Synthesis and biological activity of a novel class nicotinic acetylcholine receptors (nAChRs) ligands structurally related to anatoxin-a (original) (raw)
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4-Oxystilbene compounds are selective ligands for neuronal nicotinic a Bungarotoxin receptors
Brit J Pharmacol, 1998
1 Starting from the structure of an old 4-oxystilbene derivate with ganglioplegic activity (MG624), we synthesized two further derivates (F2 and F3) and two stereoisomers of F3 (F3A and F3B), and studied their selective eect on neuronal nicotinic acetylcholine receptor (AChR) subtypes. 2 MG 624, F3, F3A and F3B inhibited of 125 I-aBungarotoxin (aBgtx) binding to neuronal chick optic lobe (COL) membranes, with nM anity, but inhibited 125 I-aBgtx binding to TE671 cell-expressed muscle-type AChR only at much higher concentrations.
The Journal of pharmacology and experimental therapeutics, 1997
We assessed the pharmacological activity of anabaseine, a toxin found in certain animal venoms, relative to nicotine and anabasine on a variety of vertebrate nicotinic receptors, using cultured cells, the Xenopus oocyte expression system, contractility assays with skeletal and smooth muscle strips containing nicotinic receptors and in vivo rat prostration assay involving direct injection into the lateral ventricle of the brain. Anabaseine stimulated every subtype of nicotinic receptor that was tested. It was the most potent frog skeletal muscle nicotinic receptor agonist. At higher concentrations it also blocked the BC3H1 (adult mouse) muscle type receptor ion channel. The affinities of the three nicotinoid compounds for rat brain membrane alpha-bungarotoxin binding sites and their potencies for stimulating Xenopus oocyte homomeric alpha7 receptors, expressed in terms of their active monocation concentrations, displayed the same rank order, anabaseine>anabasine> nicotine. Alth...
Marine Drugs
Three major forms of the nicotinic agonist toxin anabaseine (cyclic iminium, cyclic imine and the monocationic open-chain ammonium-ketone) co-exist in almost equal concentrations at physiological pH. We asked the question: Which of these forms is pharmacologically active? First, we investigated the pH dependence of anabaseine inhibition of [3H]-methylcarbamylcholine binding at rat brain α4β2 nicotinic acetylcholine receptors (nAChRs). These experiments indicated that one or both monocationic forms interact with the orthosteric binding site for ACh. However, since they occur at equal concentrations near physiological pH, we employed another approach, preparing a stable analog of each form and examining its agonist activities and binding affinities at several vertebrate brain and neuromuscular nAChRs. Only 2-(3-pyridyl)-1,4,5,6-tetrahydropyrimidine monohydrogen chloride (PTHP), the cyclic iminium analog, displayed nAChR potencies and binding affinities similar to anabaseine. The cycli...
4-Oxystilbene compounds are selective ligands for neuronal nicotinic α Bungarotoxin receptors
British Journal of Pharmacology, 1998
1Starting from the structure of an old 4-oxystilbene derivate with ganglioplegic activity (MG624), we synthesized two further derivates (F2 and F3) and two stereoisomers of F3 (F3A and F3B), and studied their selective effect on neuronal nicotinic acetylcholine receptor (AChR) subtypes.2MG 624, F3, F3A and F3B inhibited of 125I-αBungarotoxin (αBgtx) binding to neuronal chick optic lobe (COL) membranes, with nM affinity, but inhibited 125I-αBgtx binding to TE671 cell-expressed muscle-type AChR only at much higher concentrations.3We immobilized the α7, β2 and β4 containing chick neuronal nicotinic AChR subtypes using anti-subunit specific antibodies. MG 624, F3, F3A and F3B inhibited 125I-αBgtx binding to the α7-containing receptors with nm affinity, but inhibited 3H-Epi binding to β2-containing receptors only at very high concentrations (more than 35 μm); their affinity for the β4-containing receptors was ten times more than for the β2-containing subtype.4Both MG624 and F3 compounds inhibited the ACh evoked currents in homomeric oocyte-expressed chick α7 receptors with an IC50 of respectively 94 and 119 nm.5High doses of both MG 624 and F3 depressed the contractile response to vagus nerve stimulation in guinea pig nerve-stomach preparations although at different IC50s (49.4 vs 166.2 μm) The effect of MG624 on rat nerve-hemidiaphragm preparations was 33 times less potent than that of F3 (IC50 486 vs 14.5 μm).6In conclusion, MG624 and F3 have a high degree of antagonist selectivity for neuronal nicotinic αBgtx receptors containing the α7 subunit.Starting from the structure of an old 4-oxystilbene derivate with ganglioplegic activity (MG624), we synthesized two further derivates (F2 and F3) and two stereoisomers of F3 (F3A and F3B), and studied their selective effect on neuronal nicotinic acetylcholine receptor (AChR) subtypes.MG 624, F3, F3A and F3B inhibited of 125I-αBungarotoxin (αBgtx) binding to neuronal chick optic lobe (COL) membranes, with nM affinity, but inhibited 125I-αBgtx binding to TE671 cell-expressed muscle-type AChR only at much higher concentrations.We immobilized the α7, β2 and β4 containing chick neuronal nicotinic AChR subtypes using anti-subunit specific antibodies. MG 624, F3, F3A and F3B inhibited 125I-αBgtx binding to the α7-containing receptors with nm affinity, but inhibited 3H-Epi binding to β2-containing receptors only at very high concentrations (more than 35 μm); their affinity for the β4-containing receptors was ten times more than for the β2-containing subtype.Both MG624 and F3 compounds inhibited the ACh evoked currents in homomeric oocyte-expressed chick α7 receptors with an IC50 of respectively 94 and 119 nm.High doses of both MG 624 and F3 depressed the contractile response to vagus nerve stimulation in guinea pig nerve-stomach preparations although at different IC50s (49.4 vs 166.2 μm) The effect of MG624 on rat nerve-hemidiaphragm preparations was 33 times less potent than that of F3 (IC50 486 vs 14.5 μm).In conclusion, MG624 and F3 have a high degree of antagonist selectivity for neuronal nicotinic αBgtx receptors containing the α7 subunit.British Journal of Pharmacology (1998) 124, 1197–1206; doi:10.1038/sj.bjp.0701957
Marine Drugs
Many organisms possess “secondary” compounds to avoid consumption or to immobilize prey. While the most abundant or active compounds are initially investigated, more extensive analyses reveal other “minor” compounds with distinctive properties that may also be of biomedical and pharmaceutical significance. Here, we present an initial in vitro investigation of the actions of two isomeric tetrahydropyridyl ring-containing anabasine analogs: isoanatabine, an alkaloid isolated from a marine worm, and anatabine, a relatively abundant minor alkaloid in commercial tobacco plants. Both compounds have a double bond that is distal to the piperidine ring nitrogen of anabasine. Racemic isoanatabine and anatabine were synthesized and their S- and R-enantiomers were isolated by chiral high pressure liquid chromatography (HPLC). Both isoanatabines displayed higher efficacies at α4β2 nicotinic acetylcholine receptors (nAChRs) relative to the anatabines; R-isoanatabine was most potent. Radioligand b...
Invertebrate Neuroscience, 1997
Naturally occurring toxins can often serve as useful chemical tools for investigating signalling processes in nervous and other systems. Tetrodotoxin and alpha-bungarotoxin are prime examples of toxins which are widely used in neurobiological research. Some toxins may also become molecular models for designing new drugs. Usually drugs are small, non-peptide molecules, as these display better bioavailability, longer durations of action and are less likely to generate immune responses. The relatively large size and conformational flexibility of peptides and protein toxins makes them more challenging molecular models for rational drug design. This article considers a marine invertebrate toxin, anabaseine, and describes how manipulation of the structure of this alkaloid has provided a drug candidate which selectively stimulates mammalian brain alpha7 nicotinic receptors. Numerous anabaseine analogs were synthesized and subjected to a variety of pharmacological, behavioral and toxcicological tests. This led to the choice of GTS-21 (also known as 3-(2,4-dimethoxybenzyl!dene)-anabaseine or DMXBA), as a drug candidate for the treatment of Alzheimer's dementia. The chemical and pharmacological properties of GTS-21 are compared with those of the initial lead compound, anabaseine.
British Journal of Pharmacology, 2009
Background and purpose: Benzylidene-anabaseines (BAs) are partial agonists of the a7 nicotinic acetylcholine receptor (nAChR) but their mechanism(s) of action are unknown. Our study explores several possibilities, including direct interactions of BAs with the nAChR channel. Experimental approach: Functional and radioligand-binding assays were used to examine the interaction of two BA analogues, 3-(2,4-dimethoxybenzylidene)-anabaseine (DMXBA) and its primary metabolite 3-(4-hydroxy-2-methoxybenzylidene)anabaseine (4OH-DMXBA) with both agonist and non-competitive antagonist (NCA)-binding sites on muscle-type nAChRs. Key results: Both BAs non-competitively inhibited ACh activation of human fetal muscle nAChRs and sterically inhibited the specific binding of the NCAs [piperidyl-3,4-3H(N)]-(N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine ([ 3 H]TCP) and [ 3 H]dizocilpine to Torpedo nAChRs in the desensitized state. These compounds modulated [ 3 H]tetracaine, [ 14 C]amobarbital and [ 3 H]TCP binding to resting nAChRs by allosteric mechanisms. Both BAs enhanced [ 3 H]TCP binding when the nAChR was initially in the resting but activatable state, suggesting that both compounds desensitized the Torpedo nAChR. Although DMXBA failed to activate human fetal muscle nAChRs, 4OH-DMXBA was found to be a partial agonist. [ 3 H]Nicotine competition-binding experiments confirmed that 4OH-DMXBA has higher affinity than DMXBA for the agonist sites, and that DMXBA is also a competitive antagonist. Conclusions and implications: 3-(4-hydroxy-2-methoxybenzylidene)-anabaseine is a partial agonist for human fetal muscle nAChRs, whereas DMXBA only has competitive and NCA activities. The NCA-binding site for BAs overlaps both the phencyclidine-and dizocilpine-binding sites in the desensitized Torpedo nAChR ion channel. The desensitizing property of BAs suggests another possible mode of non-competitive inhibition in addition to direct channel-blocking mechanisms.
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.).
The Journal of pharmacology and experimental therapeutics, 1998
3-(2,4-dimethoxybenzylidene)anabaseine (GTS-21) is a selective partial agonist for rat alpha-7 nicotine receptors with reportedly much lower efficacy for human alpha-7 receptors. Because this drug improves memory-related performance in nonhuman primates, and is presently in a clinical trial for Alzheimer's disease, we investigated the potential effects of its primary human metabolite, 3-(4-hydroxy, 2-methoxy-benzylidene)anabaseine) on human as well as rat nicotinic acetylcholine receptor. 4OH-GTS-21 exhibited a similar level of efficacy for both rat and human alpha-7 receptors expressed in Xenopus oocytes. It displaced high affinity [125I]alpha-bungarotoxin binding to human SK-N-SH cell-membranes (Ki 0.17 microM) and rat PC12 cell-membranes (Ki 0.45 microM). GTS-21 also displaced [125I]alpha-bungarotoxin binding to PC12 cell membranes with high potency (Ki 0.31 microM), but was much less potent in this regard in SK-N-SH cells (23 microM). 4OH-GTS-21 produced less residual inhibi...
Journal of Biological Chemistry, 1999
We have investigated the molecular determinants responsible for ␣-bungarotoxin (␣Bgtx) binding to nicotinic acetylcholine receptors through chimeric analysis of two homologous ␣ subunits, one highly sensitive to ␣Bgtx block (␣1) and the other, ␣Bgtx-insensitive (␣3). By replacing rat ␣3 residues 184-191 with the corresponding region from the Torpedo ␣1 subunit, we introduced a cluster of five ␣1 residues (Trp-184, Trp-187, Val-188, Tyr-189, and Thr-191) into the ␣3 subunit. Functional activity and ␣Bgtx sensitivity were assessed following co-expression in Xenopus oocytes of the chimeric ␣3 subunit (␣3/␣1[5]) with either rat 2 or 4 subunits. Agonist-evoked responses of ␣3/␣1[5]-containing receptors were blocked by ␣Bgtx with nanomolar affinity (IC 50 values: 41 nM for ␣3/␣1[5]2 and 19 nM for ␣3/␣1[5]4). Furthermore, receptors containing the single point mutation ␣3K189Y acquire significant sensitivity to ␣Bgtx block (IC 50 values: 186 nM for ␣3K189Y2 and 179 nM for ␣3K189Y4). Another ␣3 chimeric subunit, ␣3/␣7[6], similar to ␣3/␣1[5] but incorporating the corresponding residues from the ␣Bgtx-sensitive ␣7 subunit, also conferred potent ␣Bgtx sensitivity to chimeric receptors when co-expressed with the 4 subunit (IC 50 value ؍ 31 nM). Our findings demonstrate that the residues between positions 184 and 191 of the ␣Bgtx-sensitive subunits ␣1 and ␣7 play a critical functional role in the interaction of ␣Bgtx with nicotinic acetylcholine receptors sensitive to this toxin.