alpha-Conotoxin ImI inhibits the alpha-bungarotoxin-resistant nicotinic response in bovine adrenal chromaffin cells

The activity of alpha-conotoxin (alpha-CTX) lml, from the vermivorous marine snail Conus imperialis, has been studied on mammalian nicotinic receptors on bovine chromaffin cells and at the rat neuromuscular junction. Synthetic alpha-CTX lml was a potent inhibitor of the neuronal[ nicotinic response in bovine adrenal chromaffin cells (IC50 = 2.5 mu M, log IC50 = 0.4 +/- 0.07), showing competitive inhibition of nicotine-evoked catecholamine secretion. (alpha-CTX lml also inhibited nicotine-evoked Ca-45(2+) uptake but not Ca-45(2+) uptake stimulated by 56 mM Kr. In contrast, alpha-CTX lml had no effect at the neuromuscular junction over the concentration range 1-20 mu M. Bovine chromaffin cells are known to contain the alpha 3 beta 4, alpha 7, and (possibly) alpha 3 beta 4 alpha 5 subtypes. However, the secretory response of bovine chromaffin cells is not inhibited by alpha-bungarotoxin, indicating that alpha 7 nicotinic receptors are not involved. We propose that alpha-CTX lml interacts selectively with the functional (alpha 3 beta 4 or alpha 3 beta 4 alpha 5) nicotinic acetylcholine receptor to inhibit the neuronal-type nicotinic response in bovine chromaffin cells.

Solution structure of alpha-conotoxin ImI by H-1 nuclear magnetic resonance

alpha-Conotoxin ImI derives from the venom of Conus imperialis and is the first and only small-peptide ligand that selectively binds to the neuronal alpha(7) homopentameric subtype of the nicotinic acetylcholine receptor (nAChR). This receptor subtype is a possible drug target for several neurological disorders. The cysteines are connected in the pairs Cys2-Cys8 and Cys3-Cys12, To date it is the only alpha-conotoxin with a 4/3 residue spacing between the cysteines, The structure of ImI has been determined by H-1 NMR spectroscopy in aqueous solution, The NMR structure is of high quality, with a backbone pairwise rmsd of 0.34 Angstrom for a family of 19 structures, and comprises primarily a series of nested beta turns. Addition of organic solvent does not perturb the solution structure. The first eight residues of ImI are identical to the larger, but related, conotoxin EpI and adopt a similar structure, despite a truncated second loop. Residues important for binding of ImI to the alpha 7 nAChR are all clustered on one face of the molecule. Once further binding data for EPI and ImI are available, the ImI structure will allow for design of novel alpha(7) nAChR-specific agonists and antagonists with a wide range of potential pharmaceutical applications.

Applications of NMR in drug design: Sructure-activity relationships in disulfide-rich peptides

NMR is a powerful technique for determining structures of biologically active molecules in solution. In recent years. our laboratory has focussed on the structure determination of small disulfide-rich proteins from both plants and animals which are valuable targets in drug design applications. This article will review these structural studies and their implications in drug design.

Unmasking the functions of the chromaffin cell α7 nicotinic receptor by using short pulses of acetylcholine and selective blockers

Methyllycaconitine (MLA), α-conotoxin ImI, and α-bungarotoxin inhibited the release of catecholamines triggered by brief pulses of acetylcholine (ACh) (100 μM, 5 s) applied to fast-superfused bovine adrenal chromaffin cells, with IC50s of 100 nM for MLA and 300 nM for α-conotoxin ImI and α-bungarotoxin. MLA (100 nM), α-conotoxin ImI (1 μM), and α-bungarotoxin (1 μM) halved the entry of 45Ca2+ stimulated by 5-s pulses of 300 μM ACh applied to incubated cells. These supramaximal concentrations of α7 nicotinic receptor blockers depressed by 30% (MLA), 25% (α-bungarotoxin), and 50% (α-conotoxin ImI) the inward current generated by 1-s pulses of 100 μM ACh, applied to voltage-clamped chromaffin cells. In Xenopus oocytes expressing rat brain α7 neuronal nicotinic receptor for acetylcholine nAChR, the current generated by 1-s pulses of ACh was blocked by MLA, α-conotoxin ImI, and α-bungarotoxin with IC50s of 0.1 nM, 100 nM, and 1.6 nM, respectively; the current through α3β4 nAChR was unaffected by α-conotoxin ImI and α-bungarotoxin, and weakly blocked by MLA (IC50 = 1 μM). The functions of controlling the electrical activity, the entry of Ca2+, and the ensuing exocytotic response of chromaffin cells were until now exclusively attributed to α3β4 nAChR; the present results constitute the first evidence to support a prominent role of α7 nAChR in controlling such functions, specially under the more physiological conditions used here to stimulate chromaffin cells with brief pulses of ACh.

Alpha-selenoconotoxins, a new class of potent alpha(7) neuronal nicotinic receptor antagonists

Disulfide bonds are important structural motifs that play an essential role in maintaining the conformational stability of many bioactive peptides. Of particular importance are the conotoxins, which selectively target a wide range of ion channels that are implicated in numerous disease states. Despite the enormous potential of conotoxins as therapeutics, their multiple disulfide bond frameworks are inherently unstable under reducing conditions. Reduction or scrambling by thiol-containing molecules such as glutathione or serum albumin in intracellular or extracellular environments such as blood plasma can decrease their effectiveness as drugs. To address this issue, we describe a new class of selenoconotoxins where cysteine residues are replaced by selenocysteine to form isosteric and non-reducible diselenide bonds. Three isoforms of alpha-conotoxin ImI were synthesized by t-butoxycarbonyl chemistry with systematic replacement of one([ Sec(2,8)] ImI or [Sec(3,12)] ImI), or both([Sec(2,3,8,12)] ImI) disulfide bonds with a diselenide bond. Each analogue demonstrated remarkable stability to reduction or scrambling under a range of chemical and biological reducing conditions. Three-dimensional structural characterization by NMR and CD spectroscopy indicates conformational preferences that are very similar to those of native ImI, suggesting fully isomorphic structures. Additionally, full bioactivity was retained at the alpha(7) nicotinic acetylcholine receptor, with each seleno-analogue exhibiting a dose-response curve that overlaps with wild-type ImI, thus further supporting an isomorphic structure. These results demonstrate that selenoconotoxins can be used as highly stable scaffolds for the design of new drugs.

alpha-conotoxin EpI, a novel sulfated peptide from Conus episcopatus that selectively targets neuronal nicotinic acetylcholine receptors

We have isolated and characterized ol-conotoxin EpI, a novel sulfated peptide from the venom of the molluscivorous snail, Conus episcopatus, The peptide was classified as an cy-conotoxin based on sequence, disulfide connectivity, and pharmacological target. EpI has ho mology to sequences of previously described cu-conotoxins, particularly PnIA, PnIB, and ImI, However, EpI differs from previously reported conotoxins in that it has a sulfotyrosine residue, identified by amino acid analysis and mass spectrometry, Native EpI was shown to coelute with synthetic EpI, The peptide sequence is consistent with most, but not all, recognized criteria for predicting tyrosine sulfation sites in proteins and peptides, The activities of synthetic EpI and its unsulfated analogue [Tyr(15)]EpI were similar. Both peptides caused competitive inhibition of nicotine action on bovine adrenal chromaffin cells (neuronal nicotinic ACh receptors) but had no effect on the rat phrenic nerve-diaphragm (muscle nicotinic ACh receptors), Both EpI and [Tyr(15)]EpI partly inhibited acetylcholine-evoked currents in isolated parasympathetic neurons of rat intracardiac ganglia, These results indicate that EPI and [Tyr(15)]EpI selectively inhibit alpha 3 beta 2 and alpha 3 beta 4 nicotinic acetylcholine receptors.

Leu(10) of alpha-conotoxin PnIB confers potency for neuronal nicotinic responses in bovine chromaffin cells

Two alpha-conotoxins PnIA and PnIB (previously reported as being mollusc specific) which differ in only two amino acid residues (AN versus LS at residues 10 and 11, respectively), show markedly different inhibition of the neuronal nicotinic acetylcholine receptor response in bovine chromaffin cells, a mammalian preparation. Whereas alpha-conotoxin PnIB completely inhibits the nicotine-evoked catecholamine release at 10 mu M, with IC50 = 0.7 mu M, alpha-conotoxin PnIA is some 30-40 times less potent. Two peptide analogues, [A10L]PnIA and [N11S]PnIA were synthesized to investigate the extent to which each residue contributes to activity. [A10L]PnIA (IC50 = 2.0 mu M) completely inhibits catecholamine release at 10 mu M whereas [N11S]PnIA shows Little inhibition. In contrast, none of the peptides inhibit muscle-type nicotinic responses in the rat hemi-diaphragm preparation. We conclude that the enhanced potency of alpha-conotoxin PnIB over alpha-conotoxin PnIA in the neuronal-type nicotinic response is principally determined by the larger, more hydrophobic leucine residue at position 10 in alpha-conotoxin PnIB. (C) 2000 Elsevier Science B.V. All rights reserved.

Determination of alpha-conotoxin binding modes on neuronal nicotinic acetylcholine receptors

alpha-Conotoxins, from cone snails, and alpha-neurotoxins, from snakes, are competitive inhibitors of nicotinic acetylcholine receptors (nAChRs) that have overlapping binding sites in the ACh binding pocket. These disulphide-rich peptides are used extensively as tools to localize and pharmacologically characterize specific nAChRs subtypes. Recently, a homology model based on the high-resolution structure of an ACh binding protein (AChBP) allowed the three-fingered alpha-neurotoxins to be docked onto the alpha7 nAChR. To investigate if alpha-conotoxins interact with the nAChR in a similar manner, we built homology models of human alpha7 and alpha3beta2 nAChRs, and performed docking simulations of alpha-conotoxins ImI, PnIB, PnIA and MII using the program GOLD. Docking revealed that alpha-conotoxins have a different mode of interaction compared with alpha-neurotoxins, with surprisingly few nAChR residues in common between their overlapping binding sites. These docking experiments show that Imi and PnIB bind to the ACh binding pocket via a small cavity located above the beta9/beta10 hairpin of the (+)alpha7 nAChR subunit. Interestingly, PnIB, PnIA and MII were found to bind in a similar location on alpha7 or alpha3beta2 receptors mostly through hydrophobic interactions, while ImI bound further from the ACh binding pocket, mostly through electrostatic interactions. These findings, which distinguish alpha-conotoxin and alpha-neurotoxin binding modes, have implications for the rational design of selective nAChR antagonists. Copyright (C) 2004 John Wiley Sons, Ltd.

Solution structure and proposed binding mechanism of a novel potassium channel toxin kappa-conotoxin PVIIA

Background: kappa-PVIIA is a 27-residue polypeptide isolated from the venom of Conus purpurascens and is the first member of a new class of conotoxins that block potassium channels. By comparison to other ion channels of eukaryotic cell membranes, voltage-sensitive potassium channels are relatively simple and methodology has been developed for mapping their interactions with small-peptide toxins, PVIIA, therefore, is a valuable new probe of potassium channel structure. This study of the solution structure and mode of channel binding of PVIIA forms the basis for mapping the interacting residues at the conotoxin-ion channel interface. Results: The three-dimensional structure of PVIIA resembles the triple-stranded beta sheet/cystine-knot motif formed by a number of toxic and inhibitory peptides. Subtle structural differences, predominantly in loops 2 and 4, are observed between PVIIA and other conotoxins with similar structural frameworks, however. Electrophysiological binding data suggest that PVIIA blocks channel currents by binding in a voltage-sensitive manner to the external vestibule and occluding the pore, Comparison of the electrostatic surface of PVIIA with that of the well-characterised potassium channel blocker charybdotoxin suggests a likely binding orientation for PVIIA, Conclusions: Although the structure of PVIIA is considerably different to that of the alpha K scorpion toxins, it has a similar mechanism of channel blockade. On the basis of a comparison of the structures of PVIIA and charybdotoxin, we suggest that Lys19 of PVIIA is the residue which is responsible for physically occluding the pore of the potassium channel.

Structure determination of the three disulfide bond isomers of alpha-conotoxin GI: A model for the role of disulfide bonds in structural stability

The three possible disulfide bonded isomers of alpha-conotoxin GI have been selectively synthesised and their structures determined by H-1 NMR spectroscopy. alpha-Conotoxin GI derives from the venom of Conus geographus and is a useful neuropharmacological tool as it selectively binds to the nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel involved in nerve signal transmission. The peptide has the sequence ECCNPACGRHYSC-NH2, and the three disulfide bonded isomers are referred to as GI(2-7;3-13), GI(2-13;3-7) and GI(2-3;7-13). The NMR structure for the native isomer GI(2-7;3-13) is of excellent quality, with a backbone pairwise RMSD of 0.16 Angstrom for a family of 35 structures, and comprises primarily a distorted 3(10),, helix between residues 5 to 11. The two non-native isomers exhibit multiple conformers in solution, with the major populated forms being different in structure both from each other and from the native form. Structure-activity relationships for the native GI(2-7;3-13) as well as the role of the disulfide bonds on folding and stability of the three isomers are examined. It is concluded that the disulfide bonds in alpha-conotoxin GI play a crucial part in determining both the structure and stability of the peptide. A trend for increased conformational heterogeneity was observed in the order of GI(2-7;3-13) < GI(2-13;3-7) < GI(2-3;7-13). It was found that the peptide bond joining Cys2 to Cys3 in GI(2-3;7-13) is predominantly trans, rather than cis as theoretically predicted. These structural data are used to interpret the varying nAChR binding of the non-native forms. A model for the binding of native GI(2-7;3-13) to the mammalian nAChR is proposed, with an alpha-subunit binding face made up of Cys2, Asn4, Pro5, Ala6 and Cys7 and a selectivity face, comprised of Arg9 and His10. These two faces orient the molecule between the alpha and delta subunits of the receptor. The structure of the CCNPAC sequence of the native GI(2-7;3-13) is compared to the structure of the identical sequence from the toxic domain of heat-stable enterotoxins, which forms part of the receptor binding region of the enterotoxins, but which has a different disulfide connectivity. (C) 1998 Academic Press Limited.

The 1.1 angstrom resolution crystal structure of [Tyr(15)]EpI, a novel alpha-conotoxin from Conus episcopatus, solved by direct methods

Conotoxins are valuable probes of receptors and ion channels because of their small size and highly selective activity. alpha-Conotoxin EpI, a 16-residue peptide from the mollusk-hunting Conus episcopatus, has the amino acid sequence GCCSDPRCNMNNPDY(SO3H)C-NH2 and appears to be an extremely potent and selective inhibitor of the alpha 3 beta 2 and alpha 3 beta 4 neuronal subtypes of the nicotinic acetylcholine receptor (nAChR). The desulfated form of EpI ([Tyr(15)]EpI) has a potency and selectivity for the nAChR receptor similar to those of EpI. Here we describe the crystal structure of [Tyr(15)]EpI solved at a resolution of 1.1 Angstrom using SnB. The asymmetric unit has a total of 284 non-hydrogen atoms, making this one of the largest structures solved de novo try direct methods. The [Tyr(15)]EpI structure brings to six the number of alpha-conotoxin structures that have been determined to date. Four of these, [Tyr(15)]EpI, PnIA, PnIB, and MII, have an alpha 4/7 cysteine framework and are selective for the neuronal subtype of the nAChR. The structure of [Tyr(15)]EpI has the same backbone fold as the other alpha 4/7-conotoxin structures, supporting the notion that this conotoxin cysteine framework and spacing give rise to a conserved fold. The surface charge distribution of [Tyr(15)]EpI is similar to that of PnIA and PnIB but is likely to be different from that of MII, suggesting that [Tyr(15)]EpI and MII may have different binding modes for the same receptor subtype.

Three-dimensional solution structure of alpha-conotoxin MII by NMR spectroscopy: Effects of solution environment on helicity

alpha-Conotoxin MII, a 16-residue polypeptide from the venom of the piscivorous cone snail Conus magus, is a potent and highly specific blocker of mammalian neuronal nicotinic acetylcholine receptors composed of alpha 3 beta 2 subunits. The role of this receptor type in the modulation of neurotransmitter release and its relevance to the problems of addiction and psychosis emphasize the importance of a structural understanding of the mode of interaction of MII with the alpha 3 beta 2 interface. Here we describe the three-dimensional solution structure of MIT determined using 2D H-1 NMR spectroscopy. Structural restraints consisting of 376 interproton distances inferred from NOEs and 12 dihedral restraints derived from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimization in the program X-PLOR. The final set of 20 structures is exceptionally well-defined with mean pairwise rms differences over the whole molecule of 0.07 Angstrom for the backbone atoms and 0.34 Angstrom for all heavy atoms. MII adopts a compact structure incorporating a central segment of alpha-helix and beta-turns at the N- and C-termini. The molecule is stabilized by two disulfide bonds, which provide cross-links between the N-terminus and both the middle and C-terminus of the structure. The susceptibility of the structure to conformational change was examined using several different solvent conditions. While the global fold of MII remains the same, the structure is stabilized in a more hydrophobic environment provided by the addition of acetonitrile or trifluoroethanol to the aqueous solution. The distribution of amino acid side chains in MII creates distinct hydrophobic and polar patches on its surface that may be important for the specific interaction with the alpha 3 beta 2 neuronal nAChR. A comparison of the structure of MII with other neuronal-specific alpha-conotoxins provides insights into their mode of interaction with these receptors.

Single amino acid substitutions in alpha-conotoxin PnIA shift selectivity for subtypes of the mammalian neuronal nicotinic acetylcholine receptor

The alpha-conotoxins, a class of nicotinic acetylcholine receptor (nAChR) antagonists, are emerging as important probes of the role played by different nAChR subtypes in cell function and communication, In this study, the native alpha-conotoxins PnIA and PnIB were found to cause concentration-dependent inhibition of the ACh-induced current in all rat parasympathetic neurons examined, with IC50 values of 14 and 33 nM, and a maximal reduction in current amplitude of 87% and 71%, respectively. The modified alpha-conotoxin [N11S]PnIA reduced the ACh-induced current with an IC50 value of 375 nM and a maximally effective concentration caused 91% block, [A10L]PnIA was the most potent inhibitor, reducing the ACh-induced current in similar to 80% of neurons, with an IC50 value of 1.4 nM and 46% maximal block of the total current, The residual current was not inhibited further by alpha-bungarotoxin, but was further reduced by the cu-conotoxins PnIA or PnIB, and by mecamylamine. H-1 NMR studies indicate that PnIA, PnIB, and the analogues, [A10L]PnIA and [N11S]PnIA, have identical backbone structures. We propose that positions 10 and II of PnIA and PnIB influence potency and determine selectivity among alpha 7 and other nAChR subtypes, including alpha 3 beta 2 and alpha 3 beta 4, Four distinct components of the nicotinic ACh-induced current in mammalian parasympathetic neurons have been dissected with these conopeptides.

Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa - 1. Isolation, characterization and chemical synthesis

A novel conotoxin belonging to the 'four-loop' structural class has been isolated from the venom of the piscivorous cone snail Conus tulipa. It was identified using a chemical-directed strategy based largely on mass spectrometric techniques. The new toxin, conotoxin TVIIA, consists of 30 amino-acid residues and contains three disulfide bonds. The amino-acid sequence was determined by Edman analysis as SCSGRDSRCOOVCCMGLMCSRGKCVSIYGE where O = 4-transl-hydroxyproline. Two under-hydroxylated analogues, [Pro10]TVIIA and [Pro10,11]TVIIA, were also identified in the venom of C. tulipa. The sequences of TVIIA and [Pro10]TVIIA were further verified by chemical synthesis and coelution studies with native material. Conotoxin TVIIA has a six cysteine/four-loop structural framework common to many peptides from Conus venoms including the omega-, delta- and kappa-conotoxins. However, TVIIA displays little sequence homology with these well-characterized pharmacological classes of peptides, but displays striking sequence homology with conotoxin GS, a peptide from Conus geographus that blocks skeletal muscle sodium channels. These new toxins and GS share several biochemical features and represent a distinct subgroup of the four-loop conotoxins.

Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa - 2. Three-dimensional solution structure

The three-dimensional solution structure of conotoxin TVIIA, a 30-residue polypeptide from the venom of the piscivorous cone snail Conus tulipa, has been determined using 2D H-1 NMR spectroscopy. TVIIA contains six cysteine residues which form a 'four-loop' structural framework common to many peptides from Conus venoms including the omega-, delta-, kappa-, and mu O-conotoxins. However, TVIIA does not belong to these well-characterized pharmacological classes of conotoxins, but displays high sequence identity with conotoxin GS, a muscle sodium channel blocker from Conus geographus. Structure calculations were based on 562 interproton distance restraints inferred from NOE data, together with 18 backbone and nine side-chain torsion angle restraints derived from spin-spin coupling constants. The final family of 20 structures had mean pairwise rms differences over residues 2-27 of 0.18 +/- 0.05 Angstrom for the backbone atoms and 1.39 +/- 0.33 Angstrom for all heavy atoms. The structure consists of a triple-stranded, antiparallel beta sheet with +2x, -1 topology (residues 7-9, 16-20 and 23-27) and several beta turns. The core of the molecule is formed by three disulfide bonds which form a cystine knot motif common to many toxic and inhibitory polypeptides. The global fold, molecular shape and distribution of amino-acid sidechains in TVIIA is similar to that previously reported for conotoxin GS, and comparison with other four-loop conotoxin structures provides further indication that TVIIA and GS represent a new and distinct subgroup of this structural family. The structure of TVIIA determined in this study provides the basis for determining a structure-activity relationship for these molecules and their interaction with target receptors.