AT-1001: a high affinity and selective α3β4 nicotinic acetylcholine receptor antagonist blocks nicotine self-administration in rats

Abstract

Genomic and pharmacologic data have suggested the involvement of the α3β4 subtype of nicotinic acetylcholine receptors (nAChRs) in drug seeking to nicotine and other drugs of abuse. In order to better examine this receptor subtype, we have identified and characterized the first high affinity and selective α3β4 nAChR antagonist, AT-1001, both in vitro and in vivo. This is the first reported compound with a Ki below 10 nM at α3β4 nAChR and >90-fold selectivity over the other major subtypes, the α4β2 and α7 nAChR. AT-1001 competes with epibatidine, allowing for [³H]epibatidine binding to be used for structure-activity studies, however, both receptor binding and ligand-induced Ca²⁺ flux are not strictly competitive because increasing ligand concentration produces an apparent decrease in receptor number and maximal Ca²⁺ fluorescence. AT-1001 also potently and reversibly blocks epibatidine-induced inward currents in HEK cells transfected with α3β4 nAChR. Importantly, AT-1001 potently and dose-dependently blocks nicotine self-administration in rats, without affecting food responding. When tested in a nucleus accumbens (NAcs) synaptosomal preparation, AT-1001 inhibits nicotine-induced [³H]dopamine release poorly and at significantly higher concentrations compared with mecamylamine and conotoxin MII. These results suggest that its inhibition of nicotine self-administration in rats is not directly due to a decrease in dopamine release from the NAc, and most likely involves an indirect pathway requiring α3β4 nAChR. In conclusion, our studies provide further evidence for the involvement of α3β4 nAChR in nicotine self-administration. These findings suggest the utility of this receptor as a target for smoking cessation medications, and highlight the potential of AT-1001 and congeners as clinically useful compounds.

Figure 1

Chemical structure of AT-1001.

Figure 2

Inhibition of [³H]epibatidine binding to α3β4-containing HEK cell membranes by AT-1001. Scatchard analysis was plotted from saturation binding to HEK cell membranes, using [³H]epibatidine concentrations ranging from 0.04 to 3.2 nM. Saturation experiments were conducted in the presence of 0 (▪) 3 nM (▴), 10 nM (▾), and 100 nM (⧫) AT-1001. Data are from a single experiment repeated twice with similar results. Kd and Bmax values were: 0.32 nM, 12 620 fmol/mg protein; 0.48 nM, 11 700 fmol/mg protein; 0.66 nM, 9723 fmol/mg protein; and 1.40 nM, 6320 fmol/mg protein for Scatchard analysis in the presence of 0, 3, 10, and 100 nM AT-1001, respectively. Therefore, increasing concentrations of AT-1001 show a decrease in Bmax and increase in Kd, suggesting non-competitive, or mixed inhibition of binding. As inhibition was not strictly competitive, a pA2 value could not be determined.

Figure 3

Inhibition of epibatidine-induced Ca²⁺ fluorescence by AT-1001 in HEK cells containing α3β4 nAChR. Ca²⁺ fluorescence induced by increasing concentrations of epibatidine was measured as described in Materials and methods section. Epibatidine dose-response experiments conducted in the presence of 0 (▪), 3 nM (▴), 30 nM (▾), and 300 nM (♦) of AT1001 induced a rightward shift in EC50 and progressive decrease in maximal effect suggesting a non-competitive mode of inhibition. Data shown are mean±SD from a single experiment conducted in quadruplicate that was repeated with similar results.

Figure 4

Patch clamp recording in α3β4-containing HEK cells. Sample trace of the blocking effect of 10 nM AT-1001 against 10 nM Epibatidine activation (66% block, n=3). Whole-cell voltage-clamp recordings were made using Axon 700A and PClamp 8.2. Experiments were conducted within 24–72 h after replating the stable cells. Cells were clamped at –60 mV and drugs were delivered via local rapid perfusion for 500 ms, as described in Materials and methods section.

Figure 5

Inhibition of nicotine-induced [³H]DA release from nucleus accumbens synaptosomes. Synaptosomes were perfused with buffer and then 10 μM nicotine was added as indicated by the vertical line at fraction 4. Nicotine-induced [³H]DA release was inhibited completely by 10 μM mecamylamine (a) and to a much lesser extent by 10 μM AT-1001 (b). Data shown are mean±SD from a single experiment conducted in triplicate that was repeated with similar results.

Figure 6

Inhibition of nicotine-induced [³H]DA release by nAChR antagonists. Data shown represents mean inhibition±SD from three experiments conducted in triplicate. Mecamylamine and conotoxin MII significantly inhibited [³H]DA at both 1 and 10 μM while DHβE and AT-1001 inhibited release only at 10 μM.

Figure 7

AT-1001 reduces nicotine self-administration in rats. (a) Acute effects of AT-1001 treatment. Each data point is the mean±SEM number of rewards earned in the 1-h session following AT-1001 treatment. Filled bars represent animals responding for nicotine (30 μg/kg/inj, left axis), solid bars are animals responding for food pellets (45 mg pellets, right axis). AT-1001 doses (mg/kg, s.c.) are indicated on the x axis. ^*p<0.05 vs vehicle, ^*** p<0.001 vs vehicle. (b) AT-1001 (3 mg/kg) does not have long-lasting effects. Base is the number of reinforced responses on the day before AT-1001 treatment. Day 1 is the number of reinforced responses on the test day of AT-1001 (3 mg/kg) treatment, while day 2 is the day following the testday. ^*p<0.05 vs base and day 2. n=6 per group.