Discovery, Synthesis, and Structure Activity of a Highly Selective α7 Nicotinic Acetylcholine Receptor Antagonist (original) (raw)
Related papers
alpha.-Conotoxin EI, A New Nicotinic Acetylcholine Receptor Antagonist with Novel Selectivity
Biochemistry, 1995
We report the isolation and characterization of a novel nicotinic acetylcholine receptor (nAChR) ligand. The toxin is an 18 amino acid peptide and is the first reported a-conotoxin from an Atlantic fish-hunting Conus. The peptide was purified from the venom of Conus emineus and is called a-conotoxin EI. The sequence diverges from that of previously isolated a-conotoxins. We demonstrate that this structural divergence has functional consequences. In Torpedo nAChRs, a-conotoxin EI selectively binds the agonist site near the a/d subunit interface in contrast to a-conotoxin MI which selectively targets the a / y agonist binding site. In mammalian nAChRs a-conotoxin EI shows high affinity for both the a/d and a / y subunit interfaces (with some preference for the a/d site), whereas a-conotoxin MI is highly selective for the d d ligand binding site. The sequence of the peptide is: Arg-Asp-Hyp-Cys-Cys-Tyr-His-Pro-Thr-Cys-Asn-Met-Ser-Asn-Pro-Gln-Ile-Cys-NH~, with disulfide bridging between Cys4-CyslO and CysS-Cysl8, analogous to those of previously described a-conotoxins. This sequence has been verified by total chemical synthesis. Thus, a-conotoxin EI is a newly-available tool with unique structure and function for characterization of nAChRs. Nicotinic acetylcholine receptors (nAChRs)' in skeletal muscle and the electric organ of Torpedo are heteropentameric ligand-gated cation channels formed by four subunits in the stoichiometry (al)& 76. Several small molecule toxins isolated from plants, coral, and gastropods as well as polypeptide toxins from predatory organisms have been isolated which target nAChRs [for review see Chiappinelli (1993)l. The availability of these toxins has played a critical role in the progressive understanding of the structure and function of the nicotinic receptor. The nAChR requires two molecules of acetylcholine to bind two separate sites for channel opening. These nonequivalent binding sites are located at the a l y and al6 subunit interfaces (Blount & Merlie, 1989). Curariform antagonists bind with 1-2 orders of magnitude higher affinity to the a l y site than to the al6 site of both mammalian muscle and Torpedo receptors + This work was supported by NIMH Scientist Development Award for Clinicians K20 MHO0929 (J.M.M.), NIH Grants GM 48677 (B.M.O.) and NS 29951 (S.N.A.), and the Smokeless Tobacco Research Council (S.N.A.).
Differential Targeting of Nicotinic Acetylcholine Receptors by Novel αA-Conotoxins
Journal of Biological Chemistry, 1997
We describe the isolation and characterization of two peptide toxins from Conus ermineus venom targeted to nicotinic acetylcholine receptors (nAChRs). The peptide structures have been confirmed by mass spectrometry and chemical synthesis. In contrast to the 12-18 residue, 4 Cys-containing ␣-conotoxins, the new toxins have 30 residues and 6 Cys residues. The toxins, named ␣A-conotoxins EIVA and EIVB, block both Torpedo and mouse ␣1-containing muscle subtype nAChRs expressed in Xenopus oocytes at low nanomolar concentrations. In contrast to ␣-bungarotoxin, ␣A-EIVA is inactive at ␣7-containing nAChRs even at micromolar concentrations. In this regard, ␣A-EIVA is similar to the previously described ␣-conotoxins (e.g. ␣-MI and ␣-GI) which also selectively target ␣1versus ␣7-containing nAChRs. However, ␣-MI and ␣-GI discriminate between the ␣/␦ versus ␣/␥ subunit interfaces of the mouse muscle nAChR with 10,000-fold selectivity. In contrast, ␣A-conotoxin EIVA blocks both the ␣/␥ site and ␣/␦ site with equally high affinity but with distinct kinetics. The ␣A-conotoxins thus represent novel probes for the ␣/␥ as well as the ␣/␦ binding sites of the nAChR.
Toxins, 2019
Nicotinic acetylcholine receptors (nAChRs) are found throughout the mammalian body and have been studied extensively because of their implication in a myriad of diseases. α-Conotoxins (α-CTxs) are peptide neurotoxins found in the venom of marine snails of genus Conus. α-CTxs are potent and selective antagonists for a variety of nAChR isoforms. Over the past 40 years, α-CTxs have proven to be valuable molecular probes capable of differentiating between closely related nAChR subtypes and have contributed greatly to understanding the physiological role of nAChRs in the mammalian nervous system. Here, we review the amino acid composition and structure of several α-CTxs that selectively target nAChR isoforms and explore strategies and outcomes for introducing mutations in native α-CTxs to direct selectivity and enhance binding affinity for specific nAChRs. This review will focus on structure-activity relationship studies involving native α-CTxs that have been rationally mutated and molec...
A New α-Conotoxin Which Targets α3β2 Nicotinic Acetylcholine Receptors
Journal of Biological Chemistry, 1996
We have isolated a 16-amino acid peptide from the venom of the marine snail Conus magus which potently blocks nicotinic acetylcholine receptors (nAChRs) composed of ␣32 subunits. This peptide, named ␣-conotoxin MII, was identified by electrophysiologically screening venom fractions against cloned nicotinic receptors expressed in Xenopus oocytes. The peptide's structure, which has been confirmed by mass spectrometry and total chemical synthesis, differs significantly from those of all previously isolated ␣-conotoxins. Disulfide bridging, however, is conserved. The toxin blocks the response to acetylcholine in oocytes expressing ␣32 nAChRs with an IC 50 of 0.5 nM and is 2-4 orders of magnitude less potent on other nAChR subunit combinations. We have recently reported the isolation and characterization of ␣-conotoxin ImI, which selectively targets homomeric ␣7 neuronal nAChRs. Yet other ␣-conotoxins selectively block the muscle subtype of nAChR. Thus, it is increasingly apparent that ␣-conotoxins represent a significant resource for ligands with which to probe structure-function relationships of various nAChR subtypes.
Febs Journal, 2007
α-Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4/7-type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [γ15E]SrIB) were compared to those exerted by the α4/7-conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch-clamp technique in mammalian cells expressing α1β1γδ, α4β2 and α3β4 nicotinic acetylcholine receptors. At high concentrations (10 µm), the peptides SrIA, SrIB and [γ15E]SrIB showed weak blocking effects only on α4β2 and α1β1γδ subtypes, but EI also strongly blocked α3β4 receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of α1β1γδ and α4β2 nicotinic acetylcholine receptors if briefly (2–15 s) applied at concentrations several orders of magnitude lower (EC50, 1.78 and 0.37 nm, respectively). These results suggest not only that the novel α-conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both α1β1γδ and α4β2 nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin–receptor interaction, as well as models with which to design highly specific therapeutic drugs.
Conotoxin αD-GeXXA utilizes a novel strategy to antagonize nicotinic acetylcholine receptors
Scientific reports, 2015
Nicotinic acetylcholine receptors (nAChRs) play essential roles in transmitting acetylcholine-mediated neural signals across synapses and neuromuscular junctions, and are also closely linked to various diseases and clinical conditions. Therefore, novel nAChR-specific compounds have great potential for both neuroscience research and clinical applications. Conotoxins, the peptide neurotoxins produced by cone snails, are a rich reservoir of novel ligands that target receptors, ion channels and transporters in the nervous system. From the venom of Conus generalis, we identified a novel dimeric nAChR-inhibiting αD-conotoxin GeXXA. By solving the crystal structure and performing structure-guided dissection of this toxin, we demonstrated that the monomeric C-terminal domain of αD-GeXXA, GeXXA-CTD, retains inhibitory activity against the α9α10 nAChR subtype. Furthermore, we identified that His7 of the rat α10 nAChR subunit determines the species preference of αD-GeXXA, and is probably part ...
Biochemistry
We report the purification and characterization of a new conotoxin from the venom of Conus radiatus. The peptide, RS-conotoxin RVIIIA (RS-RVIIIA), is biochemically unique with respect to its amino acid sequence, post-translational modification, and molecular targets. In comparison to other nicotinic antagonists from Conus venoms, RS-RVIIIA exhibits an unusually broad targeting specificity for nicotinic acetylcholine receptor (nAChR) subtypes, as assayed by electrophysiology. The toxin is paralytic to mice and fish, consistent with its nearly irreversible block of the neuromuscular nAChR. Similar to other antagonists of certain neuronal nAChRs, the toxin also elicits seizures in mice upon intracranial injection. The only previously characterized conotoxin from the S superfamily, σ-conotoxin GVIIIA, is a specific competitive antagonist of the 5-HT 3 receptor; thus, RS-RVIIIA defines a novel family of nicotinic antagonists within the S superfamily. All previously characterized competitive conotoxin nAChR antagonists have been members of the A superfamily of conotoxins. Our working hypothesis is that the particular group of fish-hunting Conus species that includes Conus radiatus uses the RS-conotoxin family to target the muscle nAChR and paralyze prey.
α4/7-conotoxin Lp1.1 is a novel antagonist of neuronal nicotinic acetylcholine receptors
Peptides, 2008
Cone snails comprise approximately 500 species of venomous molluscs which have evolved the ability to generate multiple toxins with varied and exquisite selectivity. α-Conotoxin is a powerful tool for defining the composition and function of nicotinic acetylcholine receptors which play a crucial role in excitatory neurotransmission and are important targets for drugs and insecticides. An α4/7 conotoxin, Lp1.1, originally identified by cDNA and genomic DNA cloning from Conus leopardus, was found devoid of the highly conserved Pro residue in the first intercysteine loop. To further study this toxin, α-Lp1.1 was chemically synthesized and refolded into its globular disulfide isomer. The synthetic Lp1.1 induced seizure and paralysis on freshwater goldfish and selectively reversibly inhibited ACh-evoked currents in Xenopus oocytes expressing rat α3β2 and α6α3β2 nAChRs. Comparing the distinct primary structure with other functionally related αconotoxins could indicate structural features in Lp1.1 that may be associated with its unique receptor recognition profile.
Analogs of -Conotoxin MII Are Selective for 6-Containing Nicotinic Acetylcholine Receptors
Molecular Pharmacology, 2004
Neuronal nicotinic acetylcholine receptors (nAChRs) both mediate direct cholinergic synaptic transmission and modulate synaptic transmission by other neurotransmitters. Novel ligands are needed as probes to discriminate among structurally related nAChR subtypes. ␣-Conotoxin MII, a selective ligand that discriminates among a variety of nAChR subtypes, fails to discriminate well between some subtypes containing the closely related ␣3 and ␣6 subunits. Structure-function analysis of ␣-conotoxin MII was performed in an attempt to generate analogs with preference for ␣6-containing [␣6* (asterisks indicate the possible presence of additional subunits)] nAChRs. Alanine substitution resulted in several analogs with decreased activity at ␣3* versus ␣6* nAChRs heterologously expressed in Xenopus laevis oocytes. From the initial analogs, a series of mutations with two alanine substitutions was synthesized. Substitution at His9 and Leu15 (MII[H9A;L15A]) resulted in a 29-fold lower IC 50 at ␣64 versus ␣34 nAChRs. The peptide had a 590-fold lower IC 50 for ␣6/␣32 versus ␣32 and a 2020-fold lower IC 50 for ␣6/␣323 versus ␣32 nAChRs. MII[H9A;L15A] had little or no activity at ␣22, ␣24, ␣34, ␣42, ␣44, and ␣7 nAChRs. Functional block by MII[H9A;L15A] of rat ␣6/␣323 nAChRs (IC 50 ϭ 2.4 nM) correlated well with the inhibition constant of MII[H9A;L15A] for [ 125 I]␣-conotoxin MII binding to putative ␣62* nAChRs in mouse brain homogenates (K i ϭ 3.3 nM). Thus, structure-function analysis of ␣-conotoxin MII enabled the creation of novel selective antagonists for discriminating among nAChRs containing ␣3 and ␣6 subunits.
Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors
Acta Pharmacologica Sinica, 2009
Cysteine-rich peptides from the venom of cone snails (Conus) target a wide variety of different ion channels. One family of conopeptides, the α-conotoxins, specifically target different isoforms of nicotinic acetylcholine receptors (nAChRs) found both in the neuromuscular junction and central nervous system. This family is further divided into subfamilies based on the number of amino acids between cysteine residues. The exquisite subtype selectivity of certain α-conotoxins has been key to the characterization of native nAChR isoforms involved in modulation of neurotransmitter release, the pathophysiology of Parkinson's disease and nociception. Structure/function characterization of α-conotoxins has led to the development of analogs with improved potency and/or subtype selectivity. Cyclization of the backbone structure and addition of lipophilic moieties has led to improved stability and bioavailability of α-conotoxins, thus paving the way for orally available therapeutics. The recent advances in phylogeny, exogenomics and molecular modeling promises the discovery of an even greater number of α-conotoxins and analogs with improved selectivity for specific subtypes of nAChRs.