Allosteric Modulation and Potential Therapeutic Applications of Heteromeric Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (nAChRs) have been implicated by behavioral, anatomical and pharmacological studies, in a wide range of neurological pathways and disorders and consequently these receptors are widely viewed as highly promising therapeutic targets for drug development. nAChRs can be either homomeric or heteromeric, with the heteromeric α4β2 subtype representing the most prevalent neuronal subtype in the CNS. α4β2 receptor ligands have been shown to be particularly efficacious in animal models of nicotine and drug addiction, cognition and neuropathic pain. Over the past decade a new drug class has emerged capable of enhancing or attenuating responses induced by agonist stimulation of nAChRs. These compounds typically act by noncompetitive mechanisms and thus are known as positive or negative allosteric modulators (PAMs and NAMs, respectively). Many of these PAMs appear similar in action to the benzodiazepine and barbiturate drug classes that act on the structurally related GABAA receptors. PAMs and NAMs have the potential to provide therapeutic modulation of nAChRs in vivo to treat nicotinic related disorders. Development of this drug class will require a thorough understanding of the structure-function relationships of these compounds, identification of binding domains and in vivo characterization. This review will discuss the primary lead compounds identified to date that allosterically potentiate or inhibit heteromeric nAChRs, including α4β2 and α3β4 subtypes, with a focus on the current understanding of these structural and functional relationships. To cite this article: Marvin K. Schulte, et al. Allosteric modulation and potential therapeutic applications of heteromeric nicotinic acetylcholine receptors. Licensed under a Creative Commons Attribution 4.0 International License which allows users including authors of articles to copy and redistribute the material in any medium or format, in addition to remix, transform, and build upon the material for any purpose, even commercially, as long as the author and original source are properly cited or credited.


Introduction
Neuronal nicotinic acetylcholine receptors (nAChRs) are members of a large, heterogeneous family of ligand-gated ion channels distributed throughout the CNS.Postsynaptic nAChRs are responsible for supporting fast transmission at cholinergic synapses, but most CNS nAChRs are located presynaptically and modulate the release of other neurotransmitters, including dopamine, norepinephrine, serotonin (5-HT), and glutamate [1,2] .Consistent with their broad distribution in the brain, nAChRs have been implicated in a wide range of cognitive processes, including arousal, sensory processing, anxiety, attention, and learning and memory [2,3] .
In the past few years, a large amount of research has REVIEW focused on the homomeric α7 nAChR, which is expressed at particularly high levels in the hippocampus and has been implicated in the etiology of Alzheimer's disease [4] .However, the most abundant neuronal nAChRs are the heteromeric nAChRs.These include α4β2 receptors, which comprise up to 80% of nAChRs in the CNS, and the α3β4 receptor, which is the predominant subtype in the autonomic nervous system and in certain brain regions (e.g., habenula) [1] , The heteromeric nAChRs have been shown to be particularly important in drug and nicotine dependence [5] , Parkinson's disease [6] , depression [7] , and pain [8][9][10] .Alongside the α7 receptors, the heteromeric nAChRs (particularly the α4β2 receptor) may also play critical roles in age-related cognitive impairment, Alzheimer's disease, and attention-deficit hyperactivity disorder (ADHD) [11, 12]   .There is also emerging evidence for involvement of heteromeric nAChRs in diseases affecting non-neuronal tissues, particularly cancer [13][14][15][16] .

Therapeutic opportunities for modulators of heteromeric nAChRs
Given the potential therapeutic value of nAChRs as drug targets in CNS disorders it is not surprising that significant efforts have been made to develop commercial therapeutic drugs that act as selective nAChR agonists and antagonists (for reviews see Arneric [17] and Decker [18] ).However, only one drug, the α4β2 partial agonist varenicline, has been approved for clinical use [19] .While efficacious, the use of nAChRs agonists and antagonists may be limited due to their direct mechanism of action.For example, activation by agonists can produce receptor desensitization during continuous exposure and produce inhibition rather than stimulation.Chronic exposure can also lead to alterations in receptor expression as is observed during long term exposure to nAChR agonists such as nicotine, which produces up-regulation [20][21][22][23][24][25] .A similar effect has been observed for nAChR antagonists [26] .
Over the past 10 years, there has been a growing interest in the use of positive and negative allosteric modulators (PAMs and NAMs, respectively) as an alternate or adjuvant therapy to direct agonists and antagonists.PAMs and NAMs typically produce little or no stimulation of receptors themselves but potentiate or attenuate the response only when an agonist is present.This has the potential advantage of preventing receptor desensitization by the drug and maintaining the control of synaptic signaling by pre-synaptic release of endogenous agonist.This is particularly true of PAMs that alter the apparent efficacy of the agonist since they effectively increase the peak synaptic current at all agonist concentrations, although prolonged open channel times can lead to channel block and produce inhibition by agonists at these concentrations.One disadvantage of PAMs is the potential of the drug to substantially slow receptor desensitization rates leading to increased receptor activation as has been observed for type II PAMs on α7 receptors [27,28] .
Allosteric modulators have been shown to have potential benefit in pain management [8] , cognitive enhancement [29] , and nicotine addiction [30] .Unfortunately, only a small number of heteromeric selective nAChR allosteric modulators have been studied sufficiently to permit a rational drug design approach.There is a substantial need for more comprehensive medicinal chemistry studies of these compounds and concurrent studies of where and how they bind to and modulate nAChRs.This review provides a general overview of PAMs and NAMs identified for the heteromeric nAChRs, and more detailed discussions of compounds for which putative binding sites have been identified and/or significant structure function studies have been conducted.

Heterogeneity and distribution of heteromeric nAChRs
The nAChRs are members of the cys-loop superfamily of receptors, which also includes GABAA and GABAC, serotonin type III (5-HT3R), and glycine (Gly) receptors.nAChRs are large pentameric membrane proteins, of molecular mass ~290 KD, formed by combinations of five identical (i.e., homomeric) or different (i.e., heteromeric) subunits.The subunits that contribute to neuronal nAChRs are α (α2-α9) and β (β2-β4) [31,32] .Different combinations of α and β subunits generate a plethora of different receptor subtypes, of which the three most prominent in the nervous system are α4β2, α3β4, and α7 [1,33] .The α4β2 and α3β4 receptor subtypes may also contain subunit substitutions (such as α5 containing receptors) that alter the receptors' response characteristics and pharmacology.α4β2 receptors exist in at least two different stoichiometries: (α4)2(β2)3, which has high affinity for acetylcholine (ACh) and nicotine, and (α4)3(β2)2, which has low affinity for acetylcholine and nicotine.These two receptors are referred to as high sensitivity (HS) and low sensitivity (LS) receptors, respectively [34] .Different stoichiometries for α3β4 receptors have also been observed in vitro, depending on the ratio of different cRNAs used, but the in vivo relevance of these in vitro observations has not been determined [35] .The α4β2 receptor is expressed throughout the CNS.It is the predominant nAChR type in the thalamus and striatum, consistent with strong evidence for α4β2's involvement in pain and Parkinson's disease [1,6,33,36,37] .The α4β2 receptor also ranks alongside the α7 receptor as one of the two most abundant nAChRs in the cerebral cortex and hippocampus [1] .In contrast, the α3β4 receptor is highly expressed in just a handful of circumscribed brain regions, including the habenula, pineal gland, and certain cranial nerve nuclei.Habenular α3β4 receptors have been found to play a critical role in addiction to nicotine and other drugs [38][39][40] .

Allosteric modulation of nAChRs: binding and mechanism
Multiple ligand binding sites are present on nAChRs.The endogenous ligand ACh and other agonists bind to the orthosteric binding sites, which are located on the extracellular domain of the receptor, at the α + /β -and α -/α + subunit interfaces of α4β2 receptors and at the α + /β -interface of α3β4 receptors [ 41-44]   .It has not yet been determined if alternate stoichiometries of the α3β4 receptor produce an orthostatic binding site at the -/α + interface of these receptors.Allosteric ligands bind at a number of different binding sites, which are unique to different classes of allosteric ligands.
Allosteric ligands typically produce no channel opening but rather alter the functional properties of the agonist-activated receptor.Allosteric ligands that potentiate agonist responses are commonly referred to as PAMs, and those that attenuate responses are known as NAMs [45,46] .Studies of the mechanisms of action of PAMs and NAMs have focused primarily on population level receptor function, although there is some data available to identify specific mechanisms at the receptor/ion channel level.In whole cell approaches, such as the commonly used Xenopus oocytes recording system, the PAM or NAM is co-applied with an agonist and produces an increase or decrease in its Imax (agonist efficacy) and/or a shift in the concentration-response curve (agonist potency).The usefulness of different modulators will likely depend on their effects on agonist potency or efficacy.Modulators that increase efficacy have the advantage of enabling potentiation even when high agonist concentrations are present in the synapse, whereas modulators that do not alter efficacy may provide little synaptic modulation but may be useful in modulation of extrasynaptic receptors where agonist concentrations are lower.
On a single channel level, changes in efficacy produced by PAMs and NAMs can reflect any number of effects including changes in the number of receptors available for activation, changes in open or closed state stability, first latency of opening, changes in receptor inactivation, frequency of opening, changes in conductance, or other alterations in receptor kinetics.Extensive characterization of the action of allosteric modulators at the single channel level is required to adequately link the chemistry of the ligands to specific receptor mechanisms, and would be of great value in drug development and classification of modulators.The use of modulators in these studies may also reveal currently hidden conformational states and provide improved models of receptor mechanism.

Non-competitive Antagonists: α3β4 and α4β2 channel blockers
Some of the earliest identified non-competitive inhibitors of nAChRs have been shown bind to the channel pore where they reduce current by blocking ion flow through the channel [47]   .Drugs like amantadine, memantine and dizocilpine fall into this class of compounds and are commonly referred to as channel blockers [48] .While sometimes referred to as NAMs, these compounds are better described as noncompetitive antagonists since they primarily inhibit ion conductance by binding in the channel lumen, whereas allosteric modulators bind to nonluminal sites to modulate receptor function.While there are a large number of channel blocking agents for nAChRs, one of the most complex appears to be mecamylamine.

Mecamylamine
Mecamylamine (Figure 1A) is noncompetitive antagonist of human neuronal nAChRs and has been used clinically as an oral hypotensive agent (Inversine) [49,50] .Mecamylamine has also been found to increase nicotine self-administration [51,52] but to reduce cocaine [53,54] and alcohol consumption [55] .Its mechanism of action is typically considered to be inhibition of the ion current through channel blockade but recent studies have revealed a much more complex pharmacology for this compound [56] .Usually available as a racemic mixture, the two enantiomers of mecamylamine, S-(+) and R-(-), have been shown to have different effects on the HS and LS subtypes of α4β2 receptors.The S-(+) enantiomer selectively potentiates HS and inhibits LS receptors while the R-(-) enantiomer inhibits the HS but not the LS subtype expressed in a human epithelial cell line [57] , although this result could not be reproduced in a Xenopus oocyte system [56] .Differential effects on different HS and LS receptors suggests that the two enantiomers of mecamylamine could be exploited as drug leads to develop subtype selective drugs with either potentiating or attenuating effects on agonist responses if specific binding site differences producing these effects can be identified.Channel blockade appears to result from the binding of mecamylamine to the channel lumen of α4β2 and α3β4 receptors [50,58] .Molecular docking studies showed that protonated mecamylamine (predominate at physiological pH) binds at the interface of the two M2 helices of the α4 and β2 subunits, towards the extracellular mouth of the receptor [59] .In an α4β2 receptor docking model, protonated mecamylamine was shown to bind to the M2 pore region between position 17' and position 20' along the ion channel lumen [60] .A similar binding position was observed for mecamylamine on hα3β4 receptor models [58] .Recent NMR and modeling studies have provided additional detail on these interactions for the S-(+) and R-(-) enantiomers and their different interactions with human HS and LS α4β2 receptors [61] .These studies confirmed the α4/β2 binding position in the extracellular mouth of the channel but also identified other luminal and non-luminal sites.The two enantiomers interact at two different luminal sites, one located at the extracellular end of the channel mouth and the other at the cytoplasmic end.Interacting residues differ between the α4 and β2 subunit, for example a cysteine (C260) on the β2 subunit is replaced by a glutamate (E266) in the identical position of the α4 subunit [61]   , thus subunit composition plays a role in the inhibitory effect.Multiple non-luminal sites that differ slightly between HS and LS receptors were also identified.A significant difference between enantiomers is seen at a site close to the α4 TM3 region that interacts with the R-(-) enantiomer but not the S-(+) [61] .This type of extremely detailed structural analysis has not been done for other modulators but is necessary if the complex effects of compounds like mecamylamine are to be understood and the different binding sites evaluated as drug targets.

Dextromethorphan (DM)
Dextromethorphan (DM, Figure 1A) is commonly used clinically for treating cough.It may also potentially be useful for the treatment of morphine addiction, as it reduces selfadministration in rats (likely due to its structural similarities with morphine) [62] .Other nAChR-related disorders including nicotine addiction are also potential targets for DM [62][63][64] .DM does not displace [ 3 H]-epibatidine in competitive binding assays; hence, it likely binds to a non-orthosteric binding site and inhibits α3β4 receptors in a noncompetitive fashion [63] .DM is also an inhibitor of the NMDA-subtype of GluRs [64] .Through affinity chromatography, DM and its enantiomer levomethorphan (LM) were found to bind with micromolar affinity to the transmembrane region of the α3β4 receptor [65] .Selectivity of DM and LM for the M2 helix of the β4 but not the β2 subunit may be due to sequence variations in the putative binding domain for DM and its analogs [66] .The β4 subunit differs from the β2 subunit at channel position 13', where a phenylalanine is present instead of valine.The presence of this phenylalanine provides a favorable binding interaction for DM [64,66] .According to docking studies, DM binds through its amino group to a serine at position 6' in the M2 region (S248), possibly by hydrogen bond or cation-π interactions.LM does not make similar interactions, possibly due to its different orientation [64,66] .

Potential lead molecules in the development of negative allosteric modulators (NAMs)
A few lead compounds have been identified that inhibit heteromeric receptors via non-channel blocking mechanisms.Like some of the proposed sites for mecamylamine, these compounds appear to bind outside the channel lumen and at locations separate from the orthosteric binding sites.As they appear to produce their effects by altering receptor conformations they are properly classified as NAMs.Structural studies of these compounds and their binding sites could yield novel NAMs and PAMs.UCI-30002, HDMP, and KAB-18 are among the best studied lead molecules in this class.

N-(1,2,3,4-tetrahydro-1-naphthyl)-4-nitroaniline (UCI-30002)
UCI-30002 is a non-selective NAM of neuronal nAChRs [67]   .Efficacy of inhibition was 100% at α7 and α34 receptors and 80% at α42 receptors.This compound has been shown to reduce nicotine self-administration in rats, an effect apparently selective for nicotine as no change in food reinforcement was observed [67] .Inhibition appears voltageindependent, suggesting that it does not involve ion channel blockade [67] .Like other non-channel blocking NAMs, UCI-30002 may be useful for exploring the allosteric binding sites on these receptors, particularly if its binding domain overlaps those utilized by PAMs.As UCI-30002's NAM activity is independent of agonist concentration, this drug should be capable of inhibiting responses even if high concentrations of ACh are present in the synapse.
HDMP (Figure 1A) displays high potency at α7 receptors (IC50=70 nM) and is significantly less potent at α4β2 and α3β4 receptors (25-30 µM) [68] .HDMP does not compete for binding with either [ 3 H]-epibatidine or [ 125 I]methyllycaconitine (MLA), indicating a noncompetitive interaction at α7 and α4β2 receptors [68] .Inhibition was voltage-independent at all three receptor types, suggesting that HDMP is not an open channel blocker.In rats, HDMP was found to be a potent antagonist of nicotine-induced analgesia in the tail-flick test but not the hot plate test [68] .These results are consistent with evidence that nicotine-induced analgesia in the hot-plate test is largely mediated by α4β2 receptors [69] , and suggest that nicotine analgesia in the tail-flick test is mediated in part by α7 receptors.The further development of HDMP analogs could lead to drugs selective for specific kinds of pain.
Since a binding site for HDMP has not been identified, a better understanding of the molecular interactions of this compound with different receptor subtypes would be valuable, as it could reveal differences among receptors that might be exploited to develop subtype-selective NAMs [68] .In addition, determining whether the allosteric binding site for HDMP overlaps with known PAM sites would improve our understanding of allosteric modulation of nAChRs.

Biphenyl-2-carboxylic acid 1-(3-phenyl-propyl)-piperidin-3-ylmethyl ester (KAB-18)
KAB-18 (Figure 1A) is the lead compound of a series of NAMs of the α4β2 receptorone of the few series of NAMs that have been subjected to extensive structure-activity relationship (SAR) analyses [70] .Also significantly, a putative binding domain for this series of compounds has been identified and characterized [71,72] .KAB-18 selectively inhibits α4β2 receptors with an IC50 around 13.5 µM, with no inhibitory effect on α3β4 at concentrations up to 100 µM.
The KAB-18 molecule contains three phenyl rings and one piperidine ring and can be roughly divided into four regions.The three phenyl rings comprise region 1 and region 2 and the piperidine ring forms region 3.A fourth region is defined by the ester bond linkage that connects regions 2 and 4.An extensive SAR study for KAB-18 identified how each region of the molecule contributes to its affinity and selectivity [70] .Modifications in regions 1 and 2 in particular appear to change its potency and selectivity.Potency can be increased by hydrogen, methyl or dimethyl substitutions on the phenyl rings in region 1, but at the cost of selectivity.Substitution of the α-carbon with a carbonyl group increases potency while retaining selectivity.In region 2, which contains two phenyl rings, addition of an oxygen between the rings removes selectivity for α42 and α34, and replacement of the oxygen with an amine reduces potency on α42 receptors but increases it on α34 receptors.Selectivity is lost if the phenyl ring is replaced by smaller methoxy, chloride or acetamide The α4β2 receptor structure is shown only to illustrate the location of the putative binding regions on the amino terminal domain of the receptor and is not intended to represent a molecular model of the receptor binding site.Only three of the five subunits are shown and illustrate (from left to right) the α4 -face, the α4 + α4 -interface, and the β2 + face.The illustration was generated based on the refined Torpedo nAChR α1 subunit structure [73] (2BG9).Deepview software (SIB Swiss Institute of Bioinformatics) was used to mutate residues to match the α4 or β2 subunit sequence, to merge the three subunits shown in the figure and to highlight the position of key binding regions and amino acids within the overall receptor.Citations to the source research studies are provided in the text descriptions for each compound.
groups.Modification of the piperidine ring (region 3) had less impact on potency and selectivity, although a 3hydroxymethyl substitution to the 4 th -position reduced selectivity and increased the potency on α4β2 [70] .Increased stability of the compound by replacement of the ester linkage with an amide had little effect on either potency or selectivity.
The importance of regions 1 and 2 of the molecule are reflected in molecular modeling studies and site directed mutagenesis [70,71] .The putative binding site for KAB-18 is just 10 Å away from the orthosteric binding site of the α4β2 receptor [72] .Selectivity is mediated by interaction with the 2 subunit, and those regions of the molecule identified by SAR as important for β2 binding make contacts at positions where the amino acids differ substantially on the 2 versus 4 subunits: 1) S112 and K78 of the β2 subunit make contacts with region 1 of KAB-18, where the equivalent 4 amino acids are arginine and isoleucine, respectively; 2) a methionine (M35) of the 2 subunit that makes contact with region 2 of the molecule is replaced by glutamine on the β4 subunit; and 3) T58 and F118 of the β2, which make contacts with regions 3 and 4, and are replaced by lysine and leucine on the 4 subunit.Portions of this model have been confirmed by mutagenesis studies with three major amino acids (Phe118, Glu60, and Thr58) on the β2 subunit side of the binding site demonstrated as important to KAB-18 NAM activity (Figure 2 and 4) [72] .

Galantamine
Galantamine is an alkaloid found in plants of the Amaryllis family [74] .Pharmaceutical synthesis of a stereospecific product was published by Lilly in 2007 [75] .Galantamine has been used clinically in the treatment of cognitive impairment in early Alzheimer's disease and explored as a potential therapeutic treatment in autism [76,77] .Galantamine was first identified as an acetylcholinesterase inhibitor.Later, it was also shown to potentiate α4β2 receptors via a shift in the agonist concentration-response curve [78,79] .The therapeutic significance of galantamine's PAM activity is unclear considering that cholinesterase inhibition is likely to be its predominant effect given the likely high concentration of ACh at cholinergic synapses, particularly in the presence of a cholinesterase inhibitor.Nonetheless, potentiation of extrasynaptic receptors could have biological impact.Galantamine's potentiation of hα4β2 receptors was confirmed using recombinant receptors expressed in HEK-293 cells, but this effect was not observed for α4β2 receptors expressed on oocytes [76,78] .Physostigmine, another acetylcholinesterase inhibitor, also potentiates α4β2 receptors, and both galantamine and physostigmine appear to bind to both the orthosteric and non-orthosteric binding sites of these receptors [80] .Physostigmine can also inhibit α4β2 receptors at higher concentrations.A structural model for the physostigmine binding site on the α3β4 receptors has also been developed [44] .
L-[-]-2,3,5,6-Tetrahydro-6-phenylimidazo [2,1b]-thiazole (Levamisole) Levamisole (Figure 1B) and the related compound pyrantel are effective anthelmintics that act via stimulation of nematode muscle nAChRs [81] .Although their use in humans has been terminated due to side effects, including severe dermatological lesions, they are still used in veterinary medicine [81][82][83] .In humans, levamisole has also been used as an adjuvant in colon cancer therapy [84] and is a common intentional contaminant of cocaine [85,86] .Levamisole was identified as a PAM of α3β2 and α34 nAChRs and produces a bell-shaped concentration-response curve typical of PAMs: it potentiates agonist-induced responses at α3β2 receptors at concentrations of 5-500 µM, and inhibits them at higher concentrations [87] .Although levamisole is capable of eliciting currents when applied alone, acting as a very weak partial agonist at α34 receptors, the very small currents were observed only at extremely high concentrations of the drug (3 mM).Potentiation is apparently mediated by noncompetitive binding of levamisole and inhibition is likely due to open channel block, but the binding site mediating the potentiating effects are unknown.It has been suggested that levamisole binds to the non-orthosteric binding clefts of α3-containing receptors, in a manner similar to that proposed for a number of other PAMs [87] .It does not appear that any further development of levamisole or its analogs has been attempted in order to optimize the potentiating effects or reduce the toxic effects of the drug.

6-Bromo-2-(1,1-dimethyl-2-propenyl)-N-1H-indole-3ethanamine (dFBr)
Desformylfulstrabromine (dFBr, Figure1B) was first extracted and purified from the marine bryozoan Flustra foliacea [88] .Initial studies of the purified compound determined that dFBr potentiated α4β2 receptors at micromolar concentrations, did not displace ligands from the orthosteric sites at less than mM concentrations, and had no effect on α3β2, α3β4, α4β4, or α7 nAChRs [88] .Subsequent total synthesis of dFBr produced a very pure form and confirmed its selectivity for α4β2 receptors [89] .dFBr displays the bell-shaped dose-response kinetics typical of other α4β2 PAMs, potentiating agonist-induced responses at concentrations of less than 1-3 µM and inhibiting these responses at higher concentrations [46,90] .dFBr is about sixteen times more potent at the HS versus the LS α4β2 stoichiometry but has equal efficacy at both types, thus it is one of the only PAMs capable of potentiating the HS subtype [34,45,46] .Partial agonist responses were potentiated to a much larger extent than those of full agonists (270% for ACh vs 760% for nicotine and 990% for cytisine), an effect that might make dFBr an effective adjuvant in therapies involving partial agonists.The inhibitory action of dFBr appears to be mediated by an open channel blocking mechanism [46] .Potentiation is due to an increase in efficacy, as is the case for NS206.However, mutagenesis studies support a binding site located at the β + /β -and/or the β + /α -interface, making it unlikely that NS206 and dFBr have the same binding sites (Figures 2 and  4) [34,46] .Single channel studies revealed that dFBr increases the probability of channel opening and decreases the probability of channel closure [88] .SAR studies have identified the gem-dimethyl group as a critical component of dFBr's ability to potentiate agonist-induced responses and the compound is very resistant to modification at this position [90] .The 6-Br appears important for dFBr's potency, but its removal does not alter the drug's efficacy.N-methylation of the primary amine also reduces potency.dFBr has been shown to alter nicotine self-administration and its ability to displace β-amyloid(1-42) from the receptor could support a potential application in the treatment of Alzheimer's disease [30,91,92] [

2-[(4-Fluorophenyl)amino]-4-methyl-5-thiazolyl]-3thienylmethanone (LY-287101)
Broad and coworkers used a high-throughput screening assay to identify three new thiazole compounds capable of potentiating nAChR subtypes [93] .These novel PAMs were selective for α4β2, α4β4, α2β4 and α7 receptors.The structure of a representative member of this group of compounds (LY-2087101) is shown in Figure 1B.All three compounds increased the amplitude of agonist-induced responses, but did not displace the agonist from its binding site, indicating noncompetitive allosteric modulation.Interestingly, these compounds acted as potentiators at low micromolar concentrations, but as partial agonists at higher concentrations (10-100 µM) and inhibitors at even higher concentrations.The partial agonist activity was blocked by the noncompetitive antagonist mecamylamine and the competitive antagonist dihydro-β-erythroidine (dHβE) indicating that the observed activity is mediated by nAChRs, but this data does not suggest whether LY-2087101 competes for binding with either mecamylamine or dHβE.The binding site for these compounds is unknown although the compounds were found not to potentiate chimeric α7/5-HT3 receptors containing an α7 extracellular domain and a 5-HT3R transmembrane domain.The authors suggested that this result may indicate that the binding site is not present on the extracellular domain or, alternatively, that the chimeric receptor is not capable of potentiation via this mechanism [93] .

3-(3-(Pyridine-3-yl)-1,2,4-oxadiazol-5-yl)benzonitrile) (NS9283)
NS9283 (Figure 1B) is a PAM that is selective for the LS stoichiometry of the α4β2 nAChR.It has been well characterized and shown to be effective in enhancing cognitive function in animal models [29,95] , and to provide benefits in animal models of movement disorders, pain and nicotine addiction [95][96][97] .Potentiation results from an increase in agonist potency rather than agonist efficacy.Mutagenesis data suggest that H142, Q150 and T152 on the α4 subunit near the ACh binding site are critical for NS9283 binding [98] .NS9283 was also found to displace [ 3 H]-epibatidine binding with high potency, and appears to bind at the unique α4 + /α4 - subunit interface of the LS α4β2 receptor (Figure 2 and 4) [98] .This interface is also known to bind nAChR agonists and regulate agonist sensitivity [99,100] .Effects of NS9283 can be blocked by alkylation of a T152C mutation, further supporting an interaction of NS9283 with the α4 + /α4 -interface [101] .
The ACh binding protein from Lymnaea stagnalis (Ls-AChBP) was co-crystalized with NS9283 to more precisely characterize interactions between the receptor and ligand [98] .The crystal structure data corroborate data from mutagenesis studies, implicating the α + /α -interface in the binding of NS9283.The principal conserved, interacting residues on the α + face include W89, W143 and W185 (interacting with the pyridine ring, oxadiazole ring and pyridine nitrogen, respectively) and W53 on the α -side (Figure 2) [98] .His142 (α + ) was also shown to be important for the interaction with NS9283 and the presence of valine in the equivalent position of the β2 subunit suggests that this single amino acid could be the primary determinant of the α4 + /α4 -versus α4 + /β2 -interface selectivity of NS9283 [98,102] .Derivatives of NS9283 have also been produced and some are selective for other variants of the receptor.Jin and coworkers [103] reported a series of novel PAMs with activity at α4α5β2 receptors.Given the potential involvement of α5containing receptors in nicotine addiction, these compounds may be valuable leads in developing more selective and therapeutically useful modulators for nicotine addiction.

3-N-Benzyloxy-3-hydroxyimino-2-oxo-6,7,8,9-tetrahydro-1H-benzo[g]indole-5-sulfonamide (NS206)
NS206 (Figure 1B) was found to be a PAM of hα4β2 nAChRs expressed in Xenopus laevis oocytes [98] .Both HS and LS α4β2 stoichiometries were potentiated with similar potency, although the amount of potentiation differed (300-400% for HS and 150% for LS α4β2 receptors).The ability to potentiate both the HS and LS subtypes of the receptor appears to be a somewhat unique trait: NS206 shares this characteristic with dFBr [34] , but not zinc or NS9283.The ability to act as a PAM at HS α4β2 receptors is therapeutically significant, as the HS receptors modulate dopamine release in the striatum, and are thought to be involved in nicotine addiction, Parkinson's disease, and other neurological disorders [98,104] .In contrast to NS9283, NS206 causes an increase in ACh efficacy rather than potency.While NS206 shows no selectivity for HS over LS receptors, it does show a high selectivity for the α4 subunit.NS206 has similar potencies at the α4β4 and α4β2 receptors, but does not potentiate either the α3β4 or α7 subtypes.Mutations of V77, I80, K160, Y165, and Y170 of an α3/α4 chimeric subunit to the equivalent residues found on α4 (V77K, I80M, K160S, Y165F, and Y170Q mutations, located in a loop between β sheet 1 and β sheet 2 of the receptor) produced a receptor responsive to NS206, suggesting that the drug's binding site lies in this region of the receptor thought to be important in transducing ligand binding to channel gating.(Figure 2) [98,105] .The mechanism of potentiation for NS206 may therefore be due to facilitation of this transduction process.NS206 is similar to the thiazole compounds described in that it binds to the putative transmembrane domain of the α4 subunit, but it has not yet been determined if these compounds have the same or overlapping binding sites.Unlike the thiazole compounds, NS206 shows no agonist properties.Despite the fact that both NS206 and NS9283 show selectivity for the α4 subunit, the two drugs show no overlap in binding sites.The potentiating effects of the two ligands are additive and their binding sites have been mapped to different receptor domains [98] .

(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
HEPES is a commonly used buffering agent and one of a group of buffers known as "Good's buffers."HEPES (Figure 1B) has been found to produce substantial potentiation of ACh responses at HS α4β2 receptors [(α4)2(β2)3] at low concentrations (up to 190% at <300 µM HEPES), and inhibition at higher concentrations, producing a bell-shaped concentration-response curve [45] .At concentrations typically used in electrophysiological experiments, HEPES's potentiating and inhibiting effects cancel out to produce no apparent functional effect.Potentiation appears specific to the HS stoichiometry of the receptor, whereas only slight inhibition was observed for the LS stoichiometry [45] .α7 nAChRs were not potentiated by HEPES.HEPES potentiation of the HS receptor was sensitive to mutation of D218 on the β2 + subunit, a residue reported to be important for zinc inhibition of the same receptors [41,45] .The involvement of D218 suggests a possible binding site for HEPES on the nonorthosteric subunit interface, and the selectivity for HS receptors suggests specifically the unique β + /β -interface (Figures 2 and 4) [45] .This unique selectivity could also make it a valuable lead for developing HS α4β2 selective drugs.Binding of HEPES at the subunit interface is supported by the presence of HEPES at the binding site in the crystal structure of an AChBP that is structurally homologous to nAChRs [106] .HEPES is a commonly used electrophysiological buffer, but the observation that it acts as a PAM at HS α4β2 receptors suggests that HEPES should be used with caution in analyzing allosteric modulators of these receptors.At concentrations typically used, HEPES may occupy critical modulatory sites or inhibit receptor potentiation, masking the effects of compounds under investigation.Since HEPES shares structural similarity with other Good's buffers [e.g.2-Hydroxyethyl)piperazine-N′-(3-propanesulfonic acid (EPPS), piperazine-N,N′-bis(2-ethanesulfonic acid PIPES, and piperazine-N,N′-bis(2-hydroxypropanesulfonic acid (POPSO)], it is possible that those buffers may also alter responses to ACh, although such effects have not yet been reported.

Endogenous PAMs and NAMs
In addition to chemically synthesized PAMs and NAMs, endogenous compounds and ions have also been found to modulate heteromeric nAChRs.While these compounds are not easily utilized as drug leads, the binding sites at which they act to modulate the receptors could serve as potential leads for drug development.These agents include proteins (Lynx-1), ions (calcium and zinc) and small molecules (steroids).

Lynx proteins
Lynx proteins are endogenous proteins belonging to the ly-6/PLAUR superfamily.The lynx proteins are structurally similar to α-bungarotoxin and share with it a critical cysteinerich three-loop structure [107,108] .The lynx-1 protein has been proposed to help maintain cholinergic tone in vivo [108,109] .Lynx-1 displays a sub-nanomolar affinity for nAChRs and appears to interact with the extracellular face of the nAChR.In this respect, lynx-1 differs from most other nAChRinteracting proteins, which bind to the intracellular loops.While lynx-1 is a GPI linked protein it remains active as a modulator even when the GPI anchor is absent.Lynx1 has been found to inhibit α4β2 receptors, although it causes an apparent increase in conductance as a result of increasing inactivation rates and decreasing ACh potency [110] .Lynx-1's binding site on the α4β2 receptor has not yet been identified, but the protein's similarity to α-bungarotoxin suggests a The α4 subunit structure is shown only to illustrate the location of the putative binding regions and is not intended to represent a molecular model of the receptor binding site.This illustration was generated based on the refined Torpedo nAChR α1 subunit structure [73] (2BG9).Deepview software (SIB Swiss Institute of Bioinformatics) was used to mutate residues to match the α4 subunit sequences and to highlight the position of key binding regions and amino acids within the overall structure of the α4 subunit.Citations to the source research studies are provided in the text descriptions for each compound.
similar binding domain at the subunit interfaces [111] .As lynx-1 has been shown to have roles in nicotine-regulated motor learning, ligands that mimic or inhibit the lynx-1's action on α4β2 receptors offer potential therapeutics for nicotine addiction [111] .

Divalent cations
nAChRs are permeable to a number of divalent cations, including Ca 2+ , Zn 2+ , Ni 2+ , Mg 2+ , Cd 2+ , Pb 2+ and Sr 2+ .Divalent cations have also been shown to modulate receptor function.Cadmium potentiates α4β4 receptors in Xenopus systems [112] .Lead was found to potentiate α3β2 receptors at high concentrations and to inhibit α4β2 and α3β4 receptors at lower concentrations [113] .Instances of potentiation by barium and strontium have also been observed for α4-containing receptors [114, 115]   .While cations are not good lead molecules for drug development, the location of ionic modulatory sites could reveal potential domains that could be targeted to produce novel modulatory agents.Effects of calcium have been observed in rat adrenal chromaffin cells and medial habenular neurons and on both α7 and α4β2 receptors [114][115][116][117] .For heteromeric receptors containing α4 subunits, data suggests that receptors can be potentiation by intracellular calcium, an effect postulated to be mediated by direct interactions of calcium with two cysteine residues (C262 and C269) within the M1-M2 loop that is located on the intracellular domain of the receptor (see Figure 3) [118] .In preliminary studies, modification of these cysteine residues by methanethiosulfonate (MTS) reagent mimics the effects of increased calcium concentrations on the intracellular side [118] .M1-M2 cysteine residues are conserved in α2, α3, α4, β2, and β4 subunits, thus receptors comprised of these subunits could be regulated in a similar manner by calcium, possibly to different degrees dependent on how many calcium binding subunits are included in each receptor subtype.
In the CNS, zinc acts as a co-neurotransmitter and is costored with Glu in synaptic vesicles in large subpopulations of glutaminergic terminals (most numerous in the cortex, hippocampus, and amygdala) and co-released with Glu in a calcium-dependent manner [119][120][121] .Vesicular zinc is required for hippocampus-and amygdala-dependent memory formation, and has been implicated in a number of neurological disorders, including Alzheimer's disease, epilepsy, autism, ADHD, and depression [119][120][121][122][123][124][125] .Zinc has been shown to act as an allosteric modulator of several members of the cys-loop family of ligand-gated ion channels, including nAChRs, GABAARs, GlyRs, and 5-HT3Rs [112, 126- 128] .
Zinc potentiates rat α4β4 and α4β2 receptors and inhibits α3β2 receptors [112,129] .The amount of potentiation varies depending on subunit composition, subunit stoichiometry, and pH [41,100,112] .LS receptors [(α4)3(β2)2] are potentiated at concentrations less than 100 µM and inhibited at higher concentrations, producing a bell-shaped dose response curve typical of α4β2 PAMs [41,112] .Potentiation of LS receptors is primarily due to a change in efficacy of ACh rather than a shift in agonist potency.This appears to result from an increase in open channel probability and burst duration rather than increased conductance or increased receptor number [129,130] .In contrast, HS receptors [(α4)2(β2)3] are inhibited, not potentiated, by zinc.Additionally, inhibition of HS receptors is voltage-dependent while inhibition of LS receptors at high zinc concentrations is voltage-independent suggesting a channel blocking mechanism of inhibition for HS receptors and non-channel blocking mechanism for LS receptors [41] .Inhibition by open channel block by zinc has been observed for other nAChRs as well [131] .
A role for histidine residues in mediating zinc potentiation was predicted based on the observation that the histidine modifying reagent diethylpyrocarbonate (DEPC) blocks zinc potentiation on α4β4 receptors but does not block inhibition of α3β2 receptors [112] .Using site directed mutagenesis and substituted cysteine accessibility method (SCAM), Hsiao and coworkers found that E59 and H162 of the α4 subunit play an important role in zinc potentiation of LS α4β2 nAChRs and noted that these residues occupy an identical position in the benzodiazepine binding site of the GABAA receptor, suggesting structural and functional similarities in the zinc potentiation sites on neuronal nAChRs and GABAA receptors [129]   .Later studies implicated H195 on the α4 subunit and D218 on the β2 subunit in the inhibiting effects of zinc at LS α4β2 receptors [41] .Moroni et al. have concluded that for LS α4β2 receptors the binding domain for zinc potentiation lies at the α + /α -interface while the inhibitory site lies at the β + /α - interface (Figures 2 and 4) [41] .The binding site for zinc has not been determined for HS receptors beyond the expectation that it binds in the channel lumen.The location of the zinc potentiating site to the α + /α -interface explains the reported selectivity of zinc for LS receptors.Zinc potentiation is distinct from that observed by calcium (described above) as the binding sites for these two cations have been mapped to different regions of the receptor.Zinc binds close an additional agonist binding domain located at the α + /α -interface of LS receptors.Since zinc appears to alter efficacy with minimal effect on agonist affinity, it seems likely that binding to this region alters gating.

Steroids
The roles of peripherally-generated steroid hormones and their metabolic derivatives in regulating physiological processes and gene expression are well known.In addition, several regions of the brain synthesize a group of steroids known as neurosteroids.Steroids have been shown to influence the functional properties of most ligand-gated ion channels, including Gly, Glu, GABA, 5-HT3A, and nAChRs in slice preparations and heterologously expressed receptors in Xenopus oocytes or mammalian cells [31,[132][133][134] .As steroids appear to act by direct binding to allosteric binding sites on these receptors, they are likely an important class of endogenous allosteric modulators for these receptors [132] .While few significant clinical studies have explored the use of steroids as nicotinic modulators in the treatment of neurological disorders, progesterone has been shown to reduce nicotine self-administration in rats [135] .
Steroid interactions with nAChRs were first reported for progesterone.
Progesterone (Figure 1A) binds noncompetitively to the neuronal α4β2 nAChR to reduce ACh-induced responses, indicating that it is a NAM of this receptor [132] .In a comprehensive study of 12 steroids, Paradiso and coworkers identified 17-β-estradiol (Figure 1B) and its homologs as PAMs of α4-containing human (but not rat) nAChRs and of α3-containing nAChRs from other species including humans [31] .Furthermore, they showed that 17-βestradiol binds to a small sequence of amino acid residues (PPWLAGMI) present at the C-terminal domain of the hα4 subunit (Figure 3).Mutations within any of the last four (AGMI) amino acids of this sequence prevented potentiation by 17-β-estradiol.The relative position of the AGMI sequence with respect to upstream proline residues is also important [31] .Using concatemers of subunits and chimeric subunits, Jin and colleagues [32] showed that transfer of the WLAGMI (estradiol-binding) domain to the C-terminal of the β2 subunit was sufficient to replicate the potentiation seen when the domain resides in its normal position in the α4 subunit.Moreover, steroid potentiation increased as additional estradiol-binding domains were inserted at the C-terminals of each subunit of the pentameric receptor; each C-terminal domain added produced a 1.6 fold increase in potentiation [134] .Single channel studies showed that 17-β-estradiol acts as a PAM on α4β2 subtypes by increasing the probability of channel open time, perhaps by stabilizing the open state conformation [32,136] .
The inhibition of hα4β2 receptors by progesterone and their potentiation by 17-β-estradiol have been shown to involve different steroid binding sites and different mechanisms.For example, the C-terminal sequence required for potentiation is not required for inhibition [31] .Independent mechanisms of potentiation and inhibition by steroids were also found for GABAA [137] and NMDA receptors [138] .

Conclusions and Perspectives
This review has presented an overview of ligands and binding sites for ligands that potentiate or attenuate agonistinduced responses at the most common heteromeric nAChRs found in the CNS.This is a relatively new area of drug development.With the exception of some early-discovered cholinesterase inhibitors that proved also to be non-selective allosteric modulators of nAChRs, most of the current series of PAMs were discovered after 2004.As a result, we are currently in a period of lead discovery for this class of drugs.Not surprisingly, the SAR studies needed to guide future drug development in this area have only recently begun to emerge.We are on the brink of a new phase in the development of PAMs and NAMs, as researchers begin to use the newly discovered leads to better understand the binding sites, mechanisms of action and usefulness of these compounds.While the developmental pathway for nAChR PAMS and NAMs may seem similar to the development of benzodiazepines and barbiturates on the GABAA receptor, the pace of discovery will be substantially increased due to the knowledge gained from studies of the GABAA receptor, our clear understanding of the nAChRs as targets for PAMs and NAMs, and the huge advances in neuropharmacology and medicinal chemistry since Librium, the first benzodiazepine, was discovered in 1955.The use of concatameric receptors to direct mutations to specific binding clefts, site directed mutagenesis and SCAM, sophisticated molecular modeling approaches, high throughput screening assays and a better fundamental understanding of nAChR mechanisms and kinetics will surely accelerate this task.
A review of the primary lead compounds for heteromeric nAChR PAMs and NAMs reveals a number of regions of the receptor that may serve as the principle modulatory sites.These include the C-terminal region (steroids), the nonorthosteric clefts (zinc, dFBr, HEPES, levamisole, NS9283, galantamine, physostigmine, and lynx-1), and the M1-M2 loop (calcium).While some binding sites are still unknown, there could be another potential site within the transmembrane regions as has been identified for α7 PAMs [139][140] .Perhaps compounds such as thiazoles or NS206 interact at this position.Regions equivalent to the GABAA receptor barbiturate site also have yet to be evaluated as potential sites for PAMs.Additionally, new classes of compounds need to be explored.If the nAChR does contain a site similar to the benzodiazepine binding domain of the GABAA receptor then it might be expected that inverse agonists and antagonists of that site could be developed.The heteromeric nAChRs are particularly complicated and rich targets for drug development since the varied stoichiometry and composition of these receptors provides ample opportunity to produce highly selective ligands.Current PAMs have already been shown to possess selectivity for different receptor stoichiometries such as the HS and LS α4β2 receptors (Figure 4).Detailed mechanistic studies of the effects of modulators on receptor kinetics are also needed to compliment binding site data and to link binding to receptor function.Modulators should be highly useful as molecular probes of mechanistic studies of receptors.
While there have been some SAR studies conducted on lead compounds (as in the cases of NS9283, thiazoles, dFBr and KAB-18), it is clear that additional development is needed for these and other promising leads.Too often, a lead compound is identified and no additional work is done to determine if potency, selectivity and/or efficacy can be improved.A better understanding of the modulator pharmacophores for different sites will provide improved appreciation of how different lead molecules interact to achieve their potency and selectivity, which is essential to further drug development in this area.This information will likely speed the development of ligands that interact at the allosteric binding sites including new PAMs for each site, inverse agonists (compounds that bind to the PAM site and attenuate responses) or antagonists (compounds that compete with the PAM but do not potentiate or attenuate responses).
Allosteric modulators for heteromeric nAChRs have enormous potential, although current studies have only begun to scratch the surface.While numerous studies have linked these receptors to human neurological disorders, the potential of PAMs and NAMs to treat these disorders has barely been explored.More studies evaluating the effects of these compounds in animal models are needed, with concurrent attention given to their bioavailability in the CNS.It is likely that heteromeric nAChR modulators will prove to be as clinically successful as the benzodiazepine and barbiturate drug classes have been in the treatment of neurological disorders, a goal potentially reachable in this second decade of development.

Figure 2 .
Figure 2. Putative amino terminal binding domains for PAMs and NAMs.The α4β2 receptor structure is shown only to illustrate the location of the putative binding regions on the amino terminal domain of the receptor and is not intended to represent a molecular model of the receptor binding site.Only three of the five subunits are shown and illustrate (from left to right) the α4 -face, the α4 + α4 -interface, and the β2 + face.The illustration was generated based on the refined Torpedo nAChR α1 subunit structure[73] (2BG9).Deepview software (SIB Swiss Institute of Bioinformatics) was used to mutate residues to match the α4 or β2 subunit sequence, to merge the three subunits shown in the figure and to highlight the position of key binding regions and amino acids within the overall receptor.Citations to the source research studies are provided in the text descriptions for each compound.

Figure 3 .
Figure 3. Binding Domains for 17-β-estradiol and Ca2+  .The α4 subunit structure is shown only to illustrate the location of the putative binding regions and is not intended to represent a molecular model of the receptor binding site.This illustration was generated based on the refined Torpedo nAChR α1 subunit structure[73] (2BG9).Deepview software (SIB Swiss Institute of Bioinformatics) was used to mutate residues to match the α4 subunit sequences and to highlight the position of key binding regions and amino acids within the overall structure of the α4 subunit.Citations to the source research studies are provided in the text descriptions for each compound.

Figure 2 .
Figure 2. Putative binding regions for PAMs proposed to bind at subunit interfaces of HS and LS α4β2 receptors.Ligands that display selectivity for different receptor interfaces on α4β2 are shown for both the LS and HS subtypes.Citations to the source research studies are provided in the text descriptions for each compound.