 125I-toxin II and showed a restricted heterogeneity in their specificity. Solid-phase immunoassays confirmed the antigenicity of these synthetic peptides and also that of two other previously described synthetic replicates of the antigenic regions of toxin II: sequences (5-14) S-S (60-64) and 50-59. The location of the four antigenic regions relative to the postulated location of the receptor-binding site of the toxin is discussed.

Identification of antigenic residues on apamin recognized by polyclonal antibodies.

The structural requirements for antigenic recognition of apamin--an 18-amino acid, disulfide-bridged peptide--by rabbit antibodies were defined using a set of 18 apamin analogs in a competition liquid-phase radioimmunoassay. Some residues contribute considerably to antigenic recognition, e.g. Ala10, Arg13, and others to a lesser extent, e.g. Arg14, Glu7 and Thr8. The N- and C-terminal moieties of apamin are less antigenically important. These findings suggest that a good part of antibody specificities are directed to the central tightly folded part of the molecule. They are consistent with the observation that in saturating conditions, labeled apamin can, on average, bind one specific Fab fragment.

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.

Bot IT2: a new scorpion toxin to study receptor site on insect sodium channels.

The insect-specific Bothus occitanus tunetanus IT2 toxin is distinguishable from other scorpion toxins by its amino acid sequence and effects on sodium conductance. The present study reveals that Bot IT2 possesses in cockroach neuronal membranes a single class of high affinity (Kd = 0.3 +/- 0.1 nM) and low capacity (Bmax = 2.4 +/- 0.5 pmol/mg) binding sites. Competitive binding experiments with several known sodium channel neurotoxins reveal that the Bot IT2 binding site is in close proximity to the other toxins.

Novel anti-platelet aggregation polypeptides from Vipera lebetina venom: isolation and characterization.

Lebetins 1 and Lebetins 2, two polypeptide groups that inhibit platelet aggregation, were isolated from Vipera lebetina venom by gel filtration and reverse phase chromatography. Amino acid sequencing indicated that the first group contains two major polypeptides of 13 and 12 residues; their molecular weight was determined by electrospray mass spectrometry. The second was composed of two peptides of 38 and 37 residues, each with one disulfide bond. Sequence analysis revealed neither RGD sequence nor homology with other proteins including known snake or tick polypeptides. Lebetins 1 were Pro and Lys rich peptides and their sequences were identical to the N-terminus of Lebetins 2. Lebetins inhibited platelet aggregation induced by thrombin, collagen and PAF-acether. The 50% concentration that inhibited human and rabbit platelet aggregation induced by thrombin was 590 nM and 125 nM for Lebetins 1 and 100 nM and 8 nM for Lebetins 2, respectively. Lebetins 1 and Lebetins 2 also inhibited fibrinogen-induced aggregation of alpha-chymotrypsin-treated platelets as well as in vivo collagen-induced thrombocytopenia in rats with half effective doses of 2 nmol/kg and 4.2 nmol/kg, respectively. Lebetins were not toxic after intravenous injection into mice and rats. These polypeptides form novel platelet inhibitors that could be used to delineate further the mechanisms of platelet aggregation and serve as a model for developing antithrombotic agents.