Anders A Jensen and Povl Krogsgaard Larsen Contents

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16.1 Alzheimer's Disease 263

16.2 Cholinergic Synaptic Mechanisms as Therapeutic Targets 266

16.3 Cholinesterases 266

16.3.1 Cholinesterase Inhibitors 266

16.3.2 Substrate Catalysis of the AChE and Ligand Binding to It 269

16.4 Muscarinic ACh Receptors 271

16.4.1 mAChR Agonists 272

16.4.2 mAChR Antagonists 273

16.4.3 Allosteric Modulators of mAChRs 274

16.4.4 Ligand Binding to the mAChR 274

16.5 Nicotinic ACh Receptors 276

16.5.1 nAChR Agonists 277

16.5.2 nAChR Antagonists 278

16.5.3 Allosteric Modulators of the nAChRs 279

16.5.4 Ligand Binding to the nAChRs 280

Bibliography 281

16.1 ALZHEIMER'S DISEASE

Alzheimer's disease (AD) is a degenerative disorder of the human central nervous system (CNS), which in most cases manifests itself in mid-to-late adult life with progressive cognitive memory and intellectual impairments, leading invariably to death usually within 7-10 years after the diagnosis. AD afflicts 2%-3% of individuals at the age of 65, with an approximate doubling of incidence for every 5 years of age afterward. While age is the dominant risk factor in AD, genetic and epidemio-logical factors are also important determinants of the development of the disorder.

Clinical diagnosis of AD is based on the progressive impairment of memory and at least one other cognitive dysfunction, be it "aphasia" (difficulty with language), "apraxia" (difficulty with complex movements), "agnosia" (difficulty with identifying objects) or impaired executive functioning (making everyday decisions), and on the ability to exclude other diseases that also cause dementia. The ability to diagnose AD is very dependent on the progression of the disease but in mid or late stages of the disease the clinical accuracy in the diagnosis is very high (~90%). However, a diagnosis of AD can only be made definitely based on a direct pathological examination of brain tissue derived from biopsy or autopsy. The typical macroscopic picture observed in brain tissue from AD patients is massive atrophy of cortical and hippocampal brain regions, and at the microscopic level a widespread cellular degeneration and loss of neocortical neurons are observed together with the pathological hallmarks of the disease: the presence of amyloid "plaques" and neurofibrillary "tangles" (Figure 16.1).

The major component of the amyloid plaques is the amyloid b (Ap40/42) peptide, which is a self-aggregating, 40/42 amino acids long peptide derived from the proteolytic cleavage of the amyloid

Normal

Alzheimer's

Normal

Alzheimer's

Plaque-

Tangle

FIGURE 16.1 Amyloid plaques and neurofibrillary tangles. (A, Reprinted courtesy of Alzheimer's disease Research, a program of the American Health Assistance Foundation. www.ahaf.org/alzheimers/.; B, Reprinted courtesy of Science Foto Library.)

Neurotoxicity

Extracellular

FIGURE 16.1 Amyloid plaques and neurofibrillary tangles. (A, Reprinted courtesy of Alzheimer's disease Research, a program of the American Health Assistance Foundation. www.ahaf.org/alzheimers/.; B, Reprinted courtesy of Science Foto Library.)

Neurotoxicity

Extracellular

Intracellular

FIGURE 16.2 The amyloidogenic pathway.

COOH

Intracellular

FIGURE 16.2 The amyloidogenic pathway.

COOH

precursor protein (APP), a transmembrane protein of unknown function. The APP is cleaved in two different regions of its extracellular amino-terminal domain by the enzymes a-secretase and b-secretase, and subsequently the remaining transmembrane protein sections, the a- and b-stubs, are cleaved by g-secretase, giving rise to the peptides p3 and Ab40/42, respectively (Figure 16.2). In particular, the Ab42 peptide undergoes oligomerization and deposition, leading to microglial and astrocytic activations, oxidative stress, and progressive synaptic injury.

The neurofibrillary tangles are bundles of paired helical filaments constituted mainly by the tau protein, a widely expressed protein from the microtubule-associated family. Under normal conditions, tau maintains microtubule stability inside the cell but in AD the protein exists in a phos-phorylated form, which aggregates into tangled clumps. The formation of the tangles reduces the number of tau proteins that are able to bind and stabilize the microtubules, which thus disintegrate, ultimately leading to cytoskeletal degeneration and neuronal death.

The complex and multifactorial pathogenesis of AD is not fully understood, and throughout the years several theories have been formed to explain the molecular mechanisms underlying the disease. In the 1970-1980s, the "cholinergic hypothesis" for AD was formulated based on the observed dramatic loss of cholinergic markers, such as choline acetyltransferase and acetylcholinesterase, in the AD brain, as this reflects a massive degeneration of cholinergic neurons. However, since the loss of cholinergic markers is a relatively a delayed event in the development of AD and since subsequent studies failed to demonstrate a causal relationship between cholinergic dysfunction and AD progression, the "cholinergic hypothesis" was abandoned as an explanation of AD's pathogenesis. Instead, it seems that the aberrant production and deposition of plaques and tangles in AD is not only a disease marker, as the pathways leading to the formation of these aggregates seem to play a causal role in the pathogenesis of AD. At present, the "amyloid cascade theory" is the dominant etiological paradigm in the AD field, and the theory is supported by a substantial amount of his-topathological, biochemical, genetic, and animal model data. However, it is still debated whether the tau tangles or the amyloid plaques are the primary cause of the neurodegeneration in AD, and the links between the b-amyloid and tau and between the formation of plaques and tangles in the disease are still elusive.

The complexity of the etiology of AD is reflected in the numerous strategies applied over the years in the attempts to develop clinical efficacious drugs against the disorder. The formulation of the "cholinergic hypothesis" spawned the clinical testing of drugs targeting the acetylcholine (ACh) neurotransmitter system in the late 1980-1990s, the overall rationale being to augment cholinergic signaling to compensate for the degeneration of cholinergic neurons. Four out of five drugs currently approved for clinical treatment of AD are acetylcholinesterase inhibitors (AChEIs), and thus this drug class still represents the predominant clinical treatment of AD (see the following). In Figure 16.3 selected structures of noncholinergic ligands studied in the context of AD treatment is shown. Several potent inhibitors of the two enzymes mediating the formation of the Ab40/42 peptide, b-secretase, and g-secretase, have been developed, for example, the g-secretase inhibitors BMS 299897 (16.1) and LY-374973 (16.2). Other lines of research have focused on the development of molecules capable of inhibiting the aggregation processes leading to the formation of plaques and tangles. In view of the excitotoxicity in AD, considerable efforts have been put into the studies of calcium channel blockers, protease inhibitors, and glutamate receptor antagonists (see Chapter 15), and the uncompetitive NMDA receptor antagonist memantine (16.3) has recently become the first noncholinergic drug for the treatment of AD to be introduced on the market (Namenda®). On the other hand augmentation of AMPA receptor signaling (see Chapter 15) by a class of compounds

XOOH

BMS 299897 (16.1)

nh2 o

Memantine (16.3)

Aniracetam (16.4)

LY-374973 (16.2)

Acetylcholine (16.5)

Choline (16.6)

FIGURE 16.3 Chemical structures of acetylcholine, choline, and some noncholinergic ligands developed for the treatment of AD.

called ampokines such as aniracetam (16.4) has also been pursued as a way to treat AD. Finally, the effects of steroids and nonsteroidal anti-inflammatory drugs (NSAIDs) on the inflammation and of antioxidants on the oxidative damages observed in AD are also being investigated.

16.2 CHOLINERGIC SYNAPTIC MECHANISMS AS THERAPEUTIC TARGETS

The neurotransmitter acetylcholine (ACh, 16.5) is found throughout the body, where it regulates a wide range of important functions. In the periphery, cholinergic signaling is, for example, of key importance for cardiac function, gastric acid secretion, gastrointestinal motility, and smooth muscle contractions. In the CNS, cholinergic neurotransmission is involved in numerous processes underlying cognitive functions, learning and memory, arousal, reward, motor control, and analgesia.

The cholinergic synapse and the complex events underlying cholinergic neurotransmission are depicted in Figure 16.4. ACh exerts its physiological effects via signaling through two distinct receptor classes: muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs), which mediate the metabolic (slow) and the fast response to ACh, respectively. Once ACh is released into the synaptic cleft, two cholinesterases, acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholin-esterase (BuChE, EC 3.1.1.8), are responsible for its conversion into choline (16.6), which subsequently is taken up into the presynaptic terminal.

16.3 CHOLINESTERASES

The events underlying cholinergic neurotransmission depicted in Figure 16.4 are not that different from those in other neurotransmitter systems, as these also involve the biosynthesis and storage of the neurotransmitter in synaptic vesicles, synaptic release, and activation of different receptor classes and reuptake by transporter systems. The only extraordinary feature of the cholin-ergic synapse is the presence of two synaptic enzymes converting the neurotransmitter into its precursor in order for it to be taken back up by the presynaptic terminal. The AChE and BuChE belong to the "a/p hydrolase fold protein" superfamily comprising serine hydrolases such as cholinesterases, carboxylesterases, and lipases. Both cholinesterases are present in cholinergic synapses in the CNS, in the parasympathic synapses in the periphery, and in the neuromuscular junction. Whereas AChE is selective for ACh hydrolysis, BuChE accommodates and degrades several other substrates, including numerous neuroactive peptides. Of the two enzymes most attention has been paid to AChE, since it is responsible for ~80% of the total cholinesterase activity in the brain and has a remarkable high turnover (in the 104 s-1 range) compared to BuChE.

16.3.1 Cholinesterase Inhibitors

The physiological significance of AChE activity is reflected by the observation that it is targeted by numerous "natural" and synthetic toxins, ranging from snake and insect venoms to pesticides and nerve gasses used in chemical warfare. The efforts in the design of AChEIs in medicinal chemistry have been greatly facilitated by the availability of crystal structures of AChE complexed with ligands. Based on the nature of their activity, AChEIs can be divided into two main classes: (1) irreversible organophosphorus inhibitors and (2) reversible inhibitors. Compounds such as dyflos (16.7) and sarin (16.8) belong to the former class, which due to the irreversible nature of their action are characterized by having a long duration of action in the body, since AChE activity only is restored after resynthesis of the enzyme.

The reversible AChEIs were the first drugs developed for the symptomatic treatment of AD, and the drug class is still dominating the field. Inhibition of synaptic cholinesterase activity has proven to be efficacious in the treatment of AD, as the effect of this amplification of the natural spatial and temporal tone of ACh-mediated signaling seems to be preferable to the constant stimulus resulting from direct activation of mAChRs or nAChRs by agonists.

Presynaptic

Acetyl CoA

fcj^lC+Ql

Presynaptic

Acetyl CoA

fcj^lC+Ql

mAChRs

mAChRs

s nAChRs

Postsynaptic

Ca2+

Hceq] acetate

FIGURE 16.4 The cholinergic synapse and the events during synaptic firing. (A) In the presynaptic terminal, acetylcholine (ACh) is synthesized by "choline acetyltransferase" (ChAT) through acetylation of the precursor choline (Cho), the source of the acetyl groups being acetyl Coenzyme A (acetyl CoA). The synthesized ACh is packaged into synaptic vesicles by uptake by the vesicular ACh transporter (VAChT). (B) Upon stimulation of the presynaptic neuron, cytoplasmic Ca2+ concentrations are elevated due to the influx of the dication trough voltage-dependent calcium channels (VDCCs), and this causes the ACh-containing vesicles to fuse with the plasma membrane and release ACh into the synaptic cleft. (C) Here, ACh exerts its effects via activation of mAChRs and nAChRs. Activation of the postsynaptic nAChRs and mAChRs elicits the fast excitatory and the slow metabolic signaling of ACh, respectively, whereas activation of presynaptic receptors inhibits or augments the synaptic release of ACh, thus constituting negative and positive feedback mechanisms. Concurrently with its binding to the receptors, ACh signaling is being terminated by "acetylcholinesterase" (AChE) and "butyrylesterase," the two enzymes in the synaptic cleft, which converts ACh back to Cho. Furthermore, the choline transporter (CHT) is transported from the inside of the presynaptic terminal to the plasma membrane. (D) Having reached the membrane, CHT transports Cho back into the presynaptic terminal, where it once again is used in the synthesis of ACh. With the degradation of ACh and the subsequent removal of Cho from the synaptic cleft, the cholinergic neuron returns to its resting state.

Donepezil (16.10)

Donepezil (16.10)

-N H Physostigmine (16.11)

Eptastigmine (16.12)

-N H Physostigmine (16.11)

Eptastigmine (16.12)

Galanthamine (16.15)

Rivastigmine (16.13)

Phenserine (16.14) H NH,

Galanthamine (16.15)

Phenserine (16.14) H NH,

Huperzine A (16.16) Huprine X (16.17)

Huperzine A (16.16) Huprine X (16.17)

Ladostigil (16.18)

Ladostigil (16.18)

Decamethonium (16.22)

t cr

Propidium (16.23) Pralidoxime (16.24)

Obidoxime (16.25)

FIGURE 16.5 Chemical structures of selected cholinesterase inhibitors (16.7-16.23) and the AChE reactivators 16.24 and 16.25.

In 1993, 9-amino-1,2,3,4-tetrahydroacrine (tacrine, 16.9) was marketed as the first AChEI against AD. It is a reversible, nonselective AChE/BuChE inhibitor, which also displays activities at monoamine oxidases, potassium channels, and mAChR and nAChR subtypes. In fact, the "dirty" profile of tacrine has been proposed to contribute to its therapeutic effects. In 1997, the piperidine-based ligand donepezil (16.10) was rationally designed and marketed for treatment of AD. Donepezil inhibits AChE in a reversible and noncompetitively manner and displays a significant selectivity for AChE over BuChE. Medicinal chemistry explorations into the "carbamate-stigmine" structure of phys-ostigmine (16.11) from the Calabar bean (Physostigma venenosum) has given rise to several important analogs, including eptastigmine (16.12), rivastigmine (16.13, marketed in 2000), and phenserine (16.14, currently in phase III trials), which all exhibit inhibitory activities at both AChE and BuChE. Galanthamine (16.15), a phenantrene alkaloid originally isolated from Galanthus nivalis, was marketed for treatment of AD in 2001. The natural product huperzine A (16.16) has been isolated from the Chinese folk medicine Huperzia serrata, and it is a potent AChEI with no activity at the BuChE.

Substantial efforts in medicinal chemistry have gone into the optimization of existing AChEIs. Developed hybrid compounds combining structural components from two AChEIs, such as the tacrine/hyperzine A hybrid, huprine X (16.17), have displayed higher inhibitory potencies, and in some cases different kinetic properties and/or AChE/BuChE selectivities, than their parent compounds. In other hybrids, substructures of AChEIs have been combined with molecular components of other drugs hereby giving rise to novel compounds, where the AChE activity has been supplemented with activities at other neurotransmitter systems. For example, rivastigmine (16.13) constitutes the template of ladostigil (16.18), where the structure has been combined with the pro-pylargyl group of the MAO-B inhibitor rasagiline, and of BCG 20-1259 (16.19), an inhibitor of both AChE and the serotonin transporter. Another popular strategy has been the development of bivalent ligands, such as the bivalent tacrine-indole ligand 16.20, which displays a low picomolar IC50 value at AChE. Finally, the observation that regulation of the synaptic ACh concentrations appears to become more and more dependent on BuChE as AD progresses has inspired the development of completely selective BuChEIs, including several cymserine analogs (16.21) (Figure 16.5).

16.3.2 Substrate Catalysis of the AChE and Ligand Binding to It

At the molecular level, AChE is a 537 amino acids long protein composed of a 12-stranded mixed b-sheet surrounded by 14 a-helices (Figure 16.6A). The hydrolysis of ACh in AChE takes place at the bottom of a long and narrow gorge lined with numerous aromatic amino acid residues that penetrates half into the enzyme. The active site is located ~20 A from the surface of the enzyme and is composed of two subsites. In the "catalytic anionic site" the choline moiety of ACh is stabilized by a cation-p interaction between the quaternary amino group of ACh and the aromatic ring system of the Trp84 residues with minor contributions from the Glu199 and Phe330 residues, whereas the "ester-atic subsite" contains a typical serine-hydrolase catalytic triad consisting of the residues Ser200, His440, and Glu327 (Figure 16.6A). In addition, binding is stabilized by interactions between the car-bonyl oxygen and the acetyl group of ACh with neighboring residues in AChE. Another binding site for ACh, the "peripheral anionic site" (PAS), is located on the surface of the enzyme at the entrance to the gorge, ~20 A above the active site and contains the Trp70, Asp72, Tyr121, Trp279, and Phe331 residues (Figure 16.6A). In addition to PAS being involved in processes important for other aspects of AChE function, binding of ACh to PAS represents the first step in the ACh catalysis, as the trapping of the substrate on its way to the active site enhances the catalytic efficiency of the process. From the PAS, ACh is transferred to the active site, where the catalysis occurs (Figure 16.7A).

The complex processes underlying ACh catalysis is reflected in the diverse binding modes displayed by the reversible AChEIs both in terms of their binding sites and the mechanisms underlying their inhibition. The "carbamoylating" inhibitors, compounds 16.11-16.14, all contain a carbamate group, which analogously to the ester group in ACh can be hydrolyzed by AChE (Figure 16.7B). Thus, these AChEIs are split into their carbamate moiety and their stigmine (16.11, 16.12, and 16.14) or dimethylamino-a-methylbenzyl (16.13) moieties interacting with the Ser200 residue and

FIGURE 16.6 AChE structure and AChEI-binding modes. (A) The 3D structure of AChE. The localization of the PAS and the residues constituting the catalytic anionic site and the esteratic site in the enzyme are indicated. (B) The active site of AChE in crystal structures of the enzymes complexes with AChEIs. The binding modes of reversible inhibitors rivastigmine, galanthamine, and donepezil to the AChE and the phosphorus AChE conjugate formed after interaction with the irreversible inhibitor sarin.

FIGURE 16.6 AChE structure and AChEI-binding modes. (A) The 3D structure of AChE. The localization of the PAS and the residues constituting the catalytic anionic site and the esteratic site in the enzyme are indicated. (B) The active site of AChE in crystal structures of the enzymes complexes with AChEIs. The binding modes of reversible inhibitors rivastigmine, galanthamine, and donepezil to the AChE and the phosphorus AChE conjugate formed after interaction with the irreversible inhibitor sarin.

the anionic site, respectively (exemplified by rivastigmine in Figure 16.6B). However, in contrast to the very fast (microseconds) hydrolysis of acetate from the Ser200 residue following the hydrolysis of ACh, the dissociation of the carbamate group is very slow (minutes) (Figure 16.7B). Thus, the inhibition exerted by this AChEI class arises from the active site in AChE being occupied and unable to bind ACh for a considerable time after the initial binding of the inhibitor. In particular, for rivastigmine (16.13) the duration of this reactivation phase is considerable, and the compound is often termed a "pseudo-irreversible" inhibitor.

Other AChEIs, such as tacrine (16.9) and galanthamine (16.15), act like "true" inhibitors, as they are not hydrolyzed by AChE but instead compete with ACh for the active site in the enzyme, interacting with residues in both the anionic and esteratic subsites (Figure 16.6B). Donepezil (16.10), on the other hand, targets the gorge connecting the active site with the surface of the enzyme, the dimethoxy-indanone and benzyl piperidine moieties of the inhibitor interacting with Trp279 in the PAS and with Trp84 and Phe330 in the anionic subsite of the active site, respectively (Figure 16.6B). Bisquaternary inhibitors, such as decamethonium (16.22), bind in a similar fashion, and the remarkably enhanced potencies displayed by bivalent ligands such as 16.20 is also attributed to their ability to target two binding sites in AChE. Finally, propidium (16.23) and fasciculin-2, a 61-amino acid peptide isolated from the venom of Dendroaspis (mamba) species, inhibit AChE noncompetitively by binding exclusively to the PAS.

Irreversible organophosphorus AChEIs such as 16.7 and 16.8 all act at the active site of AChE, as they form covalently attached phosphorus conjugates with the Ser200 residue in the esteratic subsite of the enzyme, thereby disrupting its catalytic mechanism (Figures 16.6B and 16.7C). Treatment of the phosphorus AChE conjugate with so-called reactivators, oxime-based medical antidotes such as pralidoxime (16.24) and obidoxime (16.25), can restore AChE function, but being unable to pass the blood-brain barrier (BBB) they cannot reverse the central effects of organophosphate poisoning.

r2 of

r2 of

FIGURE 16.7 Substrate catalysis of the AChE and inhibition of it. (A) Catalysis of ACh in the AChE. (B) Inhibition of AChE by a reversible carbamoylating AChEI. (C) Formation of the phosphorus AChE conjugate by organophosphorus AChEIs, and the following "aging" and oxime reactivation processes. Rj, O-alkyl or amid; R2, alkyl, O-alkyl or amid; L, leaving group.

16.4 MUSCARINIC ACh RECEPTORS

The mAChRs belong to family A of the superfamily of G-protein coupled receptors (GPCRs) (see Chapter 12). Hence, the muscarinic component of cholinergic signaling is mediated by intracellular second messenger cascades initiated by the coupling of the activated mAChRs to G-proteins and other intracellular proteins such as p-arrestins. Five mAChR subtypes have been identified, termed Mj-M5. The Mj, M3, and M5 mAChRs are coupled to Gaq-proteins and the resulting stimulation of phospholipase C and intracellular release of Ca2+, whereas the M2 and M4 subtypes are coupled to Gao and Gai proteins associated with inhibition of adenylate cyclase and a reduction of the intracel-lular levels of cAMP.

The mAChRs are expressed abundantly in both the CNS and in the peripheral tissues. In recent years, studies of "mAChR knock out mice," where the expression of one of the five mAChR subtypes have been eliminated have shed light on the functions of the respective mAChRs and the therapeutic perspectives in selective targeting of these individual mAChRs.

The M2 mAChR is widely expressed in the CNS and in the peripheral nervous system (PNS). Besides being the far most abundant mAChR in the heart, where it mediates the cholinergic regulation of the heart rate, the M2 and M3 receptors are the key subtypes when it comes to the cholinergic component of the smooth muscle contraction. In the CNS, M2, and the other Gi-coupled mAChR, M4, function as autoreceptors and heteroreceptors. In contrast to the widespread distribution of M2, M4 is predominantly centrally expressed, and besides its potential as a target for the treatment of pain, the subtype has been shown to regulate striatal dopamine release.

Similar to the M2 mAChR, the M3 subtype is expressed throughout the CNS and the PNS and in organs innervated by parasympathetic nerves. M3 knockout mice display a substantial reduction in body weight due to a reduced food intake compared to wild-type mice, and this phenotype has been ascribed to their lack of M3 receptors in hypothalamus, a key brain region for regulation of appetite. M1 is the most abundantly expressed mAChR subtype in the forebrain, where it is predominantly expressed at the postsynaptic termini. Although the Mj knockout mice do not exhibit significantly impaired cognitive impairment, substantial pharmacological evidence indicate that the receptor is involved in cognitive processes underlying learning and memory. Furthermore, M1 knockout mice exhibit epileptic symptoms, and inhibition of the receptor could hold prospects in the treatment of Parkinson's disease. The fifth mAChR subtype, M5, is expressed in considerably lower levels than the other mAChRs in both the CNS and PNS.

16.4.1 mAChR Agonists

The compounds 16.26-16.30 in Figure 16.8 are classical mAChR agonists displaying no significant selectivity for any of the five subtypes. Muscarine (16.26), which like ibotenic acid and muscimol (see Chapter 15) is a constituent of Amanita muscaria, has defined the mAChR family because of its selectivity for these receptors over the nAChRs. Stabilization of the ester moiety of ACh as a carbamate group yields carbachol (16.27), which not only is nonselective when it comes to the mAChRs but also is equipotent as a nAChR agonist. The naturally occurring heterocyclic agonist pilocarpine (16.28) is widely used as topical miotic for the control of elevated intraocular pressure associated with glaucoma. However, the bioavailability of pilocarpine is low, and the compound does not appear to penetrate the BBB. The potent agonist oxotremorine (16.29) has been used extensively as a lead compound for structure-activity studies giving rise to a plethora of mAChR ligands spanning the entire efficacy range from full agonists over partial agonist to antagonists.

Because of the high expression levels of the postsynaptic M1 mAChRs in the brain regions affected in AD, substantial medicinal chemistry efforts have been put into the development of agonists selectively targeting this subtype. Arecoline (16.31), a constituent in areca nuts (the seeds of Areca catechu), is a cyclic "reverse ester" bioisostere of ACh, containing a tertiary amino group. 16.31 is only partially protonated at physiological pH, which is an advantage in terms of BBB penetration and CNS availability. A considerable number of analogs of 16.31 have been developed, including xanomeline (16.32), where the labile ester moiety of 16.31 has been replaced with the more metabolically stable thiadiazole. 16.32 has become the prototypic "M1 selective agonist" but in functional assays the compound only exhibits a preference for M1 over the other subtypes. In support of this, the in vitro profile of 16.32 has not been translated into an acceptable safety margin in patients, and due to cholinergic side effects clinical development of the compound for treatment of AD has been discontinued. Interestingly, in these clinical trials 16.32 have been found to improve the behavioral disturbances and hallucinations often observed in AD patients, an effect that subsequently has been ascribed to an effect on dopaminergic signaling via the M4 subtype. Several other "M1-selective/preferring" agonists, including 16.33-16.35, have been in development for the treatment of AD but most of them have been faced with serious cholinergic side effects in their clinical development, suggesting that they might not be sufficiently subtype-selective. AC-42 (16.36) is a partial M1 agonist identified in a high-throughput screening, and it bears no significant structural resemblance with ACh or other orthosteric mAChR ligands (Figure 16.8).

Muscarine (16.26) Carbachol (16.27) Pilocarpine (16.28) Oxotremorine (16.29)

Muscarine (16.26) Carbachol (16.27) Pilocarpine (16.28) Oxotremorine (16.29)

FIGURE 16.8 Chemical structures of mAChR agonists.

FIGURE 16.8 Chemical structures of mAChR agonists.

In contrast to its quite potent Mj activity, AC-42 exhibits no agonist or antagonist activities at any of the other four mAChR subtypes, and thus, it is the only completely Mj-selective agonist reported to date. An AC-42 analog is currently in clinical trials for the treatment of glaucoma.

16.4.2 mAChR Antagonists

Analogous to the few true subtype-selective mAChR agonists available, it has been quite difficult to develop subtype-selective antagonists. The alkaloids atropine (16.37) and scopolamine (16.38) found in "the deadly nightshade" Atropa belladonna are potent mAChR antagonists, and like N-methylscopolamine (NMS, 16.39) and quinuclidinyl benzilate (QNB, 16.40) they have been highly important tools in the explorations of mAChR function over the years (Figure 16.9). In contrast to these nonselective antagonists, pirenzepine (16.42) has displayed higher binding affinities for mAChRs in the brain over mAChRs in the heart, suggesting a preference for M1 over M2 subtypes, although this "selectivity" has been less impressive when the compound has been tested at the cloned mAChRs. Similarly, so-called M2 selective antagonists such as AF-DX-116 (16.43), with its remarkable structural similarity to 16.42, and SCH 57790 (16.44) only display 20-40-fold M2/M1 selectivity in binding assays. In contrast, the three peptides ml-toxin, m2-toxin (16.45), and m4-toxin isolated from the venom of the green mamba, Dendroaspis augusticeps, are potent and highly selective antagonists of M1, M2, and M4, respectively. In recent years, however, the modest M2 preference of 16.44 has formed the basis for extensive SAR studies, and this work has resulted in numerous small molecular weight compounds, including 16.46, displaying subnanomolar binding affinities to M2 and >1000-fold selectivity over the other mAChRs. Several of these have entered clinical trials for AD, where the major question will be whether the desired effects of inhibition of presynaptic M2 autoreceptors in the CNS can be separated from the unwanted side effects caused by antagonism of peripheral M2 receptors.

Atropine (16.37)

O OH

Scopolamine (16.38)

O OH

O OH

Atropine (16.37)

O OH

Scopolamine (16.38)

O OH

Methoctramine (16.41)

Methoctramine (16.41)

SCH 57790 (16.44)

Pirenzepine (16.42) AF-DX-116 (16.43)

SCH 57790 (16.44)

Pirenzepine (16.42) AF-DX-116 (16.43)

FIGURE 16.9 Chemical structures of competitive mAChR antagonists. (Part of the figure is reprinted from Krajewski, J.L. et al., Mol. Pharmacol, 60, 725, 2001. With permission.)

m2-toxin (16.45)

FIGURE 16.9 Chemical structures of competitive mAChR antagonists. (Part of the figure is reprinted from Krajewski, J.L. et al., Mol. Pharmacol, 60, 725, 2001. With permission.)

16.4.3 Allosteric Modulators of mAChRs

The difficulties connected with the development of agonists or competitive antagonists capable of discriminating between mAChR subtypes have prompted the search for and identification of "allosteric modulators" of the receptors (Chapter 12). As can be seen from the examples given in Figure 16.10, these compounds (16.47-16.51) are structurally completely different from ACh and other orthosteric mAChR ligands in a complete manner. Interestingly, several of these allosteric modulators also target other receptors or enzymes.

16.4.4 Ligand Binding to the mAChR

A considerable insight into the ligand-binding modes of mAChR ligands to their receptors has, over the years, been gained from mutagenesis studies. The orthosteric site in the mAChR (and other family A GPCRs) is localized in a cavity formed by transmembrane regions (TMs) 3, 5, 6, and 7 (Figure 16.11A). All orthosteric mAChR ligands contain a quaternifed ammonium group or an amino group

Alcuronium (16.48)

Gallamine (16.47)

Alcuronium (16.48)

FIGURE 16.10 Chemical structures of allosteric modulators of mAChRs.

FIGURE 16.10 Chemical structures of allosteric modulators of mAChRs.

Domains involved in_

(A)

Domains involved in_

Domains involved in orthosteric ligand binding 1

FIGURE 16.11 (A) Binding mode of ACh to the orthosteric site of the M1 mAChR (observed from the extracellular side). (B) Localization of orthosteric and allosteric binding sites in the mAChR.

Domains involved in orthosteric ligand binding 1

FIGURE 16.11 (A) Binding mode of ACh to the orthosteric site of the M1 mAChR (observed from the extracellular side). (B) Localization of orthosteric and allosteric binding sites in the mAChR.

that is positively charged at physiological pH, and orthosteric ligand binding to the mAChR is centered in the ionic and cation-p interactions formed by this group to an aspartate residue in TM3 and aromatic residues in TM6 and TM7. This key interaction is supplemented by hydrogen bonds and van der Waals interactions between residues in TM3, TM5, TM6, and TM7 of the mAChR and other parts of the orthosteric ligand.

The wide range of structurally diverse allosteric mAChR modulators all seem to bind to a general allosteric site localized just above the orthosteric site in the receptor, constituted by the upper helix turns of the seven TMs and their three interconnecting extracellular loops (Figure 16.11B). Finally, in agreement with its novel structure and selectivity profile, the M1-selective agonist AC-42 has been demonstrated to bind to an allosteric site in this receptor composed of residues in amino-terminal domain, TM1, and TM7 of the receptor (Figure 16.11B).

16.5 NICOTINIC ACh RECEPTORS

The nAChRs belong to the superfamily of ligand-gated ion channels termed "Cys-loop receptors" (Chapter 12). The receptors are complexes composed of five subunits forming an ion pore through which Na+ and Ca2+ ions can enter the cell when the receptor is activated, resulting in depolarization of the neuron and increased intracellular Ca2+ concentrations.

To date 17 different nAChR subunits have been identified (Figure 16.12A). The "muscle-type nAChR" is composed of aj, Pj, 8, and g/e subunits and is localized postsynaptically at the neuromuscular junction (Figure 16.12B). The receptor is a key mediator of the electrical transmission across the anatomical gap between the motor nerve and the skeletal muscle, thus creating the skeletal muscle tone. Hence, antagonists of this receptor are used clinically as muscle relaxants during anesthesia. The "neuronal nAChRs" are heteromeric or homomeric complexes composed of the a2-aj0 and P2-P4 subunits (Figure 16.12B), and they are located at presynaptic and postsynaptic densities in autonomic ganglia and in cholinergic neurons throughout the CNS. Equally important to the overall contribution of nAChRs to cholinergic neurotransmission are the roles of nAChRs as autoreceptors and heteroreceptors regulating the synaptic release of ACh and other important neu-rotransmitters such as dopamine, noradrenalin, serotonin, glutamate, and GABA.

The 12 neuronal nAChR subunits display considerable different expression patterns in the CNS, and this combined with the ability of the subunits to assemble into a vast number of different combinations characterized by significantly different pharmacological profiles give rise to a large degree of heterogeneity in the native receptor populations. The two predominant physiological neuronal nAChRs are the heteromeric a4p2 subtype and the homomeric a7 receptor. The a4p2 subtype constitutes >90% of the high-affinity binding sites for nicotine in the brain and is the most obvious nAChR candidate in the treatment of AD and nicotine addiction. The homomeric a7 nAChR is characterized by its low binding affinities for the classical nAChR ligands, by fast desensitization kinetics, and by a remarkable high Ca2+ conductance. In recent years, the a7 nAChR has attracted considerable attention as a drug target for the treatment of states of inflammation, the fibromyalgia syndrome and various forms of pain, and modulation of a7 signaling appears to be beneficial in particular for the cognitive and sensory impairments observed in schizophrenia. Although the two major neuronal nAChR subtypes have attracted most of the attention in terms of development of nAChR-based therapeutics, several of the "minor" subtypes are also interesting targets. For example, the a6 subunit is localized exclusively in the midbrain, and a6-containing nAChR subtypes have been shown to regulate synaptic dopamine release in striatum making them interesting in relation to Parkinson's disease (Chapter 17).

(A) (B) [ai> Pi, ô, Y, £] [a2-as, P2-P4] [a7, a9]

FIGURE 16.12 The nAChR family. (A) A phylogenetic tree over the nAChR family. (B) The multiple nAChR complexes formed by the 17 subunits. The localization of the orthosteric sites in the respective receptors is indicated.

(A) (B) [ai> Pi, ô, Y, £] [a2-as, P2-P4] [a7, a9]

FIGURE 16.12 The nAChR family. (A) A phylogenetic tree over the nAChR family. (B) The multiple nAChR complexes formed by the 17 subunits. The localization of the orthosteric sites in the respective receptors is indicated.

16.5.1 nAChR Agonists

As in the case of the AChE and mAChR fields, medicinal chemistry development of nAChR ligands has been greatly influenced by the plethora of ligands isolated from natural sources. From a therapeutic perspective, there has been most interest in ligands augmenting nAChR signaling (i.e., agonists or allosteric potentiators). The classical nAChR agonists 16.52-16.57 have all been isolated from natural sources, and while they are selective for the nAChRs over the mAChRs, they are rather nonselective within the nAChR family (Figure 16.13). Because of their potent agonism and high BBB penetration, (S)-nicotine (16.52) and (±)-epibatidine (16.53) have been the lead compounds for the vast majority of nAChR agonists developed over the years. Modifications of the linker between the pyridine and pyrrolidine rings and introduction of new ring systems in 16.52 have resulted in several potent nAChR agonists, such as compounds 16.58-16.60. Ring-opening of the pyrrolidine ring in (S)-nicotine has given rise to ispronicline (16.61) and several other potent analogs. Interestingly, these agonists display significant functional selectivity for the a4b2 over all other nAChR subtypes and have entered clinical development as cognitive enhancers and analgesics. The 5-ethynyl nicotine analog, altinicline (16.62), is a partial agonist characterized by a modest functional preference for the a4b2 nAChR over b4-containing subtypes. 16.62 is a highly efficacious stimulant of dopamine release in nucleus accumbens and striatum and has been in clinical trial for Parkinson's disease.

(±)-Epibatidine (16.53) was originally isolated from the skin of the Ecuadorian frog Epipedobates tricolor and is by far the most potent of the classical nAChR agonists. The therapeutic interest in the compound was founded in studies showing that it is a nonaddictive analgesic, blocking pain 200 times more effectively than morphine. Because of the severe hypertension and neuromuscular paralysis observed upon epibatidine administration, however, several epibatidine analogs have been developed in the hope to isolate the analgesic effects from the side effects. Methylation of the basic amino group and substitution of the 2-chloropyridine ring in 16.53 with a 2-(pyridazin-4-yl) ring

Lobeline (16.57)

ABT-089 (16.58) ABT-418 (16.59) Tebanicline (16.60)

Lobeline (16.57)

ABT-089 (16.58) ABT-418 (16.59) Tebanicline (16.60)

Cl hN

Cl hN

Varenicline (16.65)

PNU-282987 (16.68)

FIGURE 16.13 Chemical structures of nAChR agonists.

Varenicline (16.65)

PNU-282987 (16.68)

FIGURE 16.13 Chemical structures of nAChR agonists.

has resulted in 16.63, which displays a significant functional preference as a potent agonist of a4b2 and muscle-type nAChRs over other nAChRs. In contrast, (±)-UB-165 (16.64), a hybrid compound between epibatidine and anatoxin-a (16.56, isolated from the alga Anabaena flos aquae), is one of the few nAChR agonists available that preferentially activate the "minor" heteromeric nAChRs without concomitant activation of the major CNS subtypes.

(-)-Cytisine (16.54) from Laburnum anagyroides is a potent partial agonist of the heteromeric nAChRs. (-)-Cytisine and its analog varenicline (16.65) have recently been launched as smoking cessation aids. Further, the complex natural source compound lobeline (16.57) from Lobelia inflata is also under clinical development for treatment of smoking dependence.

Although the homomeric a7 nAChR represents a low-affinity binding site for ACh and the natural source compounds 16.52-16.56, it has been possible to develop potent and a7 selective agonists from some of these leads. The toxin anabaseine (16.55) is isolated from marine worms and certain ant species, and it is a rather nonselective agonist displaying a somewhat higher efficacy at a7 nAChR than at the heteromeric nAChRs. Introduction of conjugated aryl substituents in the 3-position of the pyridine ring of anabaseine has increased this selectivity, as exemplified by the prototypic a7 agonist GTS-21 (16.66). The quinuclidine (1-azabicyclo[2.2.2]octane) ring system forms the scaffold in several a7-selective agonists, including AR-R-17779 (16.67) and PNU-282987 (16.68), of which the latter has entered clinical trials for schizophrenia. Finally, replacement of the pyrrolidine ring in 16.52 with a azabicyclo[3.2.2.]nonane ring has provided the potent and selective a7 agonist TC-1698 (16.69).

16.5.2 nAChR Antagonists

As described for the nAChR agonists, several competitive nAChR antagonists have been obtained from natural sources. The peptide toxin, a-bungarotoxin (16.70) from the Taiwan banded krait (Bungarus multicinctus) is a potent competitive antagonist of a7 and muscle-type nAChRs, and methyllycaconitine (16.71), isolated from Delphinum and Consolida species, is a highly selective a7 antagonist. In contrast to the selectivity of these two compounds, other classical competitive nAChR antagonists, including dihydro-b-erythroidine (DHbE, 16.72), are far less discriminative between different nAChR subtypes (Figure 16.14).

In addition to the agonists derived from (S)-nicotine and (±)-epibatidine, several antagonists have emerged. Introduction of n-alkyl groups ranging from methyl to dodecyl (C12H25) at the pyridine nitrogen of (S)-nicotine have produced several potent albeit nonselective antagonists (exemplified by 16.73). Furthermore, the pyridyl ether A-186253 (16.74), a 16.58 analog, displays high selectivity

a-Bungarotoxin (16.70)

a-Bungarotoxin (16.70)

Cl N

Cl N

FIGURE 16.14 Chemical structures of competitive nAChR antagonists and 3D structures of a-bungarotoxin and four a-conotoxins.

for native a4pf nAChRs versus a3p* and a7 receptors in binding assays. The epibatidine analog 16.75 is an antagonist with a K value of 1 pM to native a4p* nAChRs, making it the nAChR ligand with the highest binding affinity published to date.

Within the last decade, several small peptides of 12-20 amino acid residues, the so-called a-conotoxins, have been isolated from a family of predatory cone snails, the Conus snails. The peptides have turned out to be very interesting pharmacological tools, as they are highly subtype-specific in their antagonism of nAChRs. The 3D structures of the a-conotoxins are established by intramolecular disulfide bonds formed by the four highly conserved cysteine residues in the peptides, which are organized in different arrangements: a3/5, a4/3, a4/7, or a4/6, the nomenclature referring to the number of residues between the conserved cysteines (Figure 16.14). The subtype-selective activities of the respective a-conotoxins arise from the differences in the nonconserved residues. So far, a-conotoxins selective for nAChR subtypes a7 (for example Iml), a3p2 (for example MII and PnIA), and a3p4 (AulB) have been identified (Figure 16.14). Considering that the entire mollusk family is estimated to contain ~50,000 neuropharmacologically active toxins, additional subtype-selective nAChR antagonists are likely to be identified in the future.

16.5.3 Allosteric Modulators of the nAChRs

As it is the case with other ligand-gated ion channels, such as GABAa and NMDA receptors (see Chapter 15), the nAChRs are highly susceptible to allosteric modulation (see Chapter 12). Several endogenous ligands, such as steroids (for example 16.76), 5-hydroxyindole (16.77), and Ca2+ and Zn2+ ions modulate signaling through the receptors (Figure 16.15). It is highly interesting to note that the Ap42 peptide has been found to be a potent noncompetitive inhibitor of a7 nAChR signaling but the significance of this inhibition for AD remains to be elucidated.

Allosteric potentiators hold several advantages to regular agonists when it comes to the augmentation of nAChR signaling. First, analogous to the AChEIs they only exert their effect when ACh is

17P -Estradiol (16.76) 5-Hydroxyindole (5-HI, 16.77) PNU-120596 (16.78)

LY-2087101 (16.80) Mecamylamine (16.81)

LY-2087101 (16.80) Mecamylamine (16.81)

10 s

FIGURE 16.15 Chemical structures of allosteric modulators of nAChRs and the potentiation of the ACh-induced a7 nAChR signaling exerted by 5-hydroxyindole (5-HI) and PNU-120596. (Part of the figure is reprinted from Bertrand, D. and Gopalakrishnan, M., Biochem. Pharmacol, 74, 1155, 2007. With permission.)

10 s

FIGURE 16.15 Chemical structures of allosteric modulators of nAChRs and the potentiation of the ACh-induced a7 nAChR signaling exerted by 5-hydroxyindole (5-HI) and PNU-120596. (Part of the figure is reprinted from Bertrand, D. and Gopalakrishnan, M., Biochem. Pharmacol, 74, 1155, 2007. With permission.)

present in the synapse, and thus the stimulation of the receptors introduced by the allosteric modulator occurs in a physiological tone. Second, since the allosteric ligands target regions less conserved than the orthosteric site in different nAChR subtypes, they are more likely to be subtype-selective than orthosteric ligands. Finally, in contrast to agonists, which for the most parts mimic the activation kinetics of the endogenous agonist, allosteric potentiators can enhance nAChR signaling in many different ways, for example, by increasing the ion conductance of the receptor, by increasing the frequency of ACh-induced ion channel openings, or by reducing the desensitization rate.

The urea analogs PNU-120596 (16.78) and NS-1738 (16.79) are selective allosteric potentiators of the a7 nAChR. Both compounds enhance the potency of as well as the maximal response elicited by ACh through the receptor, having no effect on receptor signaling in the absence of ACh. In addition to these effects, 16.78 also suppress the desensitization of a7 (Figure 16.15) and can restore the activity in an already desensitized receptor. In contrast to the subtype-selective activities of these potentiators, LY-2087101 (16.80) potentiates the signaling of a2b4, a4b4, a4b2, and a7 nAChRs but not that of the muscle-type, a3b2, and a3b4 subtypes. Interestingly, the AChEIs physostigmine (16.11) and galanthamine (16.15) have also been shown to potentiate the ACh-evoked responses through several nAChR subtypes.

16.5.4 Ligand Binding to the nAChRs

The orthosteric sites of the nAChR are situated in the extracellular amino-terminal domain of the pentameric receptor complex, more specifically at the interfaces between a- and b-subunits in the heteromeric nAChR and between two a-subunits in the homomeric nAChR (Figure 16.16). Thus, the heteromeric and homomeric nAChRs contain two and five orthosteric sites, respectively (Figure 16.12). Similarly to the mAChR ligands, agonists, and almost all competitive antagonists

Orthosteric ligands

Allosteric ligands in

Orthosteric ligands in

Steroids i > Mecamylamine 1 >

FIGURE 16.16 Ligand binding to the nAChR. Right: The binding modes of the agonist epibatidine and the competitive antagonist MLA to the orthosteric site in the amino-terminal domain of the receptor. (Part of the figure is reprinted from Hansen, S.B. et al., EMBO J, 24, 3635, 2005. With permission.) Left: Allosteric modulators targeting the amino-terminal and ion channel domains of the nAChR complex. (Part of the figure is reprinted from Jensen, A.A. et al., J. Med. Chem., 48, 4705, 2005. With permission.)

Galanthamine PNU-282987 Ca2+ Zn2+ Aß42

Steroids i > Mecamylamine 1 >

FIGURE 16.16 Ligand binding to the nAChR. Right: The binding modes of the agonist epibatidine and the competitive antagonist MLA to the orthosteric site in the amino-terminal domain of the receptor. (Part of the figure is reprinted from Hansen, S.B. et al., EMBO J, 24, 3635, 2005. With permission.) Left: Allosteric modulators targeting the amino-terminal and ion channel domains of the nAChR complex. (Part of the figure is reprinted from Jensen, A.A. et al., J. Med. Chem., 48, 4705, 2005. With permission.)

of the nAChRs possess a positively charged amino group, either in the form of a quaternary amino group or a protonated tertiary or secondary amino group (Figures 16.13 and 16.14). This amino group docks into an "aromatic box" formed by five aromatic residues facing the interface, predominantly from the a-subunit side, where the group forms a strong cation-p interaction with a tryptophan (W) residue, while the other four aromatic residues ensures optimal spatial orientation of the ligand for binding (exemplified by the agonist epibatidine and the competitive antagonist MLA in Figure 16.16). The so-called complementary binding component, i.e., the interactions between receptor and the rest of the ligand molecule, predominantly takes place to b-subunit side of the orthosteric site (Figure 16.16). Since the five aromatic residues constituting the "primary binding component" are highly conserved throughout the nAChR subunits, subtype-selectivity of orthosteric ligands most often arise from this "complementary binding component."

Galanthamine and physostigmine are believed to bind to an allosteric site in the amino-terminal domain of the a-subunit in the nAChR complex, thereby increasing the receptors affinity for the orthosteric agonist and/or the probability of ion channel opening. The potentiation and inhibition of nAChR signaling exerted by Ca2+ and the Ap42 peptide, respectively, also arise from binding to this domain (Figure 16.16). Conversely, the noncompetitive antagonist mecamylamine (16.81) binds to a site situated deep into the ion channel of the nAChR, where it blocks the influx of cations upon activation of the receptor. Finally, the modulation of nAChR function by steroids appears to originate from a binding site involving the small extracellular carboxy-termini of the nAChR subunits.

BIBLIOGRAPHY

Bertrand, D.; Gopalakrishnan, M. Allosteric modulation of nicotinic acetylcholine receptors. Biochem.

Pharmacol. 2007, 74(8), 1155-1163. Clader, J. W.; Wang, Y. Muscarinic receptor agonists and antagonists in the treatment of Alzheimer's disease.

Curr. Pharm. Des. 2005, 11, 3353-3361. Colletier, J. P.; Fournier, D.; Greenblatt, H. M.; Stojan, J.; Sussman, J. L.; Zaccai, G.; Silman, I.; Weik, M. Structural insights into substrate traffic and inhibition in acetylcholinesterase. EMBO J. 2006, 25, 2746-2756.

Eglen, R. M.; Choppin, A.; Watson, N. Therapeutic opportunities from muscarinic receptor research. Trends

Pharmacol. Sci. 2001, 22, 409-414. Hansen, S. B.; Sulzenbacher, G.; Huxford, T.; Marchot, P.; Taylor, P.; Bourne, Y. Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations. EMBO J. 2005, 24, 3635-3646. Jensen, A. A.; Fr0lund, B.; Liljefors, T.; Krogsgaard-Larsen, P. Neuronal nicotinic acetylcholine receptors: Structural revelations, target identifications and therapeutic inspirations. J. Med. Chem. 2005, 48, 4705-4745.

Krajewski, J. L.; Dickerson, I. M.; Potter, L. T. Site-directed mutagenesis of m1-toxin1: Two amino acids responsible for stable toxin binding to M1 muscarinic receptors. Mol. Pharmacol. 2001, 60, 725-731.

Lu, Z. L.; Saldanha, J. W.; Hulme, E. C. Seven-transmembrane receptors: Crystals clarify. Trends Pharmacol. Sci. 2002, 23, 140-145.

Masters, C. L.; Cappai, R.; Barnham, K. J.; Villemagne, V. L. Molecular mechanisms for Alzheimer's disease:

Implications for neuroimaging and therapeutics. J. Neurochem. 2006, 97, 1700-1725. Paterson, D.; Nordberg, A. Neuronal nicotinic receptors in the human brain. Prog. Neurobiol. 2000, 61, 75-111.

Romanelli, M. N.; Gratteri, P.; Guandalini, L.; Martini, E.; Bonaccini, C.; Gualtieri, F. Central nicotinic receptors: Structure, function, ligands, and therapeutic potential. Chem. Med. Chem. 2

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