Functional Characterization of AT-1001, an α3β4 Nicotinic Acetylcholine Receptor Ligand, at Human α3β4 and α4β2 nAChR

Abstract

Introduction: Genome-wide association studies linking the α3, β4, and α5 nicotinic acetylcholine receptor (nAChR) subunits to nicotine dependence suggest that α3β4* nAChR may be targets for smoking cessation pharmacotherapies. We previously reported that AT-1001, a selective α3β4* nAChR ligand binds with high affinity to rat α3β4 and human α3β4α5 nAChR, antagonizes epibatidine-induced activation of rat α3β4 nAChR in HEK cells and potently inhibits nicotine selfadministration in rats. Methods: Two-electrode voltage clamp was used for functional characterization of AT-1001 at recombinant human α3β4 and α4β2 nAChR expressed in Xenopus oocytes. Results: Concentration-response curves show that AT-1001 is a partial agonist at human α3β4 nAChR, evoking up to 35% of the maximal acetylcholine (ACh) response (50% effective concentration [EC 50 ] = 0.37 μM). AT-1001 showed very little agonist activity at the α4β2 nAChR, evoking only 6% of the ACh response (EC 50 = 1.5 μM). Pre-and co-application of various concentrations of AT-1001 with 50 μM ACh revealed a complex pattern of activation-inhibition by AT-1001 at α3β4 nAChR, which was best fitted by a 2-site equation. At α4β2 nAChR, co-exposure of AT-1001 with ACh only showed inhibition of ACh current with a shallower curve. Conclusions: AT-1001 is a partial agonist at the human α3β4 nAChR and causes desensitization at concentrations at which it evokes an inward current, resulting in an overall functional antagonism of α3β4 nAChR. AT-1001 does not significantly activate or desensitize α4β2 nAChR at the same concentrations as at the α3β4 nAChR, but does inhibit ACh responses at α4β2 nAChR at higher concentrations. A combination of these mechanisms may underlie the inhibition of nicotine selfadministration by AT-1001, suggesting that AT-1001 and compounds from this class may have clinical potential for smoking cessation pharmacotherapy.

AT-1001 Is a Partial Agonist with High Affinity and Selectivity at Human and Ratα3β4 Nicotinic Cholinergic Receptors

“…Our results confirm earlier studies that AT-1001 binds with high affinity and selectivity to a3b4 nAChRs (Toll et al, 2012) and that it displays partial agonist activity (Zaveri et al, 2015). In HEK cells heterologously expressing the rat versions of the a3b4 and a4b2 nAChR subtypes, we found that AT-1001 displays 41-fold selectivity for the a3b4 subtype, which also is in general agreement with the initial report (Toll et al, 2012).…”

“…The difference between the efficacies found in these two types of assays may be related to receptor desensitization, which is more evident during the much longer stimulation period required in the 86 Rb 1 efflux assay than in patch-clamp measurements (see below). In our patch-clamp measurements of human a3b4 nAChRs expressed in HEK cells, AT-1001 displayed a potency of 0.4 mM, which is nearly identical to the potency found in a recent study in Xenopus oocytes expressing human a3b4 nAChRs (Zaveri et al, 2015). The potency of AT-1001 was 3.5-fold higher at the human than the rat a3b4 receptor in patchclamp measurements and 15-fold higher in 86 Rb 1 efflux assays.…”

“…Recently, a new compound, AT-1001 [N-(2-bromophenyl)-9-methyl-9-azabicyclo [3.3.1] nonan-3-amine], was found to be a selective, high-affinity ligand at rat a3b4 nAChRs (Toll et al, 2012;Zaveri et al, 2015). Furthermore, AT-1001 was reported to be a potent antagonist or partial agonist at a3b4 nAChRs (Toll et al, 2012;Zaveri et al, 2015), and it effectively reduced nicotine self-administration in rats (Toll et al, 2012).…”

“…Furthermore, AT-1001 was reported to be a potent antagonist or partial agonist at a3b4 nAChRs (Toll et al, 2012;Zaveri et al, 2015), and it effectively reduced nicotine self-administration in rats (Toll et al, 2012). Rat and human nAChR subunits are, in general, very similar, but an important difference between the two species is found within the extracellular D loop of the b4 subunits (Young et al, 2007).…”

How Does Nicotine Take You Captive? Neurobiology of Tobacco Addiction

“…α4β2 insan beyninde en çok bulunan reseptör tipidir ve nikotin bağımlı-lığında anahtar rolü oluşturduğu düşünülmektedir. α4β2 agonisti olan vareniklin, sistisin, dianiklin gibi moleküller nAChR'lerini duyarsız hale getirir (2,8) . β2 alt ünite geni çıkarılan farelerin nikotin alma isteklerinin kaybolduğu, bu genin ventral tegmental alanda (VTA) tekrar sağlandığında nikotin aşermelerinin geri geldiği gösterilmiştir (9) .…”