Neuronal Nicotinic Acetylcholine Receptors

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Nicotinic acetylcholine receptors (nAChRs) consist of transmembrane proteins of pentameric structure and are members of a superfamily of ligand-gated ion channels, which include 5-hydroxytryptamine₃, glycine, and several -aminobutyric acid receptors. nAChRs are widely distributed throughout the body, modulating the function of the CNS, peripheral nervous system, cardiovascular and immune systems (Sargent, 1993). In the CNS, nAChRs are located mainly presynaptically, modulating synaptic activity by regulation of neurotransmitter release (Wonnacott, 1997). Remarkable subtype diversity exists among nAChRs, although the exact subunit composition, stoichiometry and arrangement of native nAChRs remains to be conclusively determined (Lukas et al., 1999). The most abundant native nAChRs in the CNS are believed to be 42 heteromeric receptors and 7 homomeric receptors. Results from [³H]nicotine binding, affinity labeling, immunoprecipitation, in situ hybridization, and gene knockout studies indicate that the 42 subtype represents the high-affinity nicotine binding site in the CNS (Marks et al., 1986; Whiting and Lindstrom, 1988; Wada et al., 1989; Picciotto et al., 1998). The 42 receptor subtype contains two ligand binding domains located at the interface of each / subunit (Lippiello, 1989). The  subunit is also believed to be an important contributor to nAChR antagonist sensitivity (Cachelin and Rust, 1995). The 7 subtype contains five ligand binding domains with potentially different affinities, and represents the major -bungarotoxin (BTX) binding site in the CNS (Orr-Urtreger et al., 1997; Rakhilin et al., 1999). The 32* receptor subtype is less prevalent in brain and is at least in part responsible for mediating DA release, although other receptor subtypes may also be involved (Crooks et al., 1995; Kulak et al., 1997; Charpantier et al., 1998).

A significant limitation in drug discovery and pharmacophore development is the current putative assignment of the specific subunit composition of native nAChRs. The pharmacological specificity for each receptor subunit combination most likely results from a unique topography at the ligand binding site. Thus, topographical variations arising from differences in amino acid sequence and from specific subunit combinations result in receptor diversity, affording the opportunity for subtype selectivity for both nAChR agonists and antagonists. Recently, exciting developments in drug discovery have focused on nAChR agonists as therapeutic agents for diseases such as cognitive dysfunction, neurodegeneration, and other CNS pathologies (Holladay et al., 1997; Lloyd and Williams, 2000). However, relatively little attention has focused on the development of nAChR antagonists as potential drug candidates (Dwoskin et al., 2000).

	Nicotinic Receptor Antagonists

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The development of selective nAChR antagonists is a relatively new field, requiring the use of functional assays to distinguish between agonist and antagonist activity and radioligand binding assays. To characterize a drug candidate as a nAChR antagonist, the drug must by definition have no intrinsic activity, i.e., no agonist efficacy at the targeted nAChR. However, receptor binding affinity and lack of intrinsic efficacy are not sufficient to establish an antagonist mechanism of action. Moreover, the drug must inhibit the response elicited by an appropriate nAChR agonist. Depending upon the functional assay used, the apparent affinity of the drug for a specific nAChR may not reflect its true affinity for the binding site on the respective nAChR protein. Competitive antagonists interact with the acetylcholine (ACh) binding site at the / subunit interface of heteromeric nAChRs, or at the / subunit interface of homomeric nAChRs, preventing allosteric transition to the open channel state. Variations in the topography and geometry of the nAChR subunit interfaces of the various subunit combinations constitute the basis for development of competitive, subtype-selective ligands.

In comparison with structure-activity relationships (SARs) of nAChR agonists, correlations of the SAR of nAChR antagonists have not been carried out, and structural similarities between many well known nAChR antagonists are not apparent. Thus, pharmacophore geometries for antagonist binding sites are ill-defined. In addition, most of the known antagonists are not nAChR subtype-selective. Previous hypotheses proposed that competitive antagonists interact with the same binding site as agonists, but via a different mode of interaction, i.e., agonists and antagonists bind differently to the different quaternary states of the nAChR receptor protein (Sheridan et al., 1986). Sheridan proposed that antagonists, although containing the appropriate agonist pharmacophore geometry, are usually large molecules that interact with the agonist binding site and extend outside the normal agonist volume. Thus, this extended area of antagonist interaction prevents the agonist-induced conformational change in the receptor protein that produces channel opening. However, the latter hypothesis is based on a simplified model involving a single pharmacophore. The complexity arising from the existence of multiple nAChR subtypes and their different physiological functions necessitates the development of subtype-selective antagonists as effective therapeutic agents with minimal untoward side effects. The elucidation of the molecular structure and subunit composition of the binding sites of native nAChRs will be crucial for the rational development of nAChR subtype-selective antagonists.

Thus, the current limited understanding of native nAChR subtype composition is a major caveat in the drug development process. High-throughput technology in drug discovery is now routine in facilitating rapid drug development in the nAChR field. However, this technology is often used without knowledge of the specific subunit composition of the native nAChR target. In many cases, recombinant receptor expression assays are utilized for high-throughput evaluation, because of their obvious practical advantages. However, these advantages are offset by the possibility that the pharmacology of the drug candidate from nAChR expression assays may not be consistent with that obtained using native receptor assays. Thus, the selection of potential clinical candidates developed using expressed nAChRs should be considered only subsequent to their assessment in native nAChR assays.

	Competitive Nicotinic Receptor Antagonists

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Dihydo--erythroidine (DHE) and Related Compounds. DHE (Fig. 1, 1), an alkaloid found in seeds of Erythrina, has been widely used as a classical, nonselective, competitive nAChR antagonist. DHE has nanomolar affinity at both 42 and 32* receptor subtypes, inhibiting [³H]nicotine binding to and DA release from rat striatal slices (IC₅₀ = 30 nM in each assay; Crooks et al., 1995). DHE blocks the agonist response at rat recombinant 42 receptors with an IC₅₀ value of 370 nM (Harvey and Luetje, 1996). Furthermore, DHE is a relatively weak antagonist at 34* (ganglionic-like) and (1)₂1 (muscle-type) receptors expressed in PC12 and TE671 cells, respectively, and has ~10-fold greater selectivity for human 44 than for human 42 nAChRs (Chavez-Noriega et al., 1997). Rat 44 and 34 nAChRs differ in sensitivity to DHE by 120-fold (IC₅₀ = 0.19 and 23.1 µM, respectively; Harvey and Luetje, 1996). Erysodine (Fig. 1, 2), another Erythrina alkaloid structurally related to DHE, is a more potent inhibitor (K_i = 5 nM) of 42 nAChRs compared with DHE (K_i = 35 nM) in the [³H]cytisine binding assay (Decker et al., 1995a). Erysodine and DHE exhibit similar low affinity (K_i = 4 and 9 µM, respectively) for the 7 subtype, inhibiting ¹²⁵I-BTX binding to rat brain membranes. Erysodine is a competitive, reversible antagonist at 32* nAChRs, inhibiting nicotine-evoked (100 nM) DA release from superfused rat striatal slices (IC₅₀ = 58 nM, equipotent with DHE; Decker et al., 1995a). Schild analysis revealed that erysodine interacts competitively and at a single site on this nAChR. Erysodine also inhibits nicotine-evoked ⁸⁶Rb⁺ efflux from 34* nAChRs expressed by IMR32 cells (IC₅₀ = 7 nM), and is 10-fold more potent than DHE. Thus, both DHE and erysodine are potent nAChR antagonists but are not subtype-selective.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Structures of natural products that are neuronal nicotinic receptor antagonists.

-Lobeline (Lobeline). Lobeline (Fig. 1, 3), a lipophilic alkaloid from Lobelia, has many nicotine-like effects, and until recently was considered to be an agonist at nAChRs (Decker et al., 1995b). However, unlike nicotine, chronic lobeline administration does not up-regulate nAChRs (Bhat et al., 1991). Lobeline binds to 42 nAChRs in the low nanomolar range and is a potent inhibitor of [³H]DHE binding (Williams and Robinson, 1984; Teng et al., 1997). Lobeline also inhibits [³H]methyllycaconitine ([³H]MLA) binding (K_i = 7 µM) (L. P. Dwoskin and P. A. Crooks, unpublished results) to rat brain membranes, indicating an interaction with the 7 nAChR subtype. More recently, lobeline has been shown to inhibit nicotine-evoked (1 µM) ⁸⁶Rb⁺ efflux from rat thalamic synaptosomes (IC₅₀ = 700 nM) and to competitively inhibit nicotine-evoked (10 µM) [³H]DA release (IC₅₀ = 200 nM) from superfused rat striatal slices (Miller et al., 2000). At high concentrations (>1.0 µM), lobeline releases DA from its presynaptic terminals in a manner insensitive to mecamylamine (i.e., not nAChR-mediated; Teng et al., 1997). Results from these more recent studies clearly indicate that lobeline acts as a competitive, nonselective antagonist at 42 and 32* nAChRs.

MLA. MLA (Fig. 1, 4), a tertiary diterpenoid alkaloid isolated from Delphinium brownii, is one of the most potent (K_i = 1 nM), selective, competitive nonpeptide antagonists at the 7 nAChR subtype (Bergmeier et al., 1999; Davies et al., 1999). Ring E analogs of MLA also exhibit similar affinity for the 7 subtype (Bergmeier et al., 1999). Recent SAR reveals that the methylsuccinimidobenzoyl portion of the molecule is necessary for high-affinity interaction with the 7 subtype, and removal of the methyl group, the methylsuccino, or the entire substituted benzoyl moiety results in a 20-, 1000-, or 2000-fold decrease, respectively, in affinity (Hardick et al., 1995). Other structural changes in the diterpenoid portion of the molecule produce little effect. MLA has 200-fold greater selectivity for 7 compared with (1)₂1 receptors, and 50- to 100-fold greater selectivity compared with 34, 42 and 32 subtypes when expressed in oocytes (Drasdo et al., 1992; Vijayaraghavan et al., 1992; Yum et al., 1996). MLA is 1000-fold more selective for rat 7 compared with rat 42 subtypes (Alkondon et al., 1992; Quik et al., 1996) and has little ability to inhibit nicotine-evoked DA release from striatal synaptosomes (Drasdo et al., 1992). However, using the more intact, striatal slice preparation, support exists for 7* receptor involvement in nicotinic agonist-evoked DA release. Specifically, MLA has been reported to partially inhibit anatoxin-a-evoked DA release from striatal slices via circuitry including glutamatergic terminal innervation (Kaiser and Wonnacott, 2000).

d-Tubocurarine. d-Tubocurarine (Fig. 1, 5), isolated from the Chondodendron tomentosum plant is a well known antagonist of (1)₂1 nAChRs, and also competitively inhibits ACh-evoked responses in rat 22 and 32 nAChRs expressed in Xenopus oocytes (Cachelin and Rust, 1995). More recently, d-tubocurarine was shown to inhibit within a 20-fold potency range, agonist response in Xenopus oocytes expressing human nAChRs, including 22, 32, 42, 24, 34, 44, and 7 nAChRs (Chavez-Noriega et al., 1997). The 44 subtype was found to be the most sensitive, followed by the 22 subtype, which had a 6-fold lower sensitivity. d-Tubocurarine has also been reported to inhibit [³H]nicotine binding to mouse striatal membranes (K_i = 67 µM) and to inhibit nicotine-evoked DA release from superfused mouse synaptosomes (>100 µM) (Grady et al., 1992), demonstrating nonselective inhibition at native 42 and 32* nAChR subtypes.

Bungarotoxin. Two very potent and selective antagonists, BTX and n-bungarotoxin (nBTX), have been isolated from the venom of Bungarus multicinctus, the Formosan banded krait. BTX selectively inhibits 7, 8, and 9 homomeric nAChRs, but not heteromeric nAChRs (McGehee and Role, 1995). BTX is a 75 amino acid peptide, with high affinity at (1)₂1 nAChRs and at the 7 nAChR (Marks et al., 1986; K_d = 1 nM). In this respect, nicotine binds to the 7 nAChR with micromolar affinity. 7 mRNA distribution in rat brain is directly correlated with ¹²⁵I-BTX binding (Clarke et al., 1985). The pharmacology of recombinant 7 homomeric receptors resembles that of native 7* BTX-sensitive receptors (Barrantes et al., 1994; Alkondon and Albuquerque, 1995; Gopalakrishnan et al., 1995; Quik et al., 1996), supporting the hypothesis that native 7* receptors represent homomeric 7 nAChRs; however, the channel properties of native and expressed 7* receptors are not identical, such that the subunit composition of native 7* receptors currently remains putative (Albuquerque et al., 1995). nBTX has received less attention than BTX, probably due to its lack of availability. Historically characterized as an 34* antagonist, nBTX potently inhibits nicotine-evoked DA release from rodent striatal tissue at 32* nAChRs (Grady et al., 1992) and inhibits binding of [³H]epibatidine at non-42 nAChRs in rat brain membranes (Houghtling et al., 1995). In Xenopus oocyte expression studies, nBTX has been shown to inhibit the 32 nAChR subtype, but also partially inhibits the 42 subtype (Luetje et al., 1990; Gotti et al., 1997); and thus, nBTX is not a subtype-selective antagonist.

Conotoxins. Venoms isolated in minute amounts from Conus snails, including -, -, and -conotoxins, represent another group of peptide neurotoxins that target nAChRs, muscle sodium channels, and neuronal calcium channels, respectively. The -conotoxins (-conotoxin MII, -conotoxin ImI, and similar homologous peptides) are small peptides of about 14 to 17 amino acids in length (McIntosh et al., 1999). -Conotoxin MII partially inhibits nicotine-evoked DA release from rat striatal synaptosomes. -Conotoxin MII has 4 orders of magnitude greater affinity for the 32* nAChR compared with the 34* subtype, i.e., specifically inhibiting 32* receptors at low concentrations (0.1-10 nM), but inhibiting additional nAChR subtypes at higher concentrations (100 nM-1 mM). Selectivity for 32* nAChRs is indicated by the lack of -conotoxin MII-induced inhibition of K⁺-stimulated DA release from striatal synaptosomes, and lack of inhibition of nicotine-evoked [³H]norepinephrine release from rat hippocampal synaptosomes. In Xenopus oocyte expression studies, -conotoxin MII blocks the 32 subtype with an affinity 2 to 4 orders of magnitude higher than at other nAChR subtype combinations. The following order of potency (IC₅₀ value) for -conotoxin MII has been reported: 32 (0.5 nM) > 7 (0.1 µM) > 42 (0.4 µM) = 22 (1 µM) = 34 (1.1 µM) > (1)₂1 (4 µM) > 44 (>4.0 µM) > 22 (>4.0 µM) (Cartier et al., 1996). The structurally related -conotoxin ImI and -conotoxin MI are selective for 7- and 1-containing receptors, respectively, and moreover, do not block nicotine-evoked DA release from rat striatal synaptosomes. -Conotoxin AuIA, AuIB, and AuIC, isolated from Conus aulicus, inhibit ACh-evoked electrophysiological currents in rat 34-expressing Xenopus oocytes. At relatively low concentrations (0.3 and 1.0 µM), these conotoxins are selective and inhibit only 34 subtypes. Other conotoxin peptides, including FAT-MII, PnIA, PnIB, PnIA A10L and Pn1AN11S, as well as modified or non-natural amino acids, are also being investigated for selective inhibition of nAChR subtypes.

Other Neurotoxins. Over 100 additional neurotoxins have been identified that act at nAChRs, including cobra toxin from the cobra Naja naja, erabutoxin from the sea snake Laticauda semifasciata, histrionicotoxin from the frog Dendrobates histrionicus, lophotoxin from coral of the genus Lophogorgia, neosurugatoxin from the Japanese ivory mollusc Babylonia japonica, and nereistoxin from the marine annelid Lumbriconereis heteropoda (Adams and Swanson, 1994). These neurotoxins have been shown to be both competitive (e.g., nereistoxin) and noncompetitive (e.g., neosurugatoxin) nAChR antagonists. Although these toxins have proven valuable for in vitro studies, most demonstrate little subtype selectivity, and their limited availability diminishes their usefulness.

N-Alkylnicotinium Iodides. N-Alkylation of the pyridino-N atom of S()-nicotine has afforded a number of N-alkylnicotinium iodides (Fig. 2, 6), which competitively inhibit native nAChRs (Crooks et al., 1995; Dwoskin et al., 1999, 2000). At low concentrations, these analogs do not have intrinsic activity in the DA release or ⁸⁶Rb⁺ efflux assays using rat striatum (i.e., 32* receptor assay) and thalamus (42 receptor assay), and moreover, act as competitive antagonists at these nAChR subtypes. The following order of potency (IC₅₀) was obtained in the nicotine-evoked (10 µM) DA release assay: NDDNI (9 nM; 6d) > NNNI (210 nM; 6b) > NONI (620 nM; 6a) > NDNI (>100 µM; 6c). In the 42 subtype assay assessed via inhibition of [³H]nicotine binding in rat striatal membranes, the following order of potency (K_i) was obtained: NDNI (93 nM) > NDDNI (140 nM) > NNNI (840 nM) > NONI (20 µM). Antagonist activity at the 42 subtype was assessed by inhibition of nicotine-evoked (1 µM) ⁸⁶Rb⁺ efflux from rat thalamic synaptosomes, and the following order of potency (IC₅₀) was obtained: NDNI (14 nM) = NNNI (9 nM) > NDDNI (40 nM) > NONI (10 µM). Thus, NONI and NDNI are selective for 32* and 42 receptors, respectively, whereas NNNI and NDDNI have inhibitory activity at both 32* and 42 receptors. The N-n-alkylnicotinium iodide series is of considerable interest, because of the high potency and selectivity at native 32* or 42 nAChR subtypes. Alkylation of the pyridyl-N atom, which converts the nicotine molecule from an agonist into an antagonist at nAChRs, alters the physicochemical properties of the molecule causing a 2-unit drop in the pK_a value of the pyrrolidine N (from 8.4 to 6.4). Consequently, the roles of the pharmacophoric nitrogen-containing moieties may be reversed, such that these analogs interact with the nAChR in a unique manner, i.e., the quaternary pyridinium center of the antagonist molecule interacts with the binding site that normally accommodates the protonated pyrrolidine nitrogen in agonist binding, and the unprotonated pyrrolidine nitrogen of the antagonist molecule substitutes for the pyridine nitrogen of the agonist at the hydrogen bonding site.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   N-Alkylnicotinium iodides and N-alkylpyridinium salts.

N-Alkylpyridinium Halides. Simple N-n-alkyl pyridinium halides (Fig. 2, 9) have also been evaluated for antagonist activity at nicotinic receptors (Crooks et al., 2000). The C₆, C₈, and C₁₀ alkylpyridinium halides had low affinity (K_i = 20-250 µM) for the [³H]nicotine binding site using rat striatal membranes, and had little or no affinity at the [³H]MLA binding site. However, in the nicotine-evoked [³H]DA release assay using superfused striatal slices, two alkylpyridinium analogs, i.e., N-n-decylpyridinium iodide (NDPI) and N-n-dodecylpyridinium iodide (NDDPI) were relatively potent (IC₅₀ = 110 and 90 nM, respectively), resembling the conformationally restricted nicotinium iodides, and exhibiting a potency 1 order of magnitude less than NDDNI (IC₅₀ = 9 nM). Thus, the minimal structural requirements that govern affinity and selectivity for the 32* receptor subtype may be simply the presence of a quaternary pyridinium moiety and a N-n-alkyl group with optimal chain length of between 10 and 12 carbons.

Analogs of Nicotine and Nornicotine. Interest has recently been focused on the design of novel nicotinoids that are structurally related to the tobacco alkaloids nicotine (10a) and nornicotine (10b). These tobacco alkaloids have been shown to have high affinity for nAChRs and to act as agonists in most receptor assays (Dwoskin et al., 1993, 1995; Teng et al., 1997). Numerous structurally related compounds (see Fig. 3) act as antagonists at nicotinic receptors; however, in most cases, their IC₅₀ values and mechanism of inhibition (i.e., whether competitive or noncompetitive) have not been determined. These structurally related compounds have been reported primarily in the patent literature, which has been reviewed recently (Dwoskin et al., 2000).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Tobacco alkaloid analogs.

Analogs of Anabaseine and Anabasine. The minor tobacco alkaloids, anabaseine (23) and anabasine (24), have high affinity for nAChRs and act as agonists in most receptor assays (Dwoskin et al., 1995). Structurally related compounds (see Fig. 3) have been reported to act as antagonists at nAChRs; however, in most cases, their IC₅₀ values and mechanism of inhibition (i.e., whether competitive or noncompetitive) have not been determined. These structurally related compounds are in the recently reviewed patent literature (Dwoskin et al., 2000).

	Therapeutic Applications of Nicotinic Receptor Antagonists

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Antagonists acting at peripheral nicotinic receptors have been used historically as clinically useful muscle relaxants during surgery and as anti-hypertensive agents. However, until very recently, there have been no examples of clinically useful nAChR antagonists. Recently, mecamylamine, a noncompetitive and nonselective channel blocker of nAChRs, as well as lobeline, a competitive nicotinic receptor antagonist, have been demonstrated to have utility as tobacco smoking cessation agents (Schneider and Olssen, 1996; Rose et al., 1998). Furthermore, preclinical results suggest that lobeline may be a potential treatment for psychostimulant abuse (Miller et al., 2000; Harrod et al., 2001). Recently, a structural variant of a nAChR subtype has been suggested to account for some forms of epilepsy (Steinlein, 1996). Treatment with nAChR antagonists may prove beneficial for such pathologies (Holladay et al., 1997). Currently, drug discovery is focusing on nAChRs as novel targets for the development of therapeutic agents for a wide variety of CNS diseases including drug addiction, neuroendocrine, neuropsychiatric, and neurological diseases, memory and learning disabilities, eating disorders, and the control of pain, as well as cardiovascular and gastrointestinal disorders. In this respect, nAChR antagonists have good potential as therapeutic agents, since they offer another means of modulating nAChR receptor function. Nicotinic agonists rapidly desensitize nAChRs, essentially inhibiting their function. Thus, one perspective is that inhibition of receptor function may be the action that confers clinical utility, indicating that nAChR antagonists could also be beneficial in the treatment of diseases for which nAChR agonists are currently being developed. For example, schizophrenia and drug abuse have both been associated with hyperactivity of CNS dopaminergic systems, and inhibition of nAChRs may be advantageous in reducing such hyperactivity. Furthermore, the availability of subtype-selective nAChR antagonists will be invaluable agents in both basic and clinical research, with regard to both the treatment and diagnosis of disease. Finally, subtype-selective antagonists will define the role of specific nAChR subtypes in both physiological function and disease states.

	Conclusions

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A vast diversity in the structure of nAChR antagonists exist. Some nAChR antagonists are structurally complex alkaloids (e.g., MLA, d-tubocurarine, and DHE). Others are analogs of the structurally less complex tobacco alkaloids (e.g., nicotine and anabasine). In addition, several potent antagonists are small peptides (e.g., the bungarotoxins and the -conotoxins). This structural diversity affords good opportunities for SAR development. One of the main goals is to achieve subtype-selective antagonists in order to reduce possible untoward side effects. Based upon current findings, it appears that subtype-selective antagonism can be achieved by appropriate modification of agonist molecules, such as nicotine or anabasine, or by varying the amino acid sequences in active peptide molecules, such as bungarotoxin and -conotoxin. Thus, there are exciting opportunities in SAR for the further development of more potent and selective antagonists. Future prospects look bright for the clinical development of this new class of therapeutic agent.