Gilles Mourier

Denmotoxin, a three-finger toxin from the colubrid snake Boiga dendrophila (Mangrove Catsnake) with bird-specific activity.

Boiga dendrophila (mangrove catsnake) is a colubrid snake that lives in Southeast Asian lowland rainforests and mangrove swamps and that preys primarily on birds. We have isolated, purified, and sequenced a novel toxin from its venom, which we named denmotoxin. It is a monomeric polypeptide of 77 amino acid residues with five disulfide bridges. In organ bath experiments, it displayed potent postsynaptic neuromuscular activity and irreversibly inhibited indirectly stimulated twitches in chick biventer cervicis nerve-muscle preparations. In contrast, it induced much smaller and readily reversible inhibition of electrically induced twitches in mouse hemidiaphragm nerve-muscle preparations. More precisely, the chick muscle α1βγδ-nicotinic acetylcholine receptor was 100-fold more susceptible compared with the mouse receptor. These data indicate that denmotoxin has a bird-specific postsynaptic activity. We chemically synthesized denmotoxin, crystallized it, and solved its crystal structure at 1.9 Å by the molecular replacement method. The toxin structure adopts a non-conventional three-finger fold with an additional (fifth) disulfide bond in the first loop and seven additional residues at its N terminus, which is blocked by a pyroglutamic acid residue. This is the first crystal structure of a three-finger toxin from colubrid snake venom and the first fully characterized bird-specific toxin. Denmotoxin illustrates the relationship between toxin specificity and the primary prey type that constitutes the snakes diet.

Natural agents targeting the α7-nicotinic-receptor in NSCLC: A promising prospective in anti-cancer drug development†

Nicotinic acetylcholine receptors (nAChR) are expressed on normal bronchial epithelial and nonsmall cell lung cancer (NSCLC) cells and are involved in cell growth regulation. Nicotine induced cell proliferation. The purpose of this study was to determine if interruption of autocrine nicotinic cholinergic signaling might inhibit A549 NSCLC cell growth. For this purpose α‐Cobratoxin (α‐CbT), a high affinity α7‐nAChR antagonist was studied. Cell growth decrease was evaluated by Clonogenic and MTT assays. Evidence of apoptosis was identified staining cell with Annexin‐V/PI. Characterization of the basal NF‐κB activity was done using the Trans‐AM NF‐κB assay colorimetric kit. “In vivo” antitumour activity was evaluated in orthotopically transplanted nude mice monitored by In vivo Imaging System technology. α‐CbT caused concentration‐dependent cell growth decrease, mitochondrial apoptosis caspases‐9 and 3‐dependent, but caspase‐2 and p53‐independent and down‐regulation of basal high levels of activated NF‐κB. α‐CbT treatment determines a significant reduction of tumor growth in nude mice orthotopically engrafted with A549‐luciferase cells (4.6% of living cells vs. 31% in untreated mice). No sign of toxicity was reported related to treatment. These findings suggest that α7‐nAChR antagonists namely α‐CbT may be useful adjuvant for treatment of NSCLC and potentially other cancers.

On the immunogenic properties of retro-inverso peptides. Total retro-inversion of T-Cell epitopes causes a loss of binding to MHC II molecules

Retro-inversion is considered an attractive approach for drug and vaccine design since it provides the modified peptides with higher resistance to proteolytic degradation. We therefore investigated in detail the effect of retro-inversion on the immunological properties of synthetic peptides. We have synthesized retro-inverso analogues of MHC II restricted peptides that thus contained the correct orientation of the side chains but an inverse main chain. Retro-inversion made the peptides unable to compete in I E(d) or I A(d) binding tests, demonstrating a very low, if any, capacity to bind to MHC II molecules. These results confirm previous structural data that hydrogen bonds between residues of MHC II molecules and the main chain of antigenic peptides play a major interacting role. In vito experiments further showed that retro-inversion of a T-cell epitope causes its inability to either sustain in vitro T-cell stimulation or to prime specific T cells. Moreover, the retro-inverso peptide was not recognized by antibodies raised against the native peptide and did not elicit antibodies when injected into BALB/c mice. Retro-inverso peptides appear to be poor immunogens as a result of their weak capacity to bind to MHC II molecules. As an advantage, they are not expected to trigger undesirable humoral responses such as hypersensitivity or allergic disease. These results also provide a molecular explanation regarding the weak immunogenicity of D-amino acids containing polypeptides.

Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the α1A-adrenoceptor

Background and purpose:  Venoms are a rich source of ligands for ion channels, but very little is known about their capacity to modulate G‐protein coupled receptor (GPCR) activity. We developed a strategy to identify novel toxins targeting GPCRs.

Fine chemical modifications at N- and C-termini enhance peptide presentation to T cells, by increasing the lifespan of both free and MHC-complexed peptides

We investigated the effect of modifying the N- and/or C-termini of the snake toxin peptide 24-36 on its presentation to T cells. Acetylation at the N-terminus as well as amidation at the C-terminus enhanced the capacity of the peptide to activate T cells. Simultaneous modifications further increased the stimulating activity, the peptide becoming approximately 100-fold more potent than the unmodified peptide. Clearly, the introduced modifications increased the lifetime of the peptide free in solution, by decreasing its proteolytic degradation, during the T cell stimulation assays. Paradoxically, however, at similar concentrations of free peptides, the modified ones, especially those having an acetylated N-terminus, were much more active than the unmodified peptide, irrespective of the experimental conditions. These observations suggested that components other than protection from proteolytic degradation should be associated with the higher stimulating activities of the modified peptides. Accordingly, chasing experiments with APC revealed that acetylation at N-terminus caused a higher persistence of the peptides at APC surface. Together, our data indicate that (i) the T cell stimulating capacity of a peptide is associated with its lifespans in the free and MHC II bound states; and (ii) these lifespans can be greatly enhanced by introducing fine chemical modifications at N- and C-termini. These data may have some implications in designing more potent peptidic immunomodulators.

Structural Model of Ligand-G Protein-coupled Receptor (GPCR) Complex Based on Experimental Double Mutant Cycle Data: MT7 SNAKE TOXIN BOUND TO DIMERIC hM1 MUSCARINIC RECEPTOR

The snake toxin MT7 is a potent and specific allosteric modulator of the human M1 muscarinic receptor (hM1). We previously characterized by mutagenesis experiments the functional determinants of the MT7-hM1 receptor interaction (Fruchart-Gaillard, C., Mourier, G., Marquer, C., Stura, E., Birdsall, N. J., and Servent, D. (2008) Mol. Pharmacol. 74, 1554–1563) and more recently collected evidence indicating that MT7 may bind to a dimeric form of hM1 (Marquer, C., Fruchart-Gaillard, C., Mourier, G., Grandjean, O., Girard, E., le Maire, M., Brown, S., and Servent, D. (2010) Biol. Cell 102, 409–420). To structurally characterize the MT7-hM1 complex, we adopted a strategy combining double mutant cycle experiments and molecular modeling calculations. First, thirty-three ligand-receptor proximities were identified from the analysis of sixty-one double mutant binding affinities. Several toxin residues that are more than 25 Å apart still contact the same residues on the receptor. As a consequence, attempts to satisfy all the restraints by docking the toxin onto a single receptor failed. The toxin was then positioned onto two receptors during five independent flexible docking simulations. The different possible ligand and receptor extracellular loop conformations were described by performing simulations in explicit solvent. All the docking calculations converged to the same conformation of the MT7-hM1 dimer complex, satisfying the experimental restraints and in which (i) the toxin interacts with the extracellular side of the receptor, (ii) the tips of MT7 loops II and III contact one hM1 protomer, whereas the tip of loop I binds to the other protomer, and (iii) the hM1 dimeric interface involves the transmembrane helices TM6 and TM7. These results structurally support the high affinity and selectivity of the MT7-hM1 interaction and highlight the atypical mode of interaction of this allosteric ligand on its G protein-coupled receptor target.

Identification of Various Allosteric Interaction Sites on M1 Muscarinic Receptor Using 125I-Met35-Oxidized Muscarinic Toxin 7

Monoiodinated, Met35-oxidized muscarinic toxin 7 (MT7ox) was synthesized, and its affinity constants for free or N-methyl scopolamine (NMS)-occupied hM1 receptor were measured directly by equilibrium and kinetic binding experiments. Identical values were obtained with the two types of assay methods, 14 pM and 0.9 nM in free or NMS-liganded receptor states, respectively, highlighting a strong negative cooperativity between this allosteric toxin and NMS. Identical results were obtained with indirect binding experiments with [3H]NMS using the ternary complex model, clearly demonstrating the reciprocal nature of this cooperativity. Furthermore, the effects of various orthosteric and allosteric agents on the dissociation kinetic of 125I-MT7ox were measured and show that, except for the MT1 toxin, all of the ligands studied [NMS, atropine, gallamine, brucine, tacrine, staurosporine, and (9S,10S,12R)-2,3,9,10,11-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester (KT5720)] interact allosterically with muscarinic toxin 7. Equilibrium binding experiments with 125I-MT7ox and [3H]NMS were conducted to reveal the effects of these ligands on the free receptor, and affinity constants (pKx values) were calculated using the allosteric ternary complex model. Our results suggest that MT7 toxin interacts with hM1 receptor at a specific allosteric site, which may partially overlap those identified previously for “classic” or “atypical” allosteric agents and highlight the potential of this new allosteric tracer in studying allosterism at muscarinic receptors.

Different Interactions between Mt7 Toxin and the Human Muscarinic M1 Receptor in its Free and N-Methylscopolamine-Occupied States.

Muscarinic MT7 toxin is a highly selective and potent antagonist of the M1 subtype of muscarinic receptor and acts by binding to an allosteric site. To identify the molecular determinants by which MT7 toxin interacts with this receptor in its free and NMS-occupied states, the effect on toxin potency of alanine substitution was evaluated in equilibrium and kinetic binding experiments as well as in functional assays. The determination of the crystallographic structure of an MT7-derivative (MT7-diiodoTyr51) allowed the selection of candidate residues that are accessible and present on both faces of the three toxin loops. The equilibrium binding data are consistent with negative cooperativity between N-methylscopolamine (NMS) and wild-type or modified MT7 and highlight the critical role of the tip of the central loop of the toxin (Arg34, Met35 Tyr36) in its interaction with the unoccupied receptor. Examination of the potency of wild-type and modified toxins to allosterically decrease the dissociation rate of [3H]NMS allowed the identification of the MT7 residues involved in its interaction with the NMS-occupied receptor. In contrast to the results with the unoccupied receptor, the most important residue for this interaction was Tyr36 in loop II, assisted by Trp10 in loop I and Arg52 in loop III. The critical role of the tips of the MT7 loops was also confirmed in functional experiments. The high specificity of the MT7-M1 receptor interaction exploits several MT7-specific residues and reveals a different mode of interaction of the toxin with the free and NMS-occupied states of the receptor.

Identification of a novel snake peptide toxin displaying high affinity and antagonist behaviour for the α2-adrenoceptors

BACKGROUND AND PURPOSE Muscarinic and adrenergic G protein‐coupled receptors (GPCRs) are the targets of rare peptide toxins isolated from snake or cone snail venoms. We used a screen to identify novel toxins from Dendroaspis angusticeps targeting aminergic GPCRs. These toxins may offer new candidates for the development of new tools and drugs.

Immunogenicity of a disulphide-containing neurotoxin: Presentation to T-cells requires a reduction step

It is known that production in a host of antibodies against a protein is associated with various molecular events. These include the stimulation of specific T-lymphocytes, a step that implies the processing of the protein into peptides by various endosomal/lysosomal enzymes, such as cathepsins. Strikingly, however, we observed in vitro that cathepsins B and D have no degrading effect on toxin alpha from Naja nigricollis, a curaremimetic toxin of 61 amino acids and four disulphides. In sharp contrast, the enzymes exert an efficient cleavage of the toxin polypeptide chain once the toxin disulphides are reduced. We also found that the fully reduced toxin and the native toxin were presented with comparable efficiency to two different T-hybridomas by antigen-presenting cells (APC). Together, the data suggest that presentation of toxin fragments to T-cells requires a reduction step of toxin disulphides and, in agreement with previous findings, that this step may be achieved by APC. We wish to suggest that this phenomenon may commonly occur for any toxic proteins that contain disulphides.