David J. Hardick

Characterisation of the binding of [3H]methyllycaconitine:a new radioligand for labelling α7-type neuronal nicotinic acetylcholine receptors

Methyllycaconitine (MLA), a norditerpenoid alkaloid isolated from Delphinium seeds, is one of the most potent non-proteinacious ligands that is selective for alpha bungarotoxin-sensitive neuronal nicotinic acetylcholine receptors (nAChR). [3H]MLA bound to rat brain membranes with high affinity (Kd = 1.86 +/- 0.31 nM) with a good ratio of specific to non-specific binding. The binding of [3H]MLA was characterised by rapid association (t 1/2 = 2.3 min) and dissociation (t 1/2 = 12.6 min) kinetics. The radioligand binding displayed nicotinic pharmacology, consistent with an interaction with alpha bungarotoxin-sensitive nAChR. The snake alpha-toxins, alpha bungarotoxin and alpha cobratoxin, displaced [3H]MLA with high affinity (Ki = 1.8 +/- 0.5 and 5.5 +/- 0.9 nM, respectively), whereas nicotine was less potent (Ki = 6.1 +/- 1.1 microM). The distribution of [3H]MLA binding sites in crudely dissected rat brain regions was identical to that of [125I] alpha bungarotoxin binding sites, with a high binding site density in hippocampus and hypothalamus, but low density in striatum and cerebellum. [3H]MLA also labelled a sub-population of binding sites which are not sensitive to the snake alpha toxins, but which did not differ significantly from the major population with respect to their other pharmacological properties or regional distribution. [3H]MLA, therefore, is a novel radiolabel for characterising alpha 7-type nAChR. A good signal to noise ratio and rapid binding kinetics provide advantages over the use of radiolabelled alpha bungarotoxin for rapid and accurate equilibrium binding assays.

Conversion of the sodium channel activator aconitine into a potent α7-selective nicotinic ligand

Methyllycaconitine (MLA) is a competitive antagonist of nicotinic acetylcholine receptors, with a remarkable preference for neuronal [125I]αBgt binding sites. We have begun to investigate the structural basis of its potency and subtype selectivity. MLA is a substituted norditerpenoid alkaloid linked to a 2‐(methylsuccinimido)benzoyl moiety. Hydrolysis of the ester bond in MLA to produce lycoctonine diminished affinity for rat brain [125I]αBgt binding sites 2500‐fold and abolished affinity for [3H]nicotine and muscle [125I]αBgt binding sites. The voltage‐gated Na+ channel activator aconitine, also a norditerpenoid alkaloid, but with significant structural differences from lycoctonine, displayed comparable weak or absent nicotinic activity. Addition of a 2‐(methylsuccinimido)benzoyl sidechain to O‐demethylated aconitine, to mimic MLA, abolished Na+ channel activation and conferred nanomolar affinity for brain [125I]αBgt binding sites, comparable to that of MLA. We propose that the ester‐linked 2‐(methylsuccinimido)benzoyl group is necessary for nicotinic potency, but α7 selectivity resides in the norditerpenoid core of the molecule.

Rapid and efficient isolation of the nicotinic receptor antagonist methyllycaconitine from delphinium:assignment of the methylsuccinimide absolute stereochemistry as S

Abstract Methyllycaconitine (MLA) has been isolated from Garden Hybrid Delphinium and purified by vacuum liquid chromatography. 13 C NMR and optical rotation have been used to characterize the absolute configuration of the methylsuccinimide moiety as S . Ligand binding assays confirmed the potency of MLA and its selectivity for α-bungarotoxin-sensitive neuronal nicotinic acetylcholine receptors.

Acylation of lycoctonine: Semi-synthesis of inuline, delsemine analogues and methyllycaconitine

Abstract Lycoctonine has been acylated to afford sequentially inuline, delsemine analogues and methyllycaconitine using isatoic anhydride followed by S -(−)-methylsuccinic anhydride. This protocol is a rapid, facile method for the regiospecific introduction of the anthranilate ester moiety found in potent nicotinic acetylcholine receptor antagonists.

Regioselective anthranoylation of demethylated aconitine: Novel analogues of aconitine inuline and methyllycaconitine

Abstract The regioselective acylation of 18- O -desmethylaconitine has been achieved with isatoic anhydride to afford the corresponding anthranilate ester, an analogue of inuline. The aniline nitrogen was then incorporated into an S -methylsuccinimide moiety, to afford a novel methyllycaconitine/aconitine hybrid alkaloid.

[3H]-Methyllycaconitine: a high affinity radioligand that labels invertebrate nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChR) of insect and other invertebrates are heterogeneous and new tools are needed to dissect their multiplicity. [(3)H]-Methyllycaconitine ([(3)H]-MLA) is a novel radioligand which is a potent antagonist at vertebrate alpha7-type nAChR. Putative invertebrate nAChR of the aphid Myzus persicae, the moths Heliothis virescens and Manduca sexta, the fly Lucilia sericata, and the squid Loligo vulgaris were investigated in radioligand binding studies with [(3)H]-MLA. Saturable binding was consistent with a single class of high affinity binding sites for each of these invertebrates, characterised by a dissociation constant, K(d), of approximately 1 nM and maximal binding capacities, B(max), between 749 and 1689 fmol/mg protein for the insects and 14,111 fmol/mg protein for squid. [(3)H]-MLA binding to M. persicae membranes was characterised in more detail. Kinetic analysis demonstrated rapid association in a biphasic manner and slow, monophasic dissociation. Displacement studies demonstrate the nicotinic character of [(3)H]-MLA binding sites. Data for all nicotinic ligands, except MLA itself, are consistent with displacement from a high and a low affinity site, indicating that displacement is occurring from two or more classes of nicotinic binding site that are not distinguished by MLA itself. Autoradiographic analysis of the distribution of [(3)H]-MLA binding sites in Manduca sexta shows discrete labelling of neuropil areas of the optic and antennal lobes.

Regioselective demethylation of aconitine

Abstract The regioselective demethylation of aconitine has been achieved with Lewis acids. Me 3 SiI afforded first 18- O -desmethyl- and then 16,18-di- O -desmethylaconitine, but AlCl 3 /NaI gave 16- O -desmethylaconitine first. The acetate and benzoate esters survived throughout these procedures. Aconitine decomposed on treatment with BBr 3 .