Crystal structure at 1.1 angstrom resolution of alpha-conotoxin PnIB: Comparison with alpha-conotoxins PnIA and GI

Conotoxins are small, cysteine-rich peptides isolated from the venom of Conus spp. of predatory marine snails, which selectively target specific receptors and ion channels critical to the functioning of the neuromuscular system. alpha-Conotoxins PnIA and PnIB are both 16-residue peptides (differing in sequence at only two positions) isolated from the molluscivorous snail Conus pennaceus. In contrast to the muscle-selective alpha-conotoxin GI from Conus geographus, PnIA and PnIB block the neuronal nicotinic acetylcholine receptor (nAChR). Here, we describe the crystal structure of PnIB, solved at a resolution of 1.1 Angstrom and phased using the Shake-and-Bake direct methods program. PnIB crystals are orthorhombic and belong to the space group P2(1)2(1)2(1) with the following unit cell dimensions: a = 14.6 Angstrom, b = 26.1 Angstrom, and c = 29.2 Angstrom. The final refined structure of alpha-conotoxin PnIB includes all 16 residues plus 23 solvent molecules and has an overall R-factor of 14.7% (R-free of 15.9%). The crystal structures of the alpha-conotoxins PnIB and PnIA are solved from different crystal forms, with different solvent contents. Comparison of the structures reveals them to be very similar, showing that the unique backbone and disulfide architecture is not strongly influenced by crystal lattice constraints or solvent interactions. This finding supports the notion that this structural scaffold is a rigid support for the presentation of important functional groups. The structures of PnIB and PnIA differ in their shape and surface charge distribution from that of GI.

The synthesis and structure of an n-terminal dodecanoic acid conjugate of a-conotoxin MII

The alpha-conotoxin MII is a 16 amino acid long peptide toxin isolated from the marine snail, Conus magus. This toxin has been found to be a highly selective and potent inhibitor of neuronal nicotinic acetylcholine receptors of the subtype alpha3beta2. To improve the bioavailability of this peptide, we have coupled to the N-terminus of conotoxin MII, 2-amino-D,L-dodecanoic acid (Laa) creating a lipidic linear peptide which was then successfully oxidised to produce the correctly folded conotoxin MII construct.

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NAIR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

Differential antagonism by conotoxin p-TIA of contractions mediated by distinct alpha(1)-adrenoceptor subtypes in rat vas deferens, spleen and aorta

The ability of the conotoxin p-TIA, a 19-amino acid peptide isolated from the marine snail Conus tulipa, to antagonize contractions induced by noradrenaline through activation of alpha(1A)-adrenoceptors in rat vas deferens, alpha(1B)-adrenoceptors in rat spleen and alpha(ID)-adrenoceptors in rat aorta, and to inhibit the binding of [I-125]HEAT (2-[[beta-(4-hydroxyphenyl)ethyl]aminomethyl]-1-tetralone) to membranes of human embryonic kidney (HEK) 293 cells expressing each of the recombinant rat alpha(1)-adrenoceptors was investigated. p-TIA (100 nM to 1 muM) antagonized the contractions of vas deferens and aorta in response to noradrenaline without affecting maximal effects and with similar potencies (pA(2)similar to7.2, n=4). This suggests that p-TIA is a competitive antagonist of alpha(1A)- and alpha(1D)-adrenoceptors with no selectivity between these subtypes. Incubation of p-TIA (30 to 300 nM) with rat spleen caused a significant reduction of the maximal response to noradrenaline, suggesting that p-TIA is a non-competitive antagonist at alpha(1B)-adrenoceptors. After receptor inactivation with phenoxybenzamine, the potency of p-TIA in inhibiting contractions was examined with similar occupancies (similar to25%) at each subtype. Its potency (pIC(50)) was 12 times higher in spleen (8.3 +/- 0.1, n=4) than in vas deferens (7.2 +/- 0.1, n=4) or aorta (7.2 0.1, n=4). In radioligand binding assays, p-TIA decreased the number of binding sites (B,,,,,,) in membranes from HEK293 cells expressing the rat alpha(1B)-adrenoceptors without affecting affinity (K-D), In contrast, in HEK293 cells expressing rat alpha(1A)- or alpha(1D)-adrenoceptors, p-TTA decreased the KD without affecting the B-max. It is concluded that p-TIA will be useful for distinguishing the role of particular alpha(1)-adrenoceptor subtypes in native tissues. (C) 2004 Elsevier B.V. All rights reserved.

Calcium-dependent glutamate release during neuronal development and synaptogenesis: different involvement of omega-agatoxin IVA- and omega-conotoxin GVIA-sensitive channels.

Hippocampal neurons maintained in primary culture recycle synaptic vesicles and express functional glutamate receptors since early stages of neuronal development. By analyzing glutamate-induced cytosolic calcium changes to sense presynaptically released neurotransmitter, we demonstrate that the ability of neurons to release glutamate in the extracellular space is temporally coincident with the property of synaptic vesicles to undergo exocytotic-endocytotic recycling. Neuronal differentiation and maturation of synaptic contacts coincide with a change in the subtype of calcium channels primarily involved in controlling neurosecretion. Whereas omega-agatoxin IVA-sensitive channels play a role in controlling neurotransmitter secretion at all stages of neuronal differentiation, omega-conotoxin GVIA-sensitive channels are primarily involved in mediating glutamate release at early developmental stages only.

Synthesis and biological evaluation of nonpeptide mimetics of co-conotoxin GVIA

A benzothiazole-derived compound (4a) designed to mimic the C-alpha-C-beta bond vectors and terminal functionalities of Lys2, TyrI3 and Arg17 in omega-conotoxin GVIA was synthesised, together with analogues (4b-d), which had each side-chain mimic systematically truncated or eliminated. The affinity of these compounds for rat brain N-type and P/Q-type voltage gated calcium channels (VGCCs) was determined. In terms of N-type channel affinity and selectivity, two of these compounds (4a and 4d) were found to be highly promising, first generation mimetics of omega-conotoxin. The fully functionalised mimetic (4a) showed low PM binding affinity to N-type VGCCs (IC50 = 1.9 muM) and greater than 20-fold selectivity for this channel sub-type over P/Q-type VGCCs, whereas the mimetic in which the guanidine-type side chain was truncated back to an amine (4d, IC50 = 4.1 muM) showed a greater than 25-fold selectivity for the N-type channel. (C) 2004 Elsevier Ltd. 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.

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the alpha-conotoxin MII

Conotoxins (CTXs), with their exquisite specificity and potency, have recently created much excitement as drug leads. However, like most peptides, their beneficial activities may potentially be undermined by susceptibility to proteolysis in vivo. By cyclizing the alpha-CTX MII by using a range of linkers, we have engineered peptides that preserve their full activity but have greatly improved resistance to proteolytic degradation. The cyclic MII analogue containing a seven-residue linker joining the N and C termini was as active and selective as the native peptide for native and recombinant neuronal nicotinic acetylcholine receptor subtypes present in bovine chromaffin cells and expressed in Xerl oocytes, respectively. Furthermore, its resistance to proteolysis against a specific protease and in human plasma was significantly improved. More generally, to our knowledge, this report is the first on the cyclization of disulfide-rich toxins. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.

beta 2 subunit contribution to 4/7 alpha-conotoxin binding to the nicotinic acetylcholine receptor

The structures of acetylcholine-binding protein ( AChBP) and nicotinic acetylcholine receptor ( nAChR) homology models have been used to interpret data from mutagenesis experiments at the nAChR. However, little is known about AChBP-derived structures as predictive tools. Molecular surface analysis of nAChR models has revealed a conserved cleft as the likely binding site for the 4/7 alpha-conotoxins. Here, we used an alpha 3 beta 2 model to identify beta 2 subunit residues in this cleft and investigated their influence on the binding of alpha-conotoxins MII, PnIA, and GID to the alpha 3 beta 2 nAChR by two-electrode voltage clamp analysis. Although a beta 2-L119Q mutation strongly reduced the affinity of all three alpha-conotoxins, beta 2-F117A, beta 2-V109A, and beta 2-V109G mutations selectively enhanced the binding of MII and GID. An increased activity of alpha-conotoxins GID and MII was also observed when the beta 2-F117A mutant was combined with the alpha 4 instead of the alpha 3 subunit. Investigation of A10L-PnIA indicated that high affinity binding to beta 2-F117A, beta 2-V109A, and beta 2-V109G mutants was conferred by amino acids with a long side chain in position 10 (PnIA numbering). Docking simulations of 4/7 alpha-conotoxin binding to the alpha 3 beta 2 model supported a direct interaction between mutated nAChR residues and alpha-conotoxin residues 6, 7, and 10. Taken together, these data provide evidence that the beta subunit contributes to alpha-conotoxin binding and selectivity and demonstrate that a small cleft leading to the agonist binding site is targeted by alpha-conotoxins to block the nAChR.

A novel conotoxin inhibitor of Kv1.6 channel and nAChR subtypes defines a new superfamily of conotoxins

Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated p114a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. p114a contains 25 amino acid residues with an amidated C-terminus, an elongated N-terminal tail (six residues), and two disulfide bonds (1-3, 2-4 connectivity) in a novel framework distinct from other conotoxins. The peptide was chemically synthesized, and its three-dimensional structure was demonstrated to be well-defined, with an R-helix and two 3(10)-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of p114a revealed a novel signal sequence, indicating that p114a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of p114a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, p114a was shown to inhibit both a K+ channel subtype (Kv1.6, IC50) 1.59 mu M) and neuronal (IC50 = 8.7 mu M for alpha 3 beta 4) and neuromuscular (IC50 = 0.54 mu M for alpha 1 beta 1 is an element of delta) subtypes of the nicotinic acetylcholine receptor ( nAChR). Similarities in sequence and structure are apparent between the middle loop of p114a and the second loop of a number of alpha-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels.

Cyclic MrIA: A stable and potent cyclic conotoxin with a novel topological fold that targets the norepinephrine transporter

Conotoxins, disulfide-rich peptides from the venom of cone snails, have created much excitement over recent years due to their potency and specificity for ion channels and their therapeutic potential. One recently identified conotoxin, MrIA, a 13-residue member of the chi-conotoxin family, inhibits the human norepinephrine transporter (NET) and has potential applications in the treatment of pain. In the current study, we show that the, beta-hairpin structure of native MrIA is retained in a synthetic cyclic version, as is biological activity at the NET. Furthermore, the cyclic version has increased resistance to trypsin digestion relative to the native peptide, an intriguing result because the cleavage site for the trypsin is not close to the cyclization site. The use of peptides as drugs is generally hampered by susceptibility to proteolysis, and so, the increase in enzymatic stability against trypsin observed in the current study may be useful in improving the therapeutic potential of MrIA. Furthermore, the structure reported here for cyclic MrIA represents a new topology among a growing number of circular disulfide-rich peptides.

mu O-conotoxin MrVIB selectively blocks Na(v)1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits

The tetroclotoxin-resistant voltage-gated sodium channel (VGSC) Na(v)1.8 is expressed predominantly by damage-sensing primary afferent nerves and is important for the development and maintenance of persistent pain states. Here we demonstrate that mu O-conotoxin MrVIB from Conus marmoreus displays substantial selectivity for Na(v)1.8 and inhibits pain behavior in models of persistent pain. In rat sensory neurons, submicromolar concentrations of MrVIB blocked tetroclotoxin-resistant current characteristic of Na(v)1.8 but not Na(v)1.9 or tetroclotoxin-sensitive VGSC currents. MrVIB blocked human Nav1.8 expressed in Xenopus oocytes with selectivity at least 10-fold greater than other VGSCs. In neuropathic and chronic inflammatory pain models, allodynia and hyperalgesia were both reduced by intrathecal infusion of MrVIB (0.03-3 nmol), whereas motor side effects occurred only at 30-fold higher doses. In contrast, the nonselective VGSC blocker lignocaine displayed no selectivity for allodynia and hyperalgesia versus motor side effects. The actions of MrVIB reveal that VGSC antagonists displaying selectivity toward Na(v)1.8 can alleviate chronic pain behavior with a greater therapeutic index than nonselective antagonists.