Discovery, Synthesis, and Structure Activity of a Highly Selective α7 Nicotinic Acetylcholine Receptor Antagonist

Abstract

Nicotinic acetylcholine receptors (nAChRs) that contain an alpha7 subunit are widely distributed in neuronal and nonneuronal tissue. These receptors are implicated in the release of neurotransmitters such as glutamate and in functions ranging from thought processing to inflammation. Currently available ligands for alpha7 nAChRs have substantial affinity for one or more other nAChR subtypes, including those with an alpha1, alpha3, alpha6, and/or alpha9 subunit. An alpha-conotoxin gene was cloned from Conus arenatus. Predicted peptides were synthesized and found to potently block alpha3-, alpha6-, and alpha7-containing nAChRs. Structure-activity information regarding conotoxins from distantly related Conus species was employed to modify the C. arenatus derived toxin into a novel, highly selective alpha7 nAChR antagonist. This ligand, alpha-CtxArIB[V11L,V16D], has low nanomolar affinity for rat alpha7 homomers expressed in Xenopus laevis oocytes, and antagonism is slowly reversible. Kinetic analysis provided insight into the mechanism of antagonism. alpha-CtxArIB interacts with five ligand binding sites per alpha7 receptor, and occupation of a single site is sufficient to block function. The peptide was also shown to be highly selective in competition binding assays in rat brain membranes. alpha-CtxArIB[V11L,V16D] is the most selective ligand yet reported for alpha7 nAChRs.

Efficient Binding of 4/7 α-Conotoxins to Nicotinic α4β2 Receptors Is Prevented by Arg185 and Pro195 in the α4 Subunit

“…The subtype selectivity of several ␣-conotoxins could be further optimized, yielding analogs that are able to differentiate even between closely related nicotinic receptor subtypes. These include ␣-conotoxins with selectivity for ␣6-containing nAChRs (MII[S4A;E11A;L15A]), ␣6␤4* nAChRs (BuIA[T5A;P6O]), and ␣7 nAChRs (ArIB[V11L;V16D]) (Whiteaker et al, 2007;Azam et al, 2008Azam et al, , 2010. Apparently, conotoxins selective for mammalian ␣4␤2 interfaces have not evolved in cone snails or up to now escaped discovery.…”

Functional Characterization of α9-Containing Cholinergic Nicotinic Receptors in the Rat Adrenal Medulla: Implication in Stress-Induced Functional Plasticity

“…However, ␣-Bgt antagonizes not only ␣7 nAChRs (IC 50 1.6 nM) (Ló pez et al, 1998), but also ␣9 and ␣9/␣10 nAChRs (IC 50 2.1 and 14 nM, respectively) (Sgard et al, 2002). Although other data support the presence of ␣7 nAChRs in adrenal chromaffin cells (Rust et al, 1994;Criado et al, 1997;Mousavi et al, 2001), their contribution to synaptic transmission merits reexamination by using, for example, the highly selective ␣7 nAChRantagonist␣-conotoxinArIB[V11L, V16D] (Whiteaker et al, 2007).…”

Conus Venom Peptide Pharmacology

“…Residues involved in MII binding on the ␣3␤2 nAChR have been identified, and these include Lys185 and Ile188 on ␣3, and Thr59, Val109, Phe117, and Leu119 on the ␤ subunit, allowing a model of the interaction to be built (Harvey et al, 1997;Dutertre et al, 2005). More recently, ArIB, which blocks both ␣7 and ␣3␤2 nAChR, was compared with other ␣-conotoxin sequences and rationally modified to increase ␣7 nAChR selectivity (Whiteaker et al, 2007). This structurefunction analysis yielded two analogs, V11L,V16A-ArIB and V11L,V16D-ArIB, which had low affinity for ␣3␤2 but retained ␣7 nAChR activity as predicted, with an iodinated form developed as a pharmacological tool (Whiteaker et al, 2008).…”