AE Succinimide, an Analogue of Methyllycaconitine, When Bound Generates a Nonconducting Conformation of the α4ß2 Nicotinic Acetylcholine Receptor.

Nicotinic acetylcholine (nACh) receptors are pentameric ligand-gated ion channels that mediate fast synaptic transmission. The α4ß2 nACh receptor is highly expressed in the brain and exists in two functional stoichiometries: the (α4)2(ß2)3 and (α4)3(ß2)2 that differ by an ACh-binding site at the α4-α4 interface of (α4)3(ß2)2 receptors. Methyllycaconitine (MLA) is an nACh receptor antagonist, and while potent at both α7 and α4ß2 nACh receptors, it has a higher selectivity for the α7 nACh receptor. The anthranilate-succinimide ester side-chain is important for its activity and selectivity. Here we identify a simplified MLA analogue that contains only the A and E ring skeleton of MLA, AE succinimide, that binds close to the channel lumen to display insurmountable inhibition at α4ß2 nACh receptors. Although inhibition by AE succinimide was found to be voltage-dependent indicating a possible pore channel blocker, substituted-cysteine accessibility experiments indicated it did not bind between 2'-16' region of the channel pore. Instead, we found that upon binding and in the presence of ACh, there is a conformational change to the channel membrane that was identified when the compound was assessed against (α4 V13'C)ß2 nACh receptors. It was found that in the 3:2 stoichiometry the two adjacent α4 subunits containing 13' cysteine mutations formed a disulfide bond and occluded ion conductance. This was reversed by treatment with the reducing agent, dithiothreitol. Thus, AE succinimide has a different mechanism of inhibition to both MLA and other AE analogues, such as AE bicyclic alcohol, in that upon binding to an as yet unidentified site, AE succinimide in the presence of ACh induces a conformational change to the channel that generates a ligand-bound closed state.

Covalent attachment of antagonists to the α7 nicotinic acetylcholine receptor: synthesis and reactivity of substituted maleimides.

The 3-methylmaleimide congeners of the natural product methyllycaconitine (MLA) and an analogue covalently attach to functional cysteine mutants of the α7 nicotinic acetylcholine receptor (nAChR).

Identifying the binding site of novel methyllycaconitine (MLA) analogs at α4ß2 nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels that mediate fast synaptic transmission. Methyllycaconitine (MLA) is a selective and potent antagonist of the α7 nAChR, and its anthranilate ester side-chain is important for its activity. Here we report the influence of structure on nAChR inhibition for a series of novel MLA analogs, incorporating either an alcohol or anthranilate ester side-chain to an azabicyclic or azatricyclic core against rat α7, α4ß2, and α3ß4 nAChRs expressed in Xenopus oocytes. The analogs inhibited ACh (EC(50)) within an IC(50) range of 2.3-26.6 µM. Most displayed noncompetitive antagonism, but the anthranilate ester analogs exerted competitive behavior at the α7 nAChR. At α4ß2 nAChRs, inhibition by the azabicyclic alcohol was voltage-dependent suggesting channel block. The channel-lining residues of α4 subunits were mutated to cysteine and the effect of azabicyclic alcohol was evaluated by competition with methanethiosulfonate ethylammonium (MTSEA) and a thiol-reactive probe in the open, closed, and desensitized states of α4ß2 nAChRs. The azabicyclic alcohol was found to compete with MTSEA between residues 6' and 13' in a state-dependent manner, but the reactive probe only bonded with 13' in the open state. The data suggest that the 13' position is the dominant binding site. Ligand docking of the azabicyclic alcohol into a (α4)(3)(ß2)(2) homology model of the closed channel showed that the ligand can be accommodated at this location. Thus our data reveal distinct pharmacological differences between different nAChR subtypes and also identify a specific binding site for a noncompetitive channel blocker.

Double-Mannich annulation of cyclic ketones using N,N-Bis(ethoxymethyl)alkylamine reagents.

[Structure: see text] N,N-Bis(ethoxymethyl)alkylamines function as effective reagents in the double-Mannich annulation of cyclic ketone substrates, providing efficient access to a series of azabicyclic ketones. These ring systems are a useful scaffold for the four-step synthesis of novel constrained homocholine analogues.