Servent D

General

Full name : Servent Denis

First name : Denis

Mail : CEA Saclay Dept d'Ingenierie et d'Etudes des Proteines - Bat 152, 91191 Gif sur Yvette

Zip Code :

City :

Country : France

Email : denis.servent@cea.fr

Phone : 33 1 69085202

Fax : 33 169089071

Website : \/\/ibitecs.cea.fr\/drf\/ibitecs\/Pages\/services\/simopro\/ltmb.aspx

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References (34)

Title : Functional characterization of multifunctional ligands targeting acetylcholinesterase and alpha 7 nicotinic acetylcholine receptor - Cieslikiewicz-Bouet_2020_Biochem.Pharmacol__114010
Author(s) : Cieslikiewicz-Bouet M , Naldi M , Bartolini M , Perez B , Servent D , Jean L , Araoz R , Renard PY
Ref : Biochemical Pharmacology , :114010 , 2020
Abstract : Alzheimer's disease (AD) is a neurodegenerative disorder associated with cholinergic dysfunction, provoking memory loss and cognitive dysfunction in elderly patients. The cholinergic hypothesis provided over the years with molecular targets for developing palliative treatments for AD, acting on the cholinergic system, namely, acetylcholinesterase and alpha7 nicotinic acetylcholine receptor (alpha7 nAChR). In our synthetic work, we used "click-chemistry" to synthesize two Multi Target Directed Ligands (MTDLs) MB105 and MB118 carrying tacrine and quinuclidine scaffolds which are known for their anticholinesterase and alpha7 nicotinic acetylcholine receptor agonist activities, respectively. Both, MB105 and MB118, inhibit human acetylcholinesterase and human butyrylcholinesterase in the nanomolar range. Electrophysiological recordings on Xenopus laevis oocytes expressing human alpha7 nAChR showed that MB105 and MB118 acted as partial agonists of the referred nicotinic receptor, albeit, with different potencies despite their similar structure. The different substitution at C-3 on the 2,3-disubtituted quinuclidine scaffold may account for the significantly lower potency of MB118 compared to MB105. Electrophysiological recordings showed that the tacrine precursor MB320 behaved as a competitive antagonist of human alpha7 nAChR, in the micromolar range, while the quinuclidine synthetic precursor MB099 acted as a partial agonist. Taken all together, MB105 behaved as a partial agonist of alpha7 nAChR at concentrations where it completely inhibited human acetylcholinesterase activity paving the way for the design of novel MTDLs for palliative treatment of AD.
ESTHER : Cieslikiewicz-Bouet_2020_Biochem.Pharmacol__114010
PubMedSearch : Cieslikiewicz-Bouet_2020_Biochem.Pharmacol__114010
PubMedID: 32360492

Title : The three-finger toxin fold: a multifunctional structural scaffold able to modulate cholinergic functions - Kessler_2017_J.Neurochem_142 Suppl 2_7
Author(s) : Kessler P , Marchot P , Silva M , Servent D
Ref : Journal of Neurochemistry , 142 Suppl 2 :7 , 2017
Abstract : Three-finger fold toxins are miniproteins frequently found in Elapidae snake venoms. This fold is characterized by three distinct loops rich in beta-strands and emerging from a dense, globular core reticulated by four highly conserved disulfide bridges. The number and diversity of receptors, channels, and enzymes identified as targets of three-finger fold toxins is increasing continuously. Such manifold diversity highlights the specific adaptability of this fold for generating pleiotropic functions. Although this toxin superfamily disturbs many biological functions by interacting with a large diversity of molecular targets, the most significant target is the cholinergic system. By blocking the activity of the nicotinic and muscarinic acetylcholine receptors or by inhibiting the enzyme acetylcholinesterase, three-finger fold toxins interfere most drastically with neuromuscular junction functioning. Several of these toxins have become powerful pharmacological tools for studying the function and structure of their molecular targets. Most importantly, since dysfunction of these receptors/enzyme is involved in many diseases, exploiting the three-finger scaffold to create novel, highly specific therapeutic agents may represent a major future endeavor. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.
ESTHER : Kessler_2017_J.Neurochem_142 Suppl 2_7
PubMedSearch : Kessler_2017_J.Neurochem_142 Suppl 2_7
PubMedID: 28326549

Title : Cyclic imine toxins from dinoflagellates: a growing family of potent antagonists of the nicotinic acetylcholine receptors - Molgo_2017_J.Neurochem_142 Suppl 2_41
Author(s) : Molgo J , Marchot P , Araoz R , Benoit E , Iorga BI , Zakarian A , Taylor P , Bourne Y , Servent D
Ref : Journal of Neurochemistry , 142 Suppl 2 :41 , 2017
Abstract : We present an overview of the toxicological profile of the fast-acting, lipophilic macrocyclic imine toxins, an emerging family of organic compounds associated with algal blooms, shellfish contamination and neurotoxicity. Worldwide, shellfish contamination incidents are expanding; therefore, the significance of these toxins for the shellfish food industry deserves further study. Emphasis is directed to the dinoflagellate species involved in their production, their chemical structures, and their specific mode of interaction with their principal natural molecular targets, the nicotinic acetylcholine receptors, or with the soluble acetylcholine-binding protein, used as a surrogate receptor model. The dinoflagellates Karenia selliformis and Alexandrium ostenfeldii / A. peruvianum have been implicated in the biosynthesis of gymnodimines and spirolides, while Vulcanodinium rugosum is the producer of pinnatoxins and portimine. The cyclic imine toxins are characterized by a macrocyclic skeleton comprising 14-27 carbon atoms, flanked by two conserved moieties, the cyclic imine and the spiroketal ring system. These phycotoxins generally display high affinity and broad specificity for the muscle type and neuronal nicotinic acetylcholine receptors, a feature consistent with their binding site at the receptor subunit interfaces, composed of residues highly conserved among all nAChRs, and explaining the diverse toxicity among animal species. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.
ESTHER : Molgo_2017_J.Neurochem_142 Suppl 2_41
PubMedSearch : Molgo_2017_J.Neurochem_142 Suppl 2_41
PubMedID: 28326551

Title : Special Issue on Toxins: New targets and new functions -
Author(s) : Benoit E , Barbier J , Popoff MR , Faure G , Molgo J , Servent D
Ref : Toxicon , 116 :1 , 2016
PubMedID: 26996493

Title : The Repellent DEET Potentiates Carbamate Effects via Insect Muscarinic Receptor Interactions: An Alternative Strategy to Control Insect Vector-Borne Diseases - Abd-Ella_2015_PLoS.One_10_e0126406
Author(s) : Abd-Ella A , Stankiewicz M , Mikulska K , Nowak W , Pennetier C , Goulu M , Fruchart-Gaillard C , Licznar P , Apaire-Marchais V , List O , Corbel V , Servent D , Lapied B
Ref : PLoS ONE , 10 :e0126406 , 2015
Abstract : Insect vector-borne diseases remain one of the principal causes of human mortality. In addition to conventional measures of insect control, repellents continue to be the mainstay for personal protection. Because of the increasing pyrethroid-resistant mosquito populations, alternative strategies to reconstitute pyrethroid repellency and knock-down effects have been proposed by mixing the repellent DEET (N,N-Diethyl-3-methylbenzamide) with non-pyrethroid insecticide to better control resistant insect vector-borne diseases. By using electrophysiological, biochemichal, in vivo toxicological techniques together with calcium imaging, binding studies and in silico docking, we have shown that DEET, at low concentrations, interacts with high affinity with insect M1/M3 mAChR allosteric site potentiating agonist effects on mAChRs coupled to phospholipase C second messenger pathway. This increases the anticholinesterase activity of the carbamate propoxur through calcium-dependent regulation of acetylcholinesterase. At high concentrations, DEET interacts with low affinity on distinct M1/M3 mAChR site, counteracting the potentiation. Similar dose-dependent dual effects of DEET have also been observed at synaptic mAChR level. Additionally, binding and in silico docking studies performed on human M1 and M3 mAChR subtypes indicate that DEET only displays a low affinity antagonist profile on these M1/M3 mAChRs. These results reveal a selective high affinity positive allosteric site for DEET in insect mAChRs. Finally, bioassays conducted on Aedes aegypti confirm the synergistic interaction between DEET and propoxur observed in vitro, resulting in a higher mortality of mosquitoes. Our findings reveal an unusual allosterically potentiating action of the repellent DEET, which involves a selective site in insect. These results open exciting research areas in public health particularly in the control of the pyrethroid-resistant insect-vector borne diseases. Mixing low doses of DEET and a non-pyrethroid insecticide will lead to improvement in the efficiency treatments thus reducing both the concentration of active ingredients and side effects for non-target organisms. The discovery of this insect specific site may pave the way for the development of new strategies essential in the management of chemical use against resistant mosquitoes.
ESTHER : Abd-Ella_2015_PLoS.One_10_e0126406
PubMedSearch : Abd-Ella_2015_PLoS.One_10_e0126406
PubMedID: 25961834

Title : Poster: Multi-target directed ligands: Electrophysiological characterization of an anticholinesterase inhibitor coupled to an agonist of alpha7 nicotinic acetylcholine receptor -
Author(s) : Araoz R , Bouet m , Bartolini M , Coquelle N , Colletier JP , Servent D , Molgo J , Jean L , Renard PY
Ref : Biochemical Pharmacology , 97 :621 , 2015
PubMedID:

Title : Poster: Cyclic imine toxins: From shellfish poisoning to neuroscience: The case of acyl derivatives -
Author(s) : Araoz R , Hess P , Pelissier F , Benoit E , Servent D , Zakarian A , Molgo J
Ref : Biochemical Pharmacology , 97 :622 , 2015
PubMedID:

Title : Marine Macrocyclic Imines, Pinnatoxins A and G: Structural Determinants and Functional Properties to Distinguish Neuronal alpha7 from Muscle alpha1(2)betagammadelta nAChRs - Bourne_2015_Structure_23_1106
Author(s) : Bourne Y , Sulzenbacher G , Radic Z , Araoz R , Reynaud M , Benoit E , Zakarian A , Servent D , Molgo J , Taylor P , Marchot P
Ref : Structure , 23 :1106 , 2015
Abstract : Pinnatoxins are macrocyclic imine phycotoxins associated with algal blooms and shellfish toxicity. Functional analysis of pinnatoxin A and pinnatoxin G by binding and voltage-clamp electrophysiology on membrane-embedded neuronal alpha7, alpha4beta2, alpha3beta2, and muscle-type alpha12betagammadelta nicotinic acetylcholine receptors (nAChRs) reveals high-affinity binding and potent antagonism for the alpha7 and alpha12betagammadelta subtypes. The toxins also bind to the nAChR surrogate, acetylcholine-binding protein (AChBP), with low Kd values reflecting slow dissociation. Crystal structures of pinnatoxin-AChBP complexes (1.9-2.2 A resolution) show the multiple anchoring points of the hydrophobic portion, the cyclic imine, and the substituted bis-spiroketal and cyclohexene ring systems of the pinnatoxins that dictate tight binding between the opposing loops C and F at the receptor subunit interface, as observed for the 13-desmethyl-spirolide C and gymnodimine A congeners. Uniquely, however, the bulky bridged EF-ketal ring specific to the pinnatoxins extends radially from the interfacial-binding pocket to interact with the sequence-variable loop F and govern nAChR subtype selectivity and central neurotoxicity.
ESTHER : Bourne_2015_Structure_23_1106
PubMedSearch : Bourne_2015_Structure_23_1106
PubMedID: 26004441

Title : Neonicotinoid binding, toxicity and expression of nicotinic acetylcholine receptor subunits in the aphid Acyrthosiphon pisum - Taillebois_2014_PLoS.One_9_e96669
Author(s) : Taillebois E , Beloula A , Quinchard S , Jaubert-Possamai S , Daguin A , Servent D , Tagu D , Thany SH , Tricoire-Leignel H
Ref : PLoS ONE , 9 :e96669 , 2014
Abstract : Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [3H]-IMI (Kd of 0.16 +/- 0.04 nM and 41.7 +/- 5.9 nM) and for the nicotinic antagonist [125I]-alpha-bungarotoxin (Kd of 0.008 +/- 0.002 nM and 1.135 +/- 0.213 nM). Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [125I]-alpha-bungarotoxin binding sites while CLT affinity was similar for both [125I]-alpha-bungarotoxin and [3H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 microg/ml) and TMX (LC50 = 0.034 microg/ml) were more toxic than CLT (LC50 = 0.118 microg/ml). The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumalpha10 and Apisumbeta1 expressions levels, whereas Apisumbeta2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies.
ESTHER : Taillebois_2014_PLoS.One_9_e96669
PubMedSearch : Taillebois_2014_PLoS.One_9_e96669
PubMedID: 24801634

Title : G protein-coupled receptors, an unexploited animal toxin targets: Exploration of green mamba venom for novel drug candidates active against adrenoceptors - Maiga_2012_Toxicon_59_487
Author(s) : Maiga A , Mourier G , Quinton L , Rouget C , Gales C , Denis C , Lluel P , Senard JM , Palea S , Servent D , Gilles N
Ref : Toxicon , 59 :487 , 2012
Abstract : At a time when pharmaceutical companies are having trouble finding new low MW drugs and when biologics are becoming more common, animal venoms could constitute an underexploited source of novel drug candidates. We looked for identifying novel animal toxins active against G protein-coupled receptors (GPCR), the most frequently exploited class of treatment targets, with the aim to develop novel research tools and drug candidates. Screening of green mamba (Dendroaspis angusticeps) venom against adrenoceptors identified two novel venom peptides. rho-Da1a shown an affinity of 0.35 nM for the alpha1a-AR while rho-Da1b displayed affinities between 14 and 73 nM for the three alpha2-ARs. These two venom peptides have sequences similar to those of muscarinic toxins and belong to the three-finger-fold protein family. alpha1a-AR is the primary target for the treatment of prostate hypertrophy. In vitro and in vivo tests demonstrated that rho-Da1a reduced prostatic muscle tone as efficiently as tamsulosin (an antagonist presently used), but with fewer cardiovascular side effects. alpha2-ARs are the prototype of GPCRs not currently used as treatment targets due to a lack of specific ligands. Blockage of these receptors increases intestinal motility, which may be compromised by abdominal surgery and reduces orthosteric hypotension. In vitro and in vivo tests demonstrated that rho-Da1b antagonizes alpha2-ARs in smooth muscles and increased heart rate and blood catecholamine concentrations. These results highlight possible exploitation of rho-Da1a and rho-Da1b in important pathologies.
ESTHER : Maiga_2012_Toxicon_59_487
PubMedSearch : Maiga_2012_Toxicon_59_487
PubMedID: 21419153

Title : Engineering of three-finger fold toxins creates ligands with original pharmacological profiles for muscarinic and adrenergic receptors - Fruchart-Gaillard_2012_PLoS.One_7_e39166
Author(s) : Fruchart-Gaillard C , Mourier G , Blanchet G , Vera L , Gilles N , Menez R , Marcon E , Stura EA , Servent D
Ref : PLoS ONE , 7 :e39166 , 2012
Abstract : Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test "loop grafting," a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the alpha(1A)-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and alpha(1A)-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles.
ESTHER : Fruchart-Gaillard_2012_PLoS.One_7_e39166
PubMedSearch : Fruchart-Gaillard_2012_PLoS.One_7_e39166
PubMedID: 22720062

Title : Muscarinic toxins - Servent_2011_Toxicon_58_455
Author(s) : Servent D , Blanchet G , Mourier G , Marquer C , Marcon E , Fruchart-Gaillard C
Ref : Toxicon , 58 :455 , 2011
Abstract : Muscarinic toxins isolated from the venom of Dendroaspis snakes may interact with a high affinity, large selectivity and various functional properties with muscarinic receptors. Therefore, these toxins are invaluable tools for studying the physiological role, molecular functioning and structural organization of the five subtypes of these G-Protein Coupled Receptors. We review the data on the most relevant results dealing with the isolation/identification, mode of action, structure/function and exploitation of these toxins and finally highlight the unresolved issues related to their pharmacological studies.
ESTHER : Servent_2011_Toxicon_58_455
PubMedSearch : Servent_2011_Toxicon_58_455
PubMedID: 21906611

Title : 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 - Marquer_2011_J.Biol.Chem_286_31661
Author(s) : Marquer C , Fruchart-Gaillard C , Letellier G , Marcon E , Mourier G , Zinn-Justin S , Menez A , Servent D , Gilquin B
Ref : Journal of Biological Chemistry , 286 :31661 , 2011
Abstract : 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 A 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.
ESTHER : Marquer_2011_J.Biol.Chem_286_31661
PubMedSearch : Marquer_2011_J.Biol.Chem_286_31661
PubMedID: 21685390

Title : Total synthesis of pinnatoxins A and G and revision of the mode of action of pinnatoxin A - Araoz_2011_J.Am.Chem.Soc_133_10499
Author(s) : Araoz R , Servent D , Molgo J , Iorga BI , Fruchart-Gaillard C , Benoit E , Gu Z , Stivala C , Zakarian A
Ref : Journal of the American Chemical Society , 133 :10499 , 2011
Abstract : Pinnatoxins belong to an emerging class of potent marine toxins of the cyclic imine group. Detailed studies of their biological effects have been impeded by unavailability of the complex natural product from natural sources. This work describes the development of a robust, scalable synthetic sequence relying on a convergent strategy that delivered a sufficient amount of the toxin for detailed biological studies and its commercialization for use by other research groups and regulatory agencies. A central transformation in the synthesis is the highly diastereoselective Ireland-Claisen rearrangement of a complex alpha,alpha-disubstituted allylic ester based on a unique mode for stereoselective enolization through a chirality match between the substrate and the lithium amide base. With synthetic pinnatoxin A, a detailed study has been performed that provides conclusive evidence for its mode of action as a potent inhibitor of nicotinic acetylcholine receptors selective for the human neuronal alpha7 subtype. The comprehensive electrophysiological, biochemical, and computational studies support the view that the spiroimine subunit of pinnatoxins is critical for blocking nicotinic acetylcholine receptor subtypes, as evidenced by analyzing the effect of a synthetic analogue of pinnatoxin A containing an open form of the imine ring. Our studies have paved the way for the production of certified standards to be used for mass-spectrometric determination of these toxins in marine matrices and for the development of tests to detect these toxins in contaminated shellfish.
ESTHER : Araoz_2011_J.Am.Chem.Soc_133_10499
PubMedSearch : Araoz_2011_J.Am.Chem.Soc_133_10499
PubMedID: 21644584

Title : Influence of MT7 toxin on the oligomerization state of the M1 muscarinic receptor - Marquer_2010_Biol.Cell_102_409
Author(s) : Marquer C , Fruchart-Gaillard C , Mourier G , Grandjean O , Girard E , le Maire M , Brown S , Servent D
Ref : Biology of the cell , 102 :409 , 2010
Abstract : BACKGROUND INFORMATION: The idea that GPCRs (G-protein-coupled receptors) may exist as homo- or hetero-oligomers, although still controversial, is now widely accepted. Nevertheless, the functional roles of oligomerization are still unclear and gaining greater insight into the mechanisms underlying the dynamics of GPCR assembly and, in particular, assessing the effect of ligands on this process seems important. We chose to focus our present study on the effect of MT7 (muscarinic toxin 7), a highly selective allosteric peptide ligand, on the oligomerization state of the hM1 (human M1 muscarinic acetylcholine receptor subtype).
RESULTS: We analysed the hM1 oligomerization state in membrane preparations or in live cells and observed the effect of MT7 via four complementary techniques: native-PAGE electrophoresis analysed by both Western blotting and autoradiography on solubilized membrane preparations of CHO-M1 cells (Chinese-hamster ovary cells expressing muscarinic M1 receptors); FRET (fluorescence resonance energy transfer) experiments on cells expressing differently tagged M1 receptors using either an acceptor photobleaching approach or a novel fluorescence emission anisotropy technique; and, finally, by BRET (bioluminescence resonance energy transfer) assays. Our results reveal that MT7 seems to protect the M1 receptor from the dissociating effect of the detergent and induces an increase in the FRET and BRET signals, highlighting its ability to affect the dimeric form of the receptor.
CONCLUSIONS: Our results suggest that MT7 binds to a dimeric form of hM1 receptor, favouring the stability of this receptor state at the cellular level, probably by inducing some conformational rearrangements of the pre-existing muscarinic receptor homodimers.
ESTHER : Marquer_2010_Biol.Cell_102_409
PubMedSearch : Marquer_2010_Biol.Cell_102_409
PubMedID: 20170475

Title : Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism - Bourne_2010_Proc.Natl.Acad.Sci.U.S.A_107_6076
Author(s) : Bourne Y , Radic Z , Araoz R , Talley TT , Benoit E , Servent D , Taylor P , Molgo J , Marchot P
Ref : Proc Natl Acad Sci U S A , 107 :6076 , 2010
Abstract : Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type alpha1(2)betagammadelta and neuronal alpha3beta2 and alpha4beta2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities governed by diffusion-limited association and slow dissociation, accounting for apparent irreversibility. Crystal structures of the phycotoxins bound to Aplysia-AChBP ( approximately 2.4A) show toxins neatly imbedded within the nest of ar-omatic side chains contributed by loops C and F on opposing faces of the subunit interface, and which in physiological conditions accommodates acetylcholine. The structures also point to three major features: (i) the sequence-conserved loop C envelops the bound toxins to maximize surface complementarity; (ii) hydrogen bonding of the protonated imine nitrogen in the toxins with the carbonyl oxygen of loop C Trp147 tethers the toxin core centered within the pocket; and (iii) the spirolide bis-spiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the se-quence-variable loop F only sparingly contributes contact points to preserve the broad receptor subtype recognition unique to phycotoxins compared with other nicotinic antagonists. These data offer unique means for detecting spiroimine toxins in shellfish and identify distinctive ligands, functional determinants and binding regions for the design of new drugs able to target several receptor subtypes with high affinity.
ESTHER : Bourne_2010_Proc.Natl.Acad.Sci.U.S.A_107_6076
PubMedSearch : Bourne_2010_Proc.Natl.Acad.Sci.U.S.A_107_6076
PubMedID: 20224036

Title : Isolation and pharmacological characterization of AdTx1, a natural peptide displaying specific insurmountable antagonism of the alpha1A-adrenoceptor - Quinton_2010_Br.J.Pharmacol_159_316
Author(s) : Quinton L , Girard E , Maiga A , Rekik M , Lluel P , Masuyer G , Larregola M , Marquer C , Ciolek J , Magnin T , Wagner R , Molgo J , Thai R , Fruchart-Gaillard C , Mourier G , Chamot-Rooke J , Menez A , Palea S , Servent D , Gilles N
Ref : British Journal of Pharmacology , 159 :316 , 2010
Abstract : 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. EXPERIMENTAL APPROACH: We studied the interactions of mamba venom fractions with alpha(1)-adrenoceptors in binding experiments with (3)H-prazosin. The active peptide (AdTx1) was sequenced by Edman degradation and mass spectrometry fragmentation. Its synthetic homologue was pharmacologically characterized by binding experiments using cloned receptors and by functional experiments on rabbit isolated prostatic smooth muscle. KEY
RESULTS: AdTx1, a 65 amino-acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. It has subnanomolar affinity (K(i)= 0.35 nM) and high specificity for the human alpha(1A)-adrenoceptor subtype. We showed high selectivity and affinity (K(d)= 0.6 nM) of radio-labelled AdTx1 in direct binding experiments and revealed a slow association constant (k(on)= 6 x 10(6).M(-1).min(-1)) with an unusually stable alpha(1A)-adrenoceptor/AdTx1 complex (t(1/2diss)= 3.6 h). AdTx1 displayed potent insurmountable antagonism of phenylephrine's actions in vitro (rabbit isolated prostatic muscle) at concentrations of 10 to 100 nM. CONCLUSIONS AND IMPLICATIONS: AdTx1 is the most specific and selective peptide inhibitor for the alpha(1A)-adrenoceptor identified to date. It displays insurmountable antagonism, acting as a potent relaxant of smooth muscle. Its peptidic nature can be exploited to develop new tools, as a radio-labelled-AdTx1 or a fluoro-labelled-AdTx1. Identification of AdTx1 thus offers new perspectives for developing new drugs for treating benign prostatic hyperplasia.
ESTHER : Quinton_2010_Br.J.Pharmacol_159_316
PubMedSearch : Quinton_2010_Br.J.Pharmacol_159_316
PubMedID: 20015090

Title : Muscarinic toxins: tools for the study of the pharmacological and functional properties of muscarinic receptors - Servent_2009_J.Neurochem_109_1193
Author(s) : Servent D , Fruchart-Gaillard C
Ref : Journal of Neurochemistry , 109 :1193 , 2009
Abstract : Muscarinic receptors mediate metabotropic actions of acetylcholine in the CNS and PNS and autocrine functions of acetylcholine in non-neuronal systems. Because of the lack of highly selective muscarinic ligands, the precise location, functional role, and roles in various diseases of the five muscarinic receptor subtypes remain unclear. Muscarinic toxins isolated from the venom of Dendroaspis snakes have a natural high affinity and selectivity, associated with roles as competitive antagonists, allosteric modulators, and potential agonists. These toxins may therefore be invaluable tools for studying muscarinic receptors. We review data on the structural and pharmacological characterization of the muscarinic toxins, focusing on recent structure-function studies on toxin-receptor interactions. We discuss the potential benefits of using these toxins for investigating muscarinic function in vivo.
ESTHER : Servent_2009_J.Neurochem_109_1193
PubMedSearch : Servent_2009_J.Neurochem_109_1193
PubMedID: 19457160

Title : Different interactions between MT7 toxin and the human muscarinic M1 receptor in its free and N-methylscopolamine-occupied states - Fruchart-Gaillard_2008_Mol.Pharmacol_74_1554
Author(s) : Fruchart-Gaillard C , Mourier G , Marquer C , Stura E , Birdsall NJ , Servent D
Ref : Molecular Pharmacology , 74 :1554 , 2008
Abstract : Muscarinic MT7 toxin is a highly selective and potent antagonist of the M(1) 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 [(3)H]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-M(1) 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.
ESTHER : Fruchart-Gaillard_2008_Mol.Pharmacol_74_1554
PubMedSearch : Fruchart-Gaillard_2008_Mol.Pharmacol_74_1554
PubMedID: 18784346

Title : How three-finger-fold toxins interact with various cholinergic receptors - Fruchart-Gaillard_2006_J.Mol.Neurosci_30_7
Author(s) : Fruchart-Gaillard C , Mourier G , Marquer C , Menez A , Servent D
Ref : Journal of Molecular Neuroscience , 30 :7 , 2006
Abstract : Three-finger-fold toxins, isolated from various snake venoms, are recognized by high affinity and various specificities by different nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs, respectively) present in peripheral, as well as central, nervous systems (Karlsson et al., 2000; Servent and Menez, 2001; Nirthanan and Gwee, 2004). The goal of our studies is (1) to identify, at the molecular level, the functional determinants involved in the various interaction profiles of nicotinic or muscarinic toxins on their respective receptors subtypes, (2) to model some of these toxin-receptor complexes using distance constraints obtained from cycle-mutant experiments, and (3) to understand how a unique scaffold (the three-finger fold) is able to support these different functional profiles and how molecular determinants have been selected during the evolution process to create these different specific properties. Finally, these structure/function analyses should be exploited to engineer non-natural peptides with new binding and functional properties useful as pharmacological tools or therapeutic agents.
ESTHER : Fruchart-Gaillard_2006_J.Mol.Neurosci_30_7
PubMedSearch : Fruchart-Gaillard_2006_J.Mol.Neurosci_30_7
PubMedID: 17192604

Title : Identification of various allosteric interaction sites on M1 muscarinic receptor using 125I-Met35-oxidized muscarinic toxin 7 - Fruchart-Gaillard_2006_Mol.Pharmacol_69_1641
Author(s) : Fruchart-Gaillard C , Mourier G , Marquer C , Menez A , Servent D
Ref : Molecular Pharmacology , 69 :1641 , 2006
Abstract : 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-diindo lo[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.
ESTHER : Fruchart-Gaillard_2006_Mol.Pharmacol_69_1641
PubMedSearch : Fruchart-Gaillard_2006_Mol.Pharmacol_69_1641
PubMedID: 16439611

Title : Pharmacological and structural characterization of the interaction of snake toxins with nicotinic acetylcholine receptors. -
Author(s) : Servent D , Fruchart-Gaillard C , Menez A
Ref : Cholinergic Mechanisms, CRC Press :27 , 2004
PubMedID:

Title : Chemical synthesis of MT1 and MT7 muscarinic toxins: critical role of Arg-34 in their interaction with M1 muscarinic receptor - Mourier_2003_Mol.Pharmacol_63_26
Author(s) : Mourier G , Dutertre S , Fruchart-Gaillard C , Menez A , Servent D
Ref : Molecular Pharmacology , 63 :26 , 2003
Abstract : Two muscarinic toxins, MT1 and MT7, were obtained by one-step solid-phase synthesis using the 9-fluorenylmethoxycarbonyl-based method. The synthetic and natural toxins, isolated from the snake venom or recombinantly expressed, display identical physicochemical properties and pharmacological profiles. High protein recovery allowed us to specify the selectivity of these toxins for various muscarinic receptor subtypes. Thus, sMT7 has a selectivity for the M1 receptor that is at least 20,000 times that for the other subtypes. The stability of the toxin-receptor complexes indicates that sMT1 interacts reversibly with the M1 receptor, unlike sMT7, which binds it quasi-irreversibly. The effect of the synthetic toxins on the atropine-induced [3H]N-methylscopolamine (NMS) dissociation confirms that sMT7 targets the allosteric site on the M1 receptor, whereas sMT1 seems interact on the orthosteric one. The great decreases in the binding potencies observed after the R34A modification in sMT1 and sMT7 toxins highlight the functional role of this conserved residue in their interactions with the M1 receptor. Interestingly, after the R34A modification, the sMT7 toxin binds reversibly on the M1 receptor. Furthermore, the potency of sMT7-R34A for the NMS-occupied receptor is lower compared with unmodified toxin, supporting the role of this residue in the allosteric interaction of sMT7. All these results and the different charge distributions observed at the two toxin surfaces of their structure models support the hypothesis that the two toxins recognize the M1 receptor differently.
ESTHER : Mourier_2003_Mol.Pharmacol_63_26
PubMedSearch : Mourier_2003_Mol.Pharmacol_63_26
PubMedID: 12488533

Title : Experimentally based model of a complex between a snake toxin and the alpha 7 nicotinic receptor - Fruchart-Gaillard_2002_Proc.Natl.Acad.Sci.U.S.A_99_3216
Author(s) : Fruchart-Gaillard C , Gilquin B , Antil-Delbeke S , Le Novere N , Tamiya T , Corringer PJ , Changeux JP , Menez A , Servent D
Ref : Proc Natl Acad Sci U S A , 99 :3216 , 2002
Abstract : To understand how snake neurotoxins interact with nicotinic acetylcholine receptors, we have elaborated an experimentally based model of the alpha-cobratoxin-alpha7 receptor complex. This model was achieved by using (i) a three-dimensional model of the alpha7 extracellular domain derived from the crystallographic structure of the homologous acetylcholine-binding protein, (ii) the previously solved x-ray structure of the toxin, and (iii) nine pairs of residues identified by cycle-mutant experiments to make contacts between the alpha-cobratoxin and alpha7 receptor. Because the receptor loop F occludes entrance of the toxin binding pocket, we submitted this loop to a dynamics simulation and selected a conformation that allowed the toxin to reach its binding site. The three-dimensional structure of the toxin-receptor complex model was validated a posteriori by an additional double-mutant experiment. The model shows that the toxin interacts perpendicularly to the receptor axis, in an equatorial position of the extracellular domain. The tip of the toxin central loop plugs into the receptor between two subunits, just below the functional receptor loop C, the C-terminal tail of the toxin making adjacent additional interactions at the receptor surface. The receptor establishes major contacts with the toxin by its loop C, which is assisted by principal (loops A and B) and complementary (loops D, F, and 1) functional regions. This model explains the antagonistic properties of the toxin toward the neuronal receptor and opens the way to the design of new antagonists.
ESTHER : Fruchart-Gaillard_2002_Proc.Natl.Acad.Sci.U.S.A_99_3216
PubMedSearch : Fruchart-Gaillard_2002_Proc.Natl.Acad.Sci.U.S.A_99_3216
PubMedID: 11867717

Title : A synthetic weak neurotoxin binds with low affinity to Torpedo and chicken alpha7 nicotinic acetylcholine receptors - Poh_2002_Eur.J.Biochem_269_4247
Author(s) : Poh SL , Mourier G , Thai R , Armugam A , Molgo J , Servent D , Jeyaseelan K , Menez A
Ref : European Journal of Biochemistry , 269 :4247 , 2002
Abstract : Weak neurotoxins from snake venom are small proteins with five disulfide bonds, which have been shown to be poor binders of nicotinic acetylcholine receptors. We report on the cloning and sequencing of four cDNAs encoding weak neurotoxins from Naja sputatrix venom glands. The protein encoded by one of them, Wntx-5, has been synthesized by solid-phase synthesis and characterized. The physicochemical properties of the synthetic toxin (sWntx-5) agree with those anticipated for the natural toxin. We show that this toxin interacts with relatively low affinity (K(d) = 180 nm) with the muscular-type acetylcholine receptor of the electric organ of T. marmorata, and with an even weaker affinity (90 microm) with the neuronal alpha7 receptor of chicken. Electrophysiological recordings using isolated mouse hemidiaphragm and frog cutaneous pectoris nerve-muscle preparations revealed no blocking activity of sWntx-5 at microm concentrations. Our data confirm previous observations that natural weak neurotoxins from cobras have poor affinity for nicotinic acetylcholine receptors.
ESTHER : Poh_2002_Eur.J.Biochem_269_4247
PubMedSearch : Poh_2002_Eur.J.Biochem_269_4247
PubMedID: 12199703

Title : Molecular characterization of the specificity of interactions of various neurotoxins on two distinct nicotinic acetylcholine receptors - Servent_2000_Eur.J.Pharmacol_393_197
Author(s) : Servent D , Antil-Delbeke S , Gaillard C , Corringer PJ , Changeux JP , Menez A
Ref : European Journal of Pharmacology , 393 :197 , 2000
Abstract : Snake curaremimetic toxins are currently classified as short-chain and long-chain toxins according to their size and their number of disulfide bonds. All these toxins bind with high affinity to muscular-type nicotinic acetylcholine receptor, whereas only long toxins recognize the alpha7 receptor with high affinity. On the basis of binding experiments with Torpedo or neuronal alpha7 receptors using wild-type and mutated neurotoxins, we characterized the molecular determinants involved in these different recognition processes. The functional sites by which long and short toxins interact with the muscular-type receptor include a common core of highly conserved residues and residues that are specific to each of toxin families. Furthermore, the functional sites through which alpha-cobratoxin, a long-chain toxin, interacts with muscular and alpha7 receptors share similarities but also marked differences. Our results reveal that the three-finger fold toxins have evolved toward various specificities by displaying distinct functional sites.
ESTHER : Servent_2000_Eur.J.Pharmacol_393_197
PubMedSearch : Servent_2000_Eur.J.Pharmacol_393_197
PubMedID: 10771013

Title : Relative spatial position of a snake neurotoxin and the reduced disulfide bond alpha (Cys192-Cys193) at the alpha gamma interface of the nicotinic acetylcholine receptor - Michalet_2000_J.Biol.Chem_275_25608
Author(s) : Michalet S , Teixeira F , Gilquin B , Mourier G , Servent D , Drevet P , Binder P , Tzartos SJ , Menez A , Kessler P
Ref : Journal of Biological Chemistry , 275 :25608 , 2000
Abstract : We determined the distances separating five functionally important residues (Gln(10), Lys(27), Trp(29), Arg(33), and Lys(47)) of a three-fingered snake neurotoxin from the reduced disulfide bond alpha(Cys(192)-Cys(193)) located at the alphagamma interface of the Torpedo nicotinic acetylcholine receptor. Each toxin position was substituted individually for a cysteine, which was then linked to a maleimido moiety through three different spacers, varying in length from 10 to 22 A. We estimated the coupling efficiency between the 15 toxin derivatives and the reduced cystine alpha(192-193) by gel densitometry of Coomassie Blue-stained gels. A nearly quantitative coupling was observed between alphaCys(192) and/or alphaCys(193) and all probes introduced at the tip of the first (position 10) and second (position 33) loops of Naja nigricollis alpha-neurotoxin. These data sufficed to locate the reactive thiolate in a "croissant-shaped" volume comprised between the first two loops of the toxin. The volume was further restrained by taking into account the absence or partial coupling of the other derivatives. Altogether, the data suggest that alphaCys(192) and/or alphaCys(193), at the alphagamma interface of a muscular-type acetylcholine receptor, is (are) located in a volume located between 11.5 and 15.5 A from the alpha-carbons at positions 10 and 33 of the toxin, under the tip of the toxin first loop and close to the second one.
ESTHER : Michalet_2000_J.Biol.Chem_275_25608
PubMedSearch : Michalet_2000_J.Biol.Chem_275_25608
PubMedID: 10807914

Title : Chemical engineering of a three-fingered toxin with anti-alpha7 neuronal acetylcholine receptor activity - Mourier_2000_Protein.Eng_13_217
Author(s) : Mourier G , Servent D , Zinn-Justin S , Menez A
Ref : Protein Engineering , 13 :217 , 2000
Abstract : Though it possesses four disulfide bonds the three-fingered fold is amenable to chemical synthesis, using a Fmoc-based method. Thus, we synthesized a three-fingered curaremimetic toxin from snake with high yield and showed that the synthetic and native toxins have the same structural and biological properties. Both were characterized by the same 2D NMR spectra, identical high binding affinity (K(d) = 22 +/- 5 pM) for the muscular acetylcholine receptor (AChR) and identical low affinity (K(d) = 2.0 +/- 0.4 microM) for alpha7 neuronal AchR. Then, we engineered an additional loop cyclized by a fifth disulfide bond at the tip of the central finger. This loop is normally present in longer snake toxins that bind with high affinity (K(d) = 1-5 nM) to alpha7 neuronal AchR. Not only did the chimera toxin still bind with the same high affinity to the muscular AchR but also it displayed a 20-fold higher affinity (K(d) = 100 nM) for the neuronal alpha7 AchR, as compared with the parental short-chain toxin. This result demonstrates that the engineered loop contributes, at least in part, to the high affinity of long-chain toxins for alpha7 neuronal receptors. That three-fingered proteins with four or five disulfide bonds are amenable to chemical synthesis opens new perspectives for engineering new activities on this fold.
ESTHER : Mourier_2000_Protein.Eng_13_217
PubMedSearch : Mourier_2000_Protein.Eng_13_217
PubMedID: 10775664

Title : Molecular determinants by which a long chain toxin from snake venom interacts with the neuronal alpha 7-nicotinic acetylcholine receptor - Antil-Delbeke_2000_J.Biol.Chem_275_29594
Author(s) : Antil-Delbeke S , Gaillard C , Tamiya T , Corringer PJ , Changeux JP , Servent D , Menez A
Ref : Journal of Biological Chemistry , 275 :29594 , 2000
Abstract : Long chain curarimimetic toxins from snake venom bind with high affinities to both muscular type nicotinic acetylcholine receptors (AChRs) (K(d) in the pm range) and neuronal alpha 7-AChRs (K(d) in the nm range). To understand the molecular basis of this dual function, we submitted alpha-cobratoxin (alpha-Cbtx), a typical long chain curarimimetic toxin, to an extensive mutational analysis. By exploring 36 toxin mutants, we found that Trp-25, Asp-27, Phe-29, Arg-33, Arg-36, and Phe-65 are involved in binding to both neuronal and Torpedo (Antil, S., Servent, D., and Menez, A. (1999) J. Biol. Chem. 274, 34851-34858) AChRs and that some of them (Trp-25, Asp-27, and Arg-33) have similar binding energy contributions for the two receptors. In contrast, Ala-28, Lys-35, and Cys-26-Cys-30 selectively bind to the alpha 7-AChR, whereas Lys-23 and Lys-49 bind solely to the Torpedo AChR. Therefore, alpha-Cbtx binds to two AChR subtypes using both common and specific residues. Double mutant cycle analyses suggested that Arg-33 in alpha-Cbtx is close to Tyr-187 and Pro-193 in the alpha 7 receptor. Since Arg-33 of another curarimimetic toxin is close to the homologous alpha Tyr-190 of the muscular receptor (Ackermann, E. J., Ang, E. T. H., Kanter, J. R., Tsigelny, I., and Taylor, P. (1998) J. Biol. Chem. 273, 10958-10964), toxin binding probably occurs in homologous regions of neuronal and muscular AChRs. However, no coupling was seen between alpha-Cbtx Arg-33 and alpha 7 receptor Trp-54, Leu-118, and Asp-163, in contrast to what was observed in a homologous situation involving another toxin and a muscular receptor (Osaka, H., Malany, S., Molles, B. E., Sine, S. M., and Taylor, P. (2000) J. Biol. Chem. 275, 5478-5484). Therefore, although occurring in homologous regions, the detailed modes of toxin binding to alpha 7 and muscular receptors are likely to be different. These data offer a molecular basis for the design of toxins with predetermined specificities for various members of the AChR family.
ESTHER : Antil-Delbeke_2000_J.Biol.Chem_275_29594
PubMedSearch : Antil-Delbeke_2000_J.Biol.Chem_275_29594
PubMedID: 10852927

Title : How do snake curaremimetic toxins discriminate between nicotinic acetylcholine receptor subtypes - Servent_1998_Toxicol.Lett_102-103_199
Author(s) : Servent D , Mourier G , Antil S , Menez A
Ref : Toxicol Lett , 102-103 :199 , 1998
Abstract : Curaremimetic toxins from snake venoms form a large family of small proteins that adopt a similar fold and which bind to Torpedo nicotinic acetylcholine receptors with high affinity. Notwithstanding its apparent homogeneity, the toxin family is subdivided into short-chain (60-62 residues and four disulfide bonds) and long-chain toxins (66-74 residues and five disulfide bonds). In agreement with this structurally-based distinction we recently showed that only long-chain toxins bind with high affinity to the neuronal nicotinic acetylcholine alpha7 receptor. We suggested that a small loop cyclized by a disulfide bond and uniquely present in long-chain toxins may act as a major discriminative element. To assess the validity of this proposal we prepared various derivatives of a long-chain toxin, using stepwise solid-phase synthesis. We found that replacement of both half cystines of the small loop by a serine caused a 35-fold affinity decrease for the neuronal receptor and only a 6-fold affinity decrease for Torpedo receptor. In addition, insertion of this loop at a homologous position of a short-chain toxin caused a 20-fold affinity increase for the neuronal receptor whereas it did not modify its affinity for the Torpedo receptor. Our findings, therefore, reveal that a small structural deviation from a toxin fold can generate exquisite discriminative recognition for some receptor subtypes.
ESTHER : Servent_1998_Toxicol.Lett_102-103_199
PubMedSearch : Servent_1998_Toxicol.Lett_102-103_199
PubMedID: 10022254

Title : Artificial toxins to explore new receptors? -
Author(s) : Menez A , Mourier G , Kessler P , Ducancel F , Bertrand D , Corringer PJ , Changeux JP , Servent D
Ref : Journal de Physiologie (Paris) , 92 :468 , 1998
PubMedID:

Title : An engineered short-chain toxin with higher affinity for the neuronalalpha7 receptor -
Author(s) : Servent D , Antil S , Mourier G , Corringer PJ , Menez A
Ref : Journal de Physiologie (Paris) , 92 :490 , 1998
PubMedID:

Title : Two novel alpha-conotoxins isolated from the venom of Conus consor: isolation, synthesis and binding on the Torpedo nicotinic acetylcholine receptor -
Author(s) : Favreau P , Legall F , Servent D , Menez A , Molgo J , Letourneux Y
Ref : Journal de Physiologie (Paris) , 92 :430 , 1998
PubMedID:

Title : Only snake curaremimetic toxins with a fifth disulfide bond have high affinity for the neuronal alpha7 nicotinic receptor - Servent_1997_J.Biol.Chem_272_24279
Author(s) : Servent D , Winckler-Dietrich V , Hu HY , Kessler P , Drevet P , Bertrand D , Menez A
Ref : Journal of Biological Chemistry , 272 :24279 , 1997
Abstract : Long chain and short chain curaremimetic toxins from snakes possess 66-74 residues with five disulfide bonds and 60-62 residues with four disulfide bonds, respectively. Despite their structural differences all of these toxins bind with high affinity to the peripheral nicotinic acetylcholine receptors (AChR). Binding experiments have now revealed that long chain toxins only, like the neuronal kappa-bungarotoxin, have a high affinity for a chimeric form of the neuronal alpha7 receptor, with Kd values ranging from about 1 to 12 nM. In contrast, all other toxins bind to the chimeric alpha7 receptor with a low affinity, with Kd values ranging between 3 and 22 microM. These results are supported by electrophysiological recordings on both the wild-type and chimeric alpha7 receptors. Amino acid sequence analyses have suggested that high affinities for the neuronal receptor are associated with the presence of the fifth disulfide at the tip of the toxin second loop. In agreement with this conclusion, we show that a long chain toxin whose fifth disulfide is reduced and then dithiopyridylated has a low affinity (Kd = 12 microM) for the neuronal alpha7 receptor, whereas it retains a high affinity (Kd = 0.35 nM) for the peripheral AChR. Thus, a long chain curaremimetic toxin having a reduced fifth disulfide bond behaves like a short chain toxin toward both the peripheral and neuronal AChR. Therefore, functional classification of toxins that bind to AChRs should probably be done by considering their activities on both peripheral and neuronal receptors.
ESTHER : Servent_1997_J.Biol.Chem_272_24279
PubMedSearch : Servent_1997_J.Biol.Chem_272_24279
PubMedID: 9305882