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From design to therapeutic efficacy: the challenge for nicotinic receptor research Bruce K.Cassels,Isabel Bermúdez,Federico Dajas,J.Andrés Abin-Carriquiry and Susan Wonnacott

S-, the principal psychoactive constituent of Nicotiana tabacum, underpins addiction to smoking. Although tobacco consumption is a leading cause of death worldwide, nicotine itself is also proposed to have potential therapeutic benefits for a diverse range of conditions. Nicotine interacts with its cognate receptors in the to exert a predominantly modulatory influence, making neuronal nicotinic receptors attractive therapeutic targets. Here, we focus on three natural products as lead compounds for drug discovery programs, nicotine, epibatidine and , and consider the aims and limitations that shape these drug discovery endeavors.

Heterogeneity of nicotinic their high relative permeability to Ca2+[2]. In contrast receptor: which subtype to target? to the well-defined roles of nAChRs in mediating Bruce K. Cassels Nicotine (Figure 1) is the classical of a fam- synaptic transmission at muscle end plates and in Department of Chemistry, Faculty of Sciences, ily of ligand-gated cation channels that also respond autonomic ganglia, neuronal nAChRs in the brain University of Chile, to acetylcholine (ACh, Figure 2) as the endogenous have rarely been shown to fulfill this function, instead Casilla 653, Santiago, ligand, hence defined as nicotinic acetylcholine being found on presynaptic terminals or at extrasy- Chile receptors (nAChRs). These pentameric receptors are naptic locations on soma and dendrites. Together e-mail:[email protected] generated from different sets of subunits expressed with their capacity for interfacing with cellular sig- Isabel Bermúdez School of Biological and in mammalian skeletal muscle (α1, β1, γ, δ, ε), neurons naling pathways, this has generated the view that Molecular Sciences, (α2–α7, β2–β4) and sensory epithelia (α9, α10). In neuronal nAChRs exert a predominantly modulatory Oxford Brookes University, Gipsy Lane, autonomic neurons, nAChRs responsible for synaptic influence in the CNS [3]. Oxford OX3 0BP, transmission comprise α3 and β4 subunits, with the A modulatory action offers attractive therapeutic UK possible addition of α5 and/or β2 subunits (hence opportunities and interest has focused on emulating Federico Dajas J. Andrés Abin-Carriquiry α3β4*, where * indicates the presence of one or more nicotine’s effects by generating agonist molecules. Instituto de Investigaciones additional types of subunit [1]). α3β4* nAChRs are However, nicotine activates all subtypes of nAChRs Biológicas Clemente Estable, α α Avenida Italia 3318, also expressed in the central nervous system (CNS) (except those comprising 9 and 10 subunits [4]), C.P.11600 Montevideo, to a limited extent, whereas the α4 subunit is exclu- albeit with different potencies. Issues for drug discov- Uruguay sively expressed in the CNS. In combination with the ery include nAChR subtype selectivity and addiction Susan Wonnacott β α β Department of Biology and 2 subunit, 4 2* nAChRs are the most abundant liability. In addition to generating with low Biochemistry, and widespread nAChR subtypes in the brain, at muscle and ganglionic nAChRs (to min- University of Bath, exhibiting high affinity for nicotine. Homomeric α7 imize peripheral side effects), is it advantageous to tar- Bath BA2 7AY, UK nAChRs are also prevalent and are distinguished by get specific neuronal nAChR subtypes (e.g. α7 versus REVIEWS

self-administration, accompanied by plenty of associated

H3C H3C cues and Pavlovian reinforcement; nicotine replacement N N therapies demonstrate the low liability associated with alternative routes of delivery. H3C O Reviews • N N Agonist,antagonist or partial agonist? Nicotine ABT-418 To achieve clinical efficacy, is activation or inhibition of

DRUG DISCOVERY TODAY OCH H 3 nicotinic signaling required? This is a challenging question O N and the answer is likely to depend on the particular nico- NH tinic contribution to each clinical target. Nicotine, the OCH H3C 3 prototype with credentials for improving several conditions, N Cl O is regarded as an agonist of muscle and neuronal nAChRs PNU-120596 but it is also a powerful desensitizing ligand that can pro- duce functional deactivation of nAChR after a few sec- onds or minutes of exposure (depending on nAChR sub- FIGURE 1 type and nicotine concentration) [8]. Functionally, nAChR Chemical structures of nicotine, its isoxazole analogue ABT-418 and α7-specific potentiator PNU-120596. Reversing the heteroatom can exist in four distinct conformations: resting, open and positions in the isoxazole ring and introducing a hydrophobic two desensitized states. The latter are refractory to activa- extension of the basic sidechain changes an agonist into a positive tion on a time scale of milliseconds or minutes depending allosteric modulator. on the desensitized state but have high affinity (pM–nM) for agonists. Chronic nicotine exposure can induce a long-

Cl lasting functional deactivation as a result of rapid and per-

CH3 sistent desensitization. Clinical prescription of drugs will + H3C N H N involve chronic administration and the interplay of acti- H3C CH3 vation and desensitization is likely to be crucial, as well as O NH O O the contribution of potentially bioactive metabolites. At Acetylcholine PNU-282987 present, modeling this relationship for human brain Cl nAChRs is simplistic, even for nicotine in relation to S N N cigarette smoking [9], and impossible to predict for novel HN N ligands. However, because cognitive enhancing and neu- O O roprotective effects of nicotine are abolished by coad- O O ministration of nAChR antagonists [5,6,10], activation of AR-R17779 Mitsubishi compound nAChRs is generally sought and attention has focused on generating small agonist ligands. Tourette’s syndrome, on FIGURE 2 the other hand, might present a case for nAChR antagonists The acetylcholine pharmacophore, held in a rigid scaffold, (see below). produces selective α7 nAChR agonists. Partial agonists provide a compromise solution that could have particular utility, for example, for smoking ces- α4β2*)? Although the answer will differ for different clin- sation. In this regard, cytisine was noted for its selective ical scenarios, some studies have implied that α7 and β2* partial agonism at α4β2* nAChR: this subtype plays a subtypes conspire together to effect neuroprotection [5,6], major role in governing mesolimbic release in in which case selectivity would be inappropriate unless response to nicotine, which underlies the reinforcing combination therapy with complementary subtype-se- properties of the drug. In fact, cytisine (Tabex®) is marketed lective drugs could be achieved. And which nAChR sub- in Eastern Europe as an aid for smokers wishing to break types to target? To date, most effort has focused on tar- their habit. It has been argued that binding of a partial geting the most prevalent subtypes, α7 or α4β2 nAChRs agonist at this receptor would provide a modicum of [7]. Less abundant subtypes with more restrictedpatterns dopamine release to compensate for the absence of nico- of localization could offer more-selective therapeutic tar- tine (providing some relief from withdrawal symptoms), gets, but the potential for nAChR heterogeneity arising from whereas occupancy of the nAChR prevents nicotine from different subunit combinations, with subtle differences in binding [11,12]. The molecular interactions that result in properties or regulation, far exceeds our current knowledge partial agonism are not understood and it is problematic of native nAChR subtypes, making informed decisions to predict the activity of any derivative. about more-complex combinations difficult or impossi- Although beyond the remit of this review, it is worth ble at present. Addiction liability is probably not a major mentioning the current interest in another class of nico- concern, unless novel compounds are likely to be smoked: tinic ligand, nAChR potentiators [13]. Such molecules cigarettes provide the optimum delivery device for nicotine produce no obvious nicotinic response alone but enhance PNU-120596 an subtypes, nAChR between Clinical targetsfornicotinicClinical drugs [ activity anti- its to addition in responses, thamine is credited with allosteric potentiation of nicotinic galan- Alzheimer’sdrug The (AD) ACh). disease nous endoge- (including agonists conventional to responses correlates withcognitiveimpairment[ sue from patients, which, in the case of AD, significantly by the tis- consistent loss of nAChRs in post-mortem brain An involvement of nAChRs in AD and PD is also supported Neurodegenerative diseases normal in individuals with pathological disease states, rather than by nAChR activation. These effects are more readily seen as the most likely component to be positively influenced cognitive function has identified attentional performance Analysis oftheabilitynAChRstimulationtoimprove Cognitive performance of a negative correlation between smoking and the incidence form of tobacco products. Epidemiological evidence shows prompted initially by the effects of nicotine taken in the The development of potential nicotinic therapeutics was familial epilepsy[ depress nAChR responses, has been advocated for treating modulator,negative to subtype-selective a searchfor A ment develop- the encourage and skills nonverbal and verbal reported improvementinattentionalcapacityandsome have patients AD in agonists nicotinic or nicotine of ramn so n ipoeet r des effects adverse or improvement no show treatment nicotine transdermal using others symptoms, PD some Although some studies demonstrate that smoking alleviates results. inconsistent generated have PD in nicotine of with equated consumption, tobacco increased show (ADHD) adolescents withattention-deficithyperactivitydisorder Patients with schizophrenia or other mental illnesses and on effect neuroprotective putative a exerting to addition in of nicotine and other agonists to evoke dopamine release, benefit fromnewnicotinicdrugs. might that conditions clinical diverse several identified have studies, animal with together observations, Such conditions. these worsens smoking quitting and colitis ulcerative and Tourette’ssyndrome improves smoking ment, schizophrenia,ADHD,ADandPD[ nists cognitive Current targets for nicotinic drug development for improved 13

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Ligand evaluation: the challenge for drug screening models NH NH The diversity of nAChRs presents a challenge for the Cl Cl N N development of relevant high throughput screens to ascer- tain drug selectivity. This is exacerbated by pharmacologi- Reviews • Epibatidine UB-165 cal differences between nAChR homologues of different species [38,39], despite a high sequence homology across

DRUG DISCOVERY TODAY Y NH species for individual subunits [40]. Heterologous expres- sion of human recombinant nAChRs has addressed the X N target species but is compromised by our limited knowl-

Deschloro-3'-arylepibatidines edge of the subunit composition of native human nAChRs. However, the expression of functional nAChRs with defined subunit combinations has contributed to the identification FIGURE 3 of putative native subunit combinations and potential ther- Chemical structures of epibatidine and synthetic analogues. apeutic targets, and to this end provides the most practical tool for studying the pharmacological and functional prop- and the development of nicotinic agonists as erties of identified nAChR. To the first approximation, phar- started only after the description of the antinociceptive macological and functional properties of heterologous and effects of epibatidine (Figure 3), which exceed those of native nAChR are similar [41–44], although biophysical [30]. Epibatidine’s antinociceptive activity is properties can be affected by expression of human nAChR compromised by serious side effects at similar doses, inXenopusoocytes [44]. An important limitation of current reflecting its relatively nonselective interaction with transient expression systems is the difficulty in obtaining nAChR subtypes; analgesia is attributed to α4β2* nAChR homogeneous populations of heteromeric nAChRs, partic- but other subtypes could also contribute [31,32]. ularly of receptors containing more than two different sub- units (e.g. α6β4β3α5 [45]). Furthermore, current stable or Smoking cessation transient expression technologies do not allow the expres- Combating nicotine addiction represents the most tangi- sion of a homogeneous nAChR population in which only ble case for a nicotinic therapy. Nicotine itself is available one of several otherwise identical subunits is mutated, as in various delivery systems for nicotine replacement ther- in many dominantly inherited channelopathies [16]. apy: although this strategy doubles the success rate of In addition to the application of heterologously expressed smokers attempting to quit the habit, approximately three nAChRs for ligand binding and electrophysiological assess- out of four have failed to give up smoking when assessed ment of novel ligands, downstream functional readouts one year later. The antagonist mecamylamine can also (e.g. changes in intracellular Ca2+) can be used [46]. Native ameliorate some nicotine-induced effects in smokers in nAChRs expressed in neuroblastoma cell lines (e.g. the clinic but results in increased craving [33]; a partial human SH-SY5Y cells) offer an alternative to heterologous agonist approach has recently been advocated [11,12]. expression but such preparations represent autonomic Intriguingly, nicotinic mechanisms might also contribute (rather than CNS) neurons. nAChR-mediated transmitter to the self-administration of other drugs, including release from brain slices has been adapted for a 96 well and [33], but the therapeutic potential of nicotinic format, to provide an assay of native (albeit ) nAChRs: drugs for treating other addictions has not been explored. this is a complex model, as several nAChR subtypes can reg- ulate transmitter release in each brain area [47]. Ulcerative colitis The interpretation of biochemical and electrophysiolog- This chronic relapsing inflammatory bowel disease is ical results, as well as the design of novel ligands, is expected largely restricted to non- or ex-smokers. Consequently, to rely increasingly on the computational modeling of transdermal nicotine [34] and nicotine enemas [35] have nAChRs (See Box 1). been tested and found to improve the symptoms. The underlying mechanism is assumed to involve nicotinic Templates for nicotinic drug design: three natural stimulation of parasympathetic innervation or possibly products modulation of local inflammatory responses [36]. However, Hereafter we focus on three natural products that have the adverse effects associated with nicotine, especially provided templates for many of the nicotinic drug dis- with transdermal delivery, encourage the development of covery programs. For a comprehensive review of nicotinic better nicotinic drugs to combat this disease. The recent ligands the reader is referred to Jensen et al.[7]. finding that α7 and α4β2 nAChRs regulate B-lymphocyte activation and immune responses suggests a possible new Nicotine therapeutic approach to a variety of other inflammatory Nicotine (Figure 1) was in the 1990s the most obvious conditions [37]. choice as a lead for the development of nicotinic receptor agonists. SARs at SARs agonists. reviewed ligands known until the year 2000 have been thoroughly on the carbon atoms leads to losses of losses to leads atoms carbon pyrrolidine the on ethyl or monofluoromethyl methyl, than other stituent with groups larger than ethyl is not tolerated and any sub- tolerated but substitutions at any other position on the on position other any at substitutions but tolerated and amethyl or ethylgroupatthesameposition iswell enhanced five-fold) to (two- slightly with compounds affords epibatidine, of carbon halogenated or bromine atom at the C-6 much greater affinity. Introduction of a fluorine, chlorine clo[2.2.1]heptane system of epibatidine, to analogues with 7-azabicy- the of case the in and, nicotine to affinities similar with compounds to cases some in leads amines However, replacement of the pyrrolidine ring by bicyclic increase. slight a causes analogue azetidine the to tion an in results its or natural the to sion affinity of more than one order of magnitude. Ring expan- homomeric particularly nAChRof domain subunits, extracellular N-terminal, the with sharessequencesimilarity novel protein proteinbinding (AChBP).This acetylcholine HEPES-bound the of structure crystal of use the recentprogress in assays, complementtoAsa ‘wet’screening Predictive [ sitehavebinding tothe published been docked cytisine and epibatidine nicotine, ACh, with nAChR of subtypes domains ligand-binding the severalof models 3D template, [ determinedvaluesexperimentally orderof forligands these reproducedepibatidine rank nicotineand the acetylcholine, [ indicatedbyCelieclearly efficacies.As ligand estimatesof providecannot theygood reasonablywell, affinities) (and AChBPreproducethe binding on structure agonist based models although receptorrolein dynamics.Thus, important toplay an areexpected receptorstatic a conformation, toforbinding crucial agonist not if which, nAChR, of domain intracellularmembrane-spanninghelicesand the absenceof communication]. personal Zapata-Torres, compounds[G. new of affinities of for useful prediction be might resultsand arereliabletoolstointerpretmodels these experimental that suggesting reproducedaffinities, properorderof the variousalso neuronalof nAChR tohuman models subtypes N with ACh)nicotine= agreement> in order(cytisine indicatedan n of membranedomains the microscopyof data Torpedo gating the of structure X-raythe of crystal use the drawbackbeen has toovercomehelp hopefully developmentwill that this recentmost a However, receptorexplain couldgating. that BOX1 81 82 78 A -methylcytisine and severalhalogenatedderivativesand their of -methylcytisine An important limitation of such modeling studies is the is studies modeling such of limitation important An ChRs –84] ] ]

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FIGURE 4 Cytisinoids exhibit a wide range of potencies and efficacies.(a)Chemical structures of 3-BrCy,5-BrCy,varenicline [12] and SSR591813 [11]. (b) nAChR-evoked [3H]dopamine release from rat striatal slices in vitro. In this assay, governed mainly by α4β2* nAChR, cytisine is a partial agonist, whereas 3-BrCy is more potent and more efficacious. 5-BrCy and 3,5-BrCy have diminished activity compared with cytisine (Abin et al., personal communication). (c) Time courses for the effects of nicotine (0.32 mg/kg s.c.; open circles) and a maximally effective dose of varenicline (1.0 mg/kg p.o.) alone (filled squares) and in combination with nicotine (triangles) on extracellular dopamine levels in the nucleus accumbens of conscious Sprague- Dawley rats.Varenicline was administered 1 h before nicotine (arrows), and effects on dopamine release are expressed as a percentage of baseline (mean of last 5 predrug basal levels) ± SEM (n = 4–6). *p<0.05: varenicline with nicotine versus nicotine alone (2-factor analysis, repeated measures, Western-Electric).Varenicline alone is less efficacious than nicotine, but it also reduces the response to nicotine, consistent with a partial agonist interaction with the nAChRs mediating this response.Part (c) reproduced,with permission,from Ref.[12].(d)Effects of SSR591813 at human α4β2 nAChRs expressed in Xenopus oocytes. Concentration response curves for epibatidine, DMPP and SSR591813 show that SSR591813 has low efficacy relative to the other agonists with respect to evoking whole cell responses. For each agonist, data points indicate the mean ± SEM of current amplitudes (n= 2–6 oocytes),normalized to 100 µM DMPP.Part (d) reproduced,with permission,from Ref.[11].Abbreviations:3-BrCy,3- bromocytisine; 5-BrCy, 5-bromocytisine; s.c., subcutaneous; p.o., per os (orally).

been performed, these compounds are less potent than and weak or negligible affinity for α7 nAChR, combined epibatidine. Unexpectedly, UB-165 (Figure 3), a synthetic with weak agonist or antagonist activities in pain, body hybrid of the efficacious α4β2* nAChR agonists epibatidine temperature or spontaneous activity tests. The high affinity 3 and anatoxin-a, is a partial agonist at this subtype [60]. (Ki = 0.029 nM versus [ H]epibatidine in rat brain cerebral Recently, a series of deschloroepibatidine analogues cortical membranes), weak agonist and potent antagonist bearing halogen or other small substituents at C-3′ were activity of N-methyl-3′-iododeschloroepibatidine have led characterized [61]. These compounds resemble C-3′- to the suggestion that its 11C and 125I analogues might arylepibatidines and deschloro-2′-substituted epibatidines be useful as ligands for in vivo studies of nAChR using (Figure 3) [62,63] in their high affinity for α4β2 nAChR positron emission tomography and SPECT, respectively [61]. ( Another partial agonist with similar topology is SSR591813 nAChR erae a brain at decreases as respect to dopamine release and has entered clinical trials autonomic as possiblenicotinicdrugs( testing for compounds novel and new of synthesis the for template a as recently very exploited been only has ity nonganglionic nAChR subtypes, increasing the lipophilic- at efficacy and affinity in changes possible of Regardless as agonist nicotinic a as recognized was cytisine Although Cytisine cytisinoids, is the potent 3-substituted from derived structure symmetrical a with However, the most structurally distant cytisine congener, of reports appeared on the affinities and functional properties later,year however,A first authors. the these by reported emitting its affinity and functional potency functional and affinity its and N- ening the effects of its derivatives in the brain. Nevertheless, height- thus penetration, barrier blood–brain improve to ( toward in several-fold reductions in affinity and functional potency ( tives were prepared in this way deriva- of moiety.number this large of A character matic which is easily accomplished as a result of the partially aro- α sine, of bromo-derivatives two including results the on groups hydrophobic introducing tion dihalogena- and decreases modest in results C-11) (or C-5 potency,at functional halogenation and whereas affinity designated in some papers as C-9) always leads to increased (also C-3 at substitution halogen that is emerges that tion muscular rat expressing cells in homomeric derivatives tested have much lower affinity and potency at tested and prepared was oxygen pyridone the of place in atom subtypes, asisalsothecaseforparentcompound. Carriquiry cytisinium Figure Figure Figure 4

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with [ were [ later [ new 12 ,72] 66 11 α 7, ]. ]. ]. form not does nicotine and bond hydrogen conventional a cationiccenterofACh isunabletoform The quaternary atom. oxygen carbonyl backbone the to bond hydrogen nated nicotine from the centroid of the Trp proto- of atom nitrogen charged the separating distance (C–H bond hydrogen nonclassical a by reinforcedfurther be might nicotine) of extent lesser a to (and epibatidine of centre of the more-positive electrostatic potential of its cationic because stronger be to expected is epibatidine in action Trp site active the to binding its from part in results tidine epiba- of potency high the that conclude authors These ring tor accep- bond hydrogen a and charge positive positioned ology used, beyond confirming the relevance of a properly potent activity, regardless of the method- for requirements structural the of understanding our to inated iod- or brominated when cytisine of nAChR neuronal for remain unexplained. Thus, the strong increases in affinity of the of nicotine and epibatidine analogues, on one hand, and atom [ quence of the electron-attracting character of the chlorine positive charge in epibatidine than in nicotine as a conse- partial greater a bear to expected is which ring, pyridine lated for(+)-and to [ regarded as a 3-substituted, ring-contracted cytisinoid isostere to ing the binding affinities of ACh, nicotine and epibatidine work of ‘’ and ‘nicotinic’ receptors by measur- frame- the within rationalized been has difference This nAChR. neuronal and neuromuscular at agonist potent than AChattheneuromuscularnAChR,epibatidineisa multi-objective geneticQSAR[ structure-affinity relationshipsandmodelsgeneratedby quantitative comparison, distance using reached were their analogues is their low affinity for A common feature of nicotine, epibatidine, cytisine, and Commonfeatures differencesand of affinity,orientation appropriate an ensuring by possibly enhances also epibatidine of system bicyclic saturated the by contributed volume the that addition in gesting sug- features, favorable are epibatidine of atom chlorine the near area the in interactions electrostatic and steric that showed analogues epibatidine of set large a on sis field analysis and comparative molecular similarity analy- 12

The different effects of substitution on the pyridine ring Whereas nicotine has 70-fold lower agonist potency agonist lower 70-fold has nicotine Whereas α the

nAChR ], α [ 4

74

[ 149 residue via a strong cation- strong a via residue 149

β ... have 78 a

2 nAChR. 3D QSAR analyses have not added much 76

α O=C) involving the hydrogen atom at C-2 of the of C-2 at atom hydrogen the involving O=C) as charged ].

at strong -pyridone ring of cytisinoids, on the other hand, ]

]. Recentl compared

is C-3

little

similar mutated

[

cation- 69 nitrogen parallel y , ( ]

−

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study

with the π

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of

76 nicotine [ nicotine 75

comparative ]. 76 ].

], thecation Similar [ π

77 amino α interaction and a and interaction

7 nAChR relative [

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]. 79 and distances

Although α ].

149 benzene

conclusions The epibatidine

acids

molecular might REVIEWS

- binding π

calcu-

inter- [

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be ].

Reviews • DRUG DISCOVERY TODAY REVIEWS

series. Introduction of a halogen atom (F, Cl, or Br) at the for rat brain high affinity nAChR binding sites, whereas C-6 position of nicotine, corresponding to the C-2′ posi- (+)- and (−)-epibatidine are almost indistinguishable [58], tion of epibatidine and neighboring the putative hydrogen is also rather surprising. However, the latter observations bond accepting site, as is the case for the C-3 position of should be regarded with caution, as the behavior of some cytisine, results in very modest (

DRUG DISCOVERY TODAY deleterious effect [76]. Removal of the C-2′chlorine atom Conclusions of (+)- or (–)-epibatidine and its replacement by fluorine, Extensive structure–activity studies taking natural nico- bromine or iodine has almost no effect on their affinities tinic agonists as the starting point have begun to reveal [58]. The functional potencies of these nicotine, epibati- some rules for enhancing agonist potency, although these dine and cytisine analogues have been assayed in widely rules are often not intuitive and improvements in nAChR differing models and are not easily comparable. Nevertheless, subtype selectivity have been more elusive. To date, few the available results also point to varying trends suggestive novel nicotinic compounds have succeeded in clinical of different binding modes or mechanisms of activation. trials, emphasizing the limitations in translation from Another striking difference is the relative insensitivity of screening models to clinical application. However, by cou- nicotine and epibatidine to the presence or absence of an pling structure–activity studies with recent advances in the N-methyl group, whereas N-methylation of cytisine leads modeling of ligand interactions with nAChR binding sites, to sixfold, 25-fold and 300-fold losses of affinity for α4β2, the future looks promising for more-successful predictive α7 and α4β4 nAChR, respectively [69]. The fact that nico- drug design to generate new nicotinic drugs for the diverse tine enantiomers differ by a factor of 30 in their affinities conditions where they could offer therapeutic benefits.

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