Structural differences determine the relative selectivity of nicotinic compounds for native alpha 4 beta 2*-, alpha 6 beta 2*-, alpha 3 beta 4*- and alpha 7-nicotine acetylcholine receptors

Abstract

Mammalian brain expresses multiple nicotinic acetylcholine receptor (nAChR) subtypes that differ in subunit composition, sites of expression and pharmacological and functional properties. Among known subtypes of receptors, alpha 4 beta 2* and alpha 6 beta 2*-nAChR have the highest affinity for nicotine (where * indicates possibility of other subunits). The alpha 4 beta 2*-nAChRs are widely distributed, while alpha 6 beta 2*-nAChR are restricted to a few regions. Both subtypes modulate release of dopamine from the dopaminergic neurons of the mesoaccumbens pathway thought to be essential for reward and addiction. alpha 4 beta 2*-nAChR also modulate GABA release in these areas. Identification of selective compounds would facilitate study of nAChR subtypes. An improved understanding of the role of nAChR subtypes may help in developing more effective smoking cessation aids with fewer side effects than current therapeutics. We have screened a series of nicotinic compounds that vary in the distance between the pyridine and the cationic center, in steric bulk, and in flexibility of the molecule. These compounds were screened using membrane binding and synaptosomal function assays, or recordings from GH4C1 cells expressing h alpha 7, to determine affinity, potency and efficacy at four subtypes of nAChRs found in brain, alpha 4 beta 2*, alpha 6 beta 2*, alpha 7 and alpha 3 beta 4*. In addition, physiological assays in gain-of-function mutant mice were used to assess in vivo activity at alpha 4 beta 2* and alpha 6 beta 2*-nAChRs. This approach has identified several compounds with agonist or partial agonist activity that display improved selectivity for alpha 6 beta 2*-nAChR.

Figure 1. Structures of compounds assayed

For Compound 1, the methylpyrrolidine ring structure of nicotine was replaced by azabicyclo[2.2.2]octane. The other compounds are related to Compound 1 in the following ways: the azabicyclo structure was modified in size to [1.2.2]heptane for Compound 2, to [3.2.2]nonane for Compound 3, and to [2.3.3.5]decane for Compound 4. An additional C was added in spacer between ring systems to generate Compound 5. An additional C as well as a 5′ halogen group were added to generate Compound 11. Additional bulky groups were added at the 5′ position of the pyridine ring for Compounds 6 and 7. The methylpyrrolidine ring of nicotine was opened up and lengthened to have 6 C between the Ns for Compound 8 and, in addition, a 5′ bulky group added for Compound 9. For Compound 10, changes include 5′ halogen groups as well as alternate ways of adding more space between the Ns.

Figure 2. Inhibition of membrane binding to assess K_i values at various subtypes of nAChR

Panel A: Inhibition of binding to α4β2*-nAChR by Compound 1. High affinity [¹²⁵I]-epibatidine binding (at 200 pM) in cortical membranes, was inhibited by 11 concentrations of Compound 1 from 0.01 nM to 1000 nM; data points are means ± sem for 6 experiments. IC₅₀ = 1.17 ± 0.14 nM (Ki = 0.46 ± 0.06 nM). Panel B: Inhibition of binding to α3β4*-nAChR by Compound 1. The data were gathered with membranes prepared from IPN, a region high in α3β4*-nAChR, using [¹²⁵I]-epibatidine with A-85380 added to block binding to β2*-nAChR. Data points are means ± sem for 3 experiments. IC₅₀ = 12.44 ± 2.20 nM (Ki = 4.4 ± 3.6 nM). Panel C: Inhibition of binding to α7-nAChR by Compound 1. [¹²⁵I]-α-bungarotoxin binding to membranes from HP was inhibited by various concentrations of Compound 1. Data points are means ± sem for 4 experiments. IC₅₀ = 30.81 ± 8.39 nM (Ki = 7.6 ± 1.9 nM). Panel D: Inhibition of binding to α6β2*-nAChR by Compound 1. [¹²⁵I]-α-CtxMII binding to membranes of combined ST, OT and SC, areas high in α6*-nAChR, was inhibited by 9 concentrations of Compound 1. Data points are means ± sem for 3 experiments. IC₅₀ = 1.9 ± 0.6 nM (Ki = 1.14 ± 0.35 nM).

Figure 3. Functional assays for agonist activity of Compound 1 at various subtypes of nAChR

Panel A: Function of α4β2*-nAChR measured by high sensitivity ⁸⁶Rb⁺ efflux from thalamic synaptosomes. EC₅₀ values were determined by either high affinity portion of a 2-site fit of data without DHβE (37 ± 25 nM), or by subtraction of the DHβE-resistant activity (inset) fit to a single site (43 ± 25 nM). Panel B: Measurement of function at α4β2*-nAChR by α-CtxMII-resistant [³H]-dopamine release and α6β2*-nAChR by α-CtxMII-sensitive [³H]-dopamine release from striatal synaptosomes. EC₅₀ values by curve fit, 34 ± 7 nM and 7.4 ± 1.3 nM, respectively. Panel C: Measurement of function at α3β4*-nAChR by [³H]ACh release from IPN synaptosomes. EC₅₀ values by curve fit, 430 ± 190 nM. Panel D: Measurement of function at α7*-nAChR by relative peak current in GH4C1 cells. EC₅₀ values by curve fit, 660 ± 370 nM. All data shown are means ± sem from 4 experiments.

Figure 4. Comparison of parameters determined by in vitro assays

Panel A compares affinity for four subtype classes of nAChRs for a number of commonly studied nicotinic compounds, varenicline, and the 11 compounds shown in Figure 1. K_i values for inhibition of various selective binding assays are plotted. Dotted lines indicate 1 nM and 1 μM. Data for commonly studied nicotinic compounds are from Whiteaker et al, 2000a; Marks et al, 1986, , ; Salminen et al, 2005; unpublished data NBF, MJM. Panel B compares potency of compounds for activation (EC₅₀ values) or inhibition (K_i values) of four subtype classes of nAChR. Dotted lines indicate 1 nM and 1 μM. Data for commonly studied nicotinic compounds are from Salminen et al, 2004; Grady et al, 2001; Marks et al, 1999; unpublished data NBF, MJM. Panel C compares efficacy for compounds as compared to nicotine for activation or inhibition of four subtype classes of nAChR. Dotted lines indicate efficacy values of 100% (equal to nicotine), 0 efficacy (no functional activity), and -100% (full antagonism). Data for commonly studied nicotinic compounds are from Salminen et al, 2004; Grady et al, 2001; Marks et al, 1999; unpublished data NBF, MJM.

Figure 5. Correlations of independent methods for assessing affinity and potency

Panel A: Comparison of potency (EC₅₀ values) for stimulating α4β2*nAChR by measurement of ⁸⁶Rb⁺ efflux from thalamic synaptosomes vs. stimulating [³H]-dopamine release resistant to α-CtxMII from striatal synaptosomes. The calculated slope is 0.98 ± 0.08, r = 0.96, and the mean ratio of the points (0.97 ± 0.14, mean ± sem, x/y) does not differ from 1. Panel B: Comparison of inhibition constants (Ki values, M) for nicotinic compounds measured by inhibition of [¹²⁵I]-αCtxMII binding to membranes prepared from mouse striatum, olfactory tubercle and superior colliculus vs. constants measured by inhibition of [¹²⁵I]-epibatidine binding to membranes prepared from striata and olfactory tubercles of α4 subunit null mutant mice. The calculated slope is 1.08 ± 0.08, r = 0.96, and the mean ratio of the points (0.88 ± 0.13, mean ± sem, x/y) does not differ from 1.

Figure 6. Physiological assays for hypothermia in α4L9'A mice and locomotor activation in α6L9'S mice by Compound 1

Panel A: Hypothermia measurements in α4L9'A mice to assay Compound 1 bioavailability and in vivo activity at α4β2* nAChRs. An averaged (n = 6 mice) whole-body temperature response in α4L9'A and WT control mice in response to an i.p. injection of Compound 1 (0.03 mg/kg) is shown. Panel B: Locomotor activation assay in α6L9'S mice to assess bioavailability and in vivo activity at α6β2* nAChRs. Average locomotor activity (n = 8 mice) for α6L9'S and WT control mice is shown in response to an i.p. injection of Compound 1 (0.01 mg/kg). All data shown are means ± SEM.