Allosteric modifiers of neuronal nicotinic acetylcholine receptors: New methods, new opportunities

Allosteric Modi¢ers of NeuronalNicotinic Acetylcholine Receptors:NewMethods, New Opportunities

Ruin Moaddel,1 Krzysztof Jozwiak,1,2 Irving W. Wainer1

1Gerontology Research Center, Laboratory of Clinical Investigations,

National Institute on Aging/NIH, Baltimore, Maryland2Department of Chemistry, Medical University of Lublin, Lublin, Poland

Published online 19 January 2007 in Wiley InterScience (www.interscience.wiley.com).

DOI 10.1002/med.20091

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Abstract: Allosteric, non-competitive inhibitors (NCIs) of neuronal nicotinic acetylcholine

receptors (nAChRs) have been shown to produce a wide variety of clinically relevant responses.

Many of the observed effects are desired as the nAChR is the therapeutic target, while others are

undesired consequences due to off-target binding at the nAChR. Thus, the determination of

whether or not a lead drug candidate is an NCI should play an important role in drug discovery

programs. However, the current experimental techniques used to identify NCIs are challenging,

expensive, and time consuming. This review focuses on an alternative approach to the investigation

of interactions between test compounds and nAChRs based upon liquid chromatographic

stationary phases containing cellular fragments from cell lines expressing nAChRs. The develop-

ment and validation of these phases as well as their use in drug discovery and pharmacophore

modeling are discussed. � 2007 Wiley Periodicals, Inc.y Med Res Rev, 27, No. 5, 723–753, 2007

Key words: affinity chromatography; molecular modeling; non-competitive inhibition; quina-

crine-binding site; ethidium-binding site

1 . I N T R O D U C T I O N

The health risks associated with tobacco and tobacco smoke have been clearly defined and well

publicized. As a result, one aspect that is often overlooked, is the fact that the tobacco plant and

tobacco smoke contain more than 4,000 compounds,1 many of which may possess beneficial

pharmacological and therapeutic properties. This possibility has been suggested by the observed

physiological effects associated with smoking that include alertness, reduced anxiety, muscle

yThis article is a U.S.Government workand, as such, is in thepublic domain in the United Statesof America.Contractgrant sponsor:National Institute on Agingof theNational Institutes of Health.Correspondence to: IrvingW. Wainer,Ph.D.,BioanalyticalandDrugDiscoveryUnit,National InstituteonAging,National Institutes

of Health,Gerontology ResearchCenter, 5600 Nathan ShockDrive,Baltimore 21224-6825,MD.E-mail:[email protected]

Medicinal Research Reviews, Vol. 27, No. 5, 723^753, 2007

� 2007 Wiley Periodicals, Inc.

relaxation, and analgesia,2 as well as specific disease-related effects, which have been observed in

Alzheimer’s disease, Parkinson’s disease, and schizophrenia.1,3

Although tobacco and tobacco smoke have been shown to contain compounds that inhibit a

number of different targets such as monoamine oxidases,4 the therapeutic responses associated with

tobacco smoking have been assumed to be primarily due to the interaction of nicotine with neuronal

nicotinic acetylcholine receptors (nAChRs). This assumption was derived from a number of clinical

observations including improved cognitive responses and memory in Alzheimer’s patients who had

received intravenous, subcutaneous, or trans-dermal doses of nicotine, the lower than expected

incidence of Parkinson’s disease in smokers, and the prevalence of cigarette smoking among patients

with schizophrenia.3 These observations have led to the initiation of drug discovery programs which

have the development of nAChR agonists as the primary focus.2,5 Some of the nAChR agonists

currently in clinical development are presented in Table I.2

An example of an nAChR-targeted drug discovery program is the development of a7 nAChR

selective agonists for the treatment of schizophrenia1,6 and Alzheimer’s disease.1,2,7 The role of the

a7 nAChR in schizophrenia has been specifically associated with a disease-related decrease in

auditory sensory gating, the ability to suppress the evoked response to the second of two auditory

stimuli.6 The first prototypical agent aimed at normalizing auditory gating is the a7-selective agonist3-[(2,4-dimethoxy)benzylidene]-anabasine.6 This compound also enhances cognitive behavior and

is under investigation for use in the treatment of Alzheimer’s disease.8

Although current drug discovery programs are primarily concentrated on the development of

nAChR agonists, nAChR antagonists have been in clinical use for over 60 years, for example, D-

tubocurarine, a competitive inhibitor of peripheral nAChRs, was introduced into clinical anesthesia

and surgicalmuscle relaxation in 1942.9 Thegrowing interest in nAChR antagonistswas reflected in a

recent review by Dwaskin and Crooks, which identified these agents as ‘‘a new direction for drug

discovery.’’10 A possible pharmacological basis for this approach is the observation that nAChR

agonists rapidly desensitize the receptors, which essentially inhibits their function. Thus, if inhibition

and not excitation is the actual therapeutic effect, competitive inhibitors or agonists that result in rapid

desensitization should work equally well.10

Table I. Compound that Act as Agonists of Neuronal Nictonic Acetylcholine Receptors, which

Are Currently in Clinical Development

Seereference [2] fordetails.1ABT, Abbott Labs; AZD, Astra Zeneca; MEM,Memory Pharmaceuticals; SSR,Sanofi Synthelabo;TC,Targacept; ACh, acetylcholine;

NR,nicotinicreceptor.2Discontinuedclinicaldevelopment intheUS.

724 * MOADDEL, JOZWIAK, AND WAINER

Competitive inhibition is only one approach to the antagonism of receptor activity. Allosteric

modulators, including non-competitive inhibitors (NCIs) have been recently suggested as a ‘‘new

generation’’ of receptor antagonists.11 Non-competitive inhibition of nAChRs is a recognized

phenomenon, and more than 50 marketed drugs (see Appendix) have been identified as NCIs of the

neuronal nAChR subtypes a3b2, a3b4, a4b2, a4b4, and a7. For most of the compounds listed in

the Appendix, the non-competitive inhibition of nAChRs was a secondary discovery, as these drugs

were primarily directed at other receptors. Indeed, NCI activity may be the source of many side

effects attributed to these compounds; for example, the impairment of cardiovascular function

observed during ketamine anesthesia has been associated with its inhibitory action on ganglionic

nAChRs.12 Thus, the screening of drug candidates for NCI activity at the nAChR, as well as other

ligand gated ion channels, may be a method for the identification of unwanted off-target

pharmacological effects before they are observed in clinical trials.

While the screening for off-targetNCI activity represents a useful component of a drug discovery

program, non-competitive inhibition of nAChRs also presents an opportunity for new drug

development. The antidepressants sertraline, paroxetine, nefazodone, and venlafaxine are potent

NCIs, and it has been suggested that nAChR subtypes in the brain could be targets for the

development of new antidepressant drugs.13 The NCIs mecamylamine and bupropion are currently

used in anti-smoking therapy14 and the use of 18-methoxycoronaridine in combination with

mecamylamine or dextromethorphan (DM) has been suggested as an approach to treat opioid and

stimulant addiction.15

A key problem in the development of a drug discovery program based on the non-competitive

inhibition of the nAChR is the difficulty of identifying and quantifying NCI activity. The standard

experimental approach to the determination of NCI activity at the nAChR involves the measurement

of concentration-dependent effects on whole cell currents16 or nicotine-induced 86Rbþ cellular

efflux.17,18 These techniques are not well suited for drug discovery programs as they are time-

consuming, expensive, exacting, and cannot be standardized for all subtypes of the nicotinic receptor.

We recently reported an alternativemethod for the identification and characterization of NCIs of

the nAChR.19,20 This approach employs liquid chromatographic stationary phases containing

immobilized cellular membranes obtained from cell lines expressing the target nAChR, non-linear

chromatography (NLC), and molecular modeling. Initial studies have demonstrated that the

technique can be used to identify NCIs,19,20 predict IC50 values,21 and estimate the rate of recovery

from functional blockade.22 This reviewwill discuss the current status of this work and suggest future

applications to drug discovery utilizing immobilized receptor and transporter-based stationary

phases and chemometric analyses.

2 . N E U R O N A L N I C O T I N I C A C E T Y L C H O L I N E R E C E P T O R S

Neuronal nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channels found

in the central and peripheral nervous systems that regulate synaptic activity. The receptor is

composed of five separate trans-membrane proteins (subunits), each containing a large extra-cellular

N-terminal domain, four membrane spanning alpha helices (M1, M2, M3, and M4) and a small C-

terminal domain23 (Fig. 1). The subunits are oriented around a central pore24,25 and the resulting

transmembrane ion channel is formed by a pentameric arrangement of the M2 helical segments

contributed by each of the five proteins26 (Fig. 1).

At present, 12 different neuronal subunits have been identified, 9 labeled alpha (2–10) and 3

labeled beta (2–4). The subunits form channels of a wide variety of homomeric and heteromeric

nAChRs.27 The most common subunit stoichiometry has been determined to be (aX)2(bY)3,(X ¼ 2–6; Y ¼ 2–4) for heteromeric subtypes and (aZ)5, (Z ¼ 7–10) for the homomeric subtypes.

However, other, more complex, combinations have also been reported.

ALLOSTERIC MODIFIERS * 725

Subtypes of the nAChR are found in different locations of the central and peripheral nervous

system, and have been associated with different pharmacological functions. In the central nervous

system, the a4b2 nAChR subtype is expressed in the thalamus, cerebral cortex, and dorsal

hippocampus, and the receptor mediates aspects of the cognition and memory, and participates in

sensory gating2 including pain, anxiety, and depression.1–3,5 The a3b4-like subtype is highly

expressed in the medial habenula, the interpeduncular nucleus, the optic tract, cerebellum, and

cranial nerve nuclei,2 and plays a role in norepinephrine release as well as cardiovascular and

gastrointestinal action. The a7 nAChR has been found in the cerebral cortex, hippocampus, and

hypothalamus,2 and plays a role in cognition, memory, and auditory processing.3,6 In addition, the a7nAChR has been identified on the surface of human macrophages and it has been suggested that this

receptor plays a role in regulation of inflammation.28 The a4b4 and a3b2 subtypes have also been

identified in the central nervous system.2

3 . A G O N I S T - B I N D I N G S I T E S A N D R E C E P T O R G A T I N G

The nAChR contains multiple-binding domains, which can accommodate different classes of

endogenous and exogenous compounds (Fig. 2). Two homologous, neurotransmitter-binding sites

Figure 1. Schematic representationof theneuronalnicotinic acetylcholine receptor showing the relationship of the receptor to theion channel, the membrane, and the arrangement of the four transmembrane-spanninghelices forming the channel.The various

regions of the channel ‘‘rings’’arealso shown.See reference [20] fordetails.


are formed by the N-terminal domains where cholinergic agonist and competitive antagonists bind.

This site has been the subject of a number of structure–activity relationship (SAR) studies due to its

recognized therapeutic importance and to the availability of rapid and facile experimental procedures

to determine binding and functional properties. These studies have been recently reviewed.29

Agonists bind to the nAChR in the resting, or low affinity state, produce conformational changes

in the internal lumen of the receptor that open the channel to the passage of cations such as sodiumand

Figure 2. Transverse schematic representation of the muscle-type AChR showing the domains involved in the specific binding of

non-competitive inhibitors fromexogenousorigin.Topand right panel: Schematic representationofasectionof the AChRat�46 —

from the membrane surface viewed from the synaptic cleft. The ethidium locus can be putatively located in the AChR vestibule

betweenbothacetylcholine (ACh)-bindingsites.The ethidiumsite includeseitheraregionnear theag interface, beingthisa submit

theonethatbears thehigh-affinity Ach-bindingsite, oraportionoftheb subunit.Bottomandleftpanel:Schematic representationof

asectionoftheAChRat the lipid^aqueous interface viewedfromthesynaptic cleft.Theaminoacidchainfromeachsubunit (a,b, g,and d) crossesthe lipidmembranefour times (M1^M4).Theperimeterof theAChRissurroundedby� 45 lipidmolecules, that is, the

annular lipid domain.The 23 empty circles around the AChR represent the phospholipidheadgroups on the extracellular leaflet of

the lipidbilayer.ThesmallblackcirclesbetweensubunitsandbetweendomainsM1/M4andM3/M4 represent thepossible locations

for non-annular lipid domains.The high-affinity quinacrine-binding site is located at the aM1transmembrane domain, near the bsubunit.Bottomandrightpanel:SchematicrepresentationoftwoM2 transmembranedomainsoftheAChRsubunit.Theaminoacid

side chains from both aM2 domains represent the transmembrane aM2 domains and the transmembrane a-helix. A consensus

exists that the interactionof certain residues from the a subunit (shownhere as filled spheres), inaddition to the respectivehomolo-

gousaminoacids fromtheother subunits (not shownforsimplicity), formaseriesofstratified ringsdisposed fromthe extracellular to

the intracellularside inthefollowingmanner:outerorextracellular, valine, leucine, serine, threonine, intermediate, andcytoplasmicor

inner ring. Inaddition, someof themare involved in thebindingof the so-called luminal non-competitive inhibitors. Inparticular, the

bindingsite for the localanestheticmeproadifen is locatedclosetothemouthofthe ionchannel, probablyat thenegativelycharged

outerorextracellular ring.Thebindingsite forcembranoidsand trifluoromethyl-iodophenyldiazirine (TID) is related toboththevaline

and the leucine ring.Finally, thebindingsite forchlorpromazine (CPZ), triphenylmethylphosphonium (TPMPþ), the local anestheticQX-222, andphencyclidine (PCP) includes the leucine, the serine, and the threonine ring.Reprinted fromreference [30].


potassium, and the process ends with the channel closed and the receptor in the desensitized, or high

affinity state. The agonist-induced gating of the nAChR has been described as an organized and

sequential movement of segments of the protein.27

The key feature of the central lumen is the existence of a series of amino acid rings, in which an

amino acid (one from each subunit) are exposed to the central pore of the channel.30 The gating is

assumed to take place at the narrowest region of the channel, identified as the valine (V) ring, number

15 in Figure 1 and Table II, and the actual gate as a hydrophobic region produced by the five isopropyl

moieties of the V molecules, which project into the central lumen. Agonist-induced conformational

changes rotate thesemoieties towards the lumenwall removing the barrier to themigration of cations

through the channel.31 The existence of isopropyl moieties at ring 15 is a common feature of all the aand b subunits, except for the b4 subunit (Table II). In this subunit, the V is replaced by a

phenylalanine (F) and the gate contains three phenyl rings and two isopropyl moieties.

4 . N O N - C O M P E T I T I V E - B I N D I N G S I T E S O N T H E n A C h R

To date, three major non-competitive-binding sites have been shown to exist on all subtypes of the

nicotinic receptor, namely, the central lumen, ethidium, and quinacrine-binding sites30 (Fig. 2).

Binding at these sites inhibits the receptor from undergoing agonist-induced gating in a non-

competitive, allosteric manner, and they have been characterized as NCI-binding sites. While other

NCI-binding sites have been identified, such as the steroid-binding site,30 the majority of the

compounds characterized as NCIs of the nAChR appear to bind at one of these three sites.

A. The Luminal-Binding Site

The luminal high affinity NCI-binding domain is located on the surface of the internal lumen that

forms the ion channel. The structure of the internal lumen has been studied by a variety of approaches

and these studies have been reviewed.30 Although most of the experimental data used to describe the

luminal domain were obtainedwith the muscle-type nAChR, abdg nAChR, it is believed that the keystructural components are common to the other members of the nAChR family.

The internal lumen of the nAChR has a high negative electrostatic potential.20 This domain can

be viewed as a cation selector in which NCIs bearing a positive charge, usually on an amine moiety,

are trapped and directed down the channel by an electrostatic gradient.32 While NCIs can bind at a

variety of sites along the central lumen, it is generally assumed that the non-competitive inhibition is

predominately a result of binding at site(s) near or at the V ring, position 15, which essentially plugs

the channel and blocks ion flux33 (Fig. 1). This mechanism has been used to describe the NCI

Table II. Sequence Alignment of M2 Transmembrane Section

Acrossdifferentsubunitsofneuronalnicotinicacetylcholinereceptors,whereresiduesonyellowbackgroundareexposedtothecenterofthe

channelandformrings, whichare coloredastherings in Figure1.ReprintedwithpermissionfromJozwiaketal.20Copyright 2004American

Chemical Society.


properties of drugs such as mecamylamine,17 DM,17 bupropion,34 phencyclidine,34 and

barbiturates.35

However, experiments utilizing NCI labeling and site-directed mutagenesis have suggested that

ligands may interact with different rings distributed along the lumen.30 The possibility of multiple-

binding sites has been reflected in the results from molecular modeling studies. For example, in

simulations of docking interactions with the a7 nAChR, pentobarbital appears to primarily dock at

ring 15,35 while other ligands primarily bound to serine (S ring, position 8) and threonine (T ring,

position 4), see Figure 1, with only minor interactions with the V ring.36

1. Molecular Models of the Luminal-Binding Site

There have been several proposed molecular models of the transmembrane domain of the a7nAChR37,38 and cryo-electron microscopy images have been used to develop models of the Torpedo

marmorata nAChR (abdg nAChR).39 We have recently reported a model of the central lumen of the

a3b4 nAChR, which can be used to describe the binding and function of NCIs to this receptor.20 Themodel was based upon a synthetic 23-mer peptide, homologous to the sequence of the putative

transmembrane M2 segment of the Torpedo californica d-nAChR subunit, which spontaneously

forms discrete ion channels in liposome bilayers.40 These channels are functionally similar to the

nAChR channel including functional blockade by known nAChR NCIs. Using frozen state NMR

techniques, the structure of the synthetic channel (Protein Data Bank id ¼ 1EQ8) was shown to

consist of fivea-helical polypeptides oriented in a fivefold symmetricalmanner forming a funnel-like

architecturewith awide opening on theN-terminal side and preserving the sequence of ‘‘rings’’ along

the channel as in natural nAChRs.

This model was used as the starting point for the development of the a3b4 nAChR, and the PDBentry 1EQ8 was modified by mutating the d–M2 residues into a3 and b4 sequences as presented inTable II. The model was further refined by energy minimization and the final model was evaluated

using Procheck. The final model of the a3b4 nAChR luminal domain is depicted in Figure 3A and B.

Figure 3A shows the stereo view of the fivea-helices forming the channel and their alignment relative

to each other. Figure 3B illustrates the location of the specific amino acid rings distributed along the

channel. The internal luminal surface has ionized residues on both ends of each helix, which appear to

be responsible for ion selection of the nAChRs. Seven rings of amino acids are clearly visible along

the channel in Figure 3B. An extracellular polar ring (E/K) at the edge of themembrane is followed in

sequence by three non-polar (L, V/F, and L) and then three polar (S, T, and intermediate (E)) rings.

The cytoplasmic ring of acidic residues (see Fig. 1) was not included in our model.

As discussed above, an important feature of the a3b4 channel is the introduction of an F at

position 15 (V/F ring). This results in the formation of a defined asymmetric hydrophobic cleft

between the a3 and the b4 helices at the interface between the isopropyl and phenyl moieties

associated with Vand F, respectively, which are depicted in Figure 4. This particular feature does not

exist in non-b4 subtypes, such as the a3b2 nAChR,where the fiveV residues at this site produced less

defined hydrophobic regions (unpublished data).

2. NCI Docking in the a3b4 nAChR Luminal Domain

Themolecularmodel of thea3b4 nAChR luminal domain suggests that there are a significant number

of possible siteswhereNCIsmight bind.A series of 17NCIs and 5 negative controlswere docked into

the model of the a3b4 nAChR using AutoDock.20 The NCIs bound deep within the channel

predominantly in the non-polar region near the V/F ring. The negative control molecules formed

scattered clusters in their final docking orientations along the channel where interactions with V/F

and L rings or G ring predominated; however, there was no single dominant binding orientation

or site.


Figure 3.


The unique pocket formed by the cleft between the phenyl ring introduced by F in the b4 subunitand the isopropyl moiety from the V in the a3 subunit was the primary-binding pocket for the

hydrophobic moieties of all of the studied NCIs. When the non-polar part of the inhibitor is included

in the hydrophobic pocket, the polar part of the ligand is able to interact with nearby polar residues

exposed to the lumen (e.g., position 8) to form hydrogen bonds. This mechanism is illustrated using

the enantiomers DM and levomethorphan (LM) (Fig. 5A and B) and is consistent with previous

docking studies with the a7 nAChR which indicate that the S at position 8 plays a role in the ligand

binding.36

Dextromethorphan (DM) and LM were chosen for the initial docking studies based upon

previous functional, chromatographic, and thermodynamic studies, which demonstrated that these

enantiomers bind enantioselectively to the a3b4 nAChR and that the observed differences were due

to an enhanced stability of the DM–a3b4 nAChR complex.20,41 In the docking simulations, the

lowest energy docked conformations of the DM and LM complexes were located at the V/F ring and

involved the insertion of the hydrophobic portion of both molecules into the hydrophobic cleft found

at this position (Fig. 5A and B).

Figure 3. Graphical representationof the finalmodelof the luminaldomainofa3b4nAChR.Thea3helicesare coloredinblue, andb4helicesare incyan.Theresidues liningthe lumenofthechannelareshownexplicitly.Chargedresiduesareshowninredandblue.Hydrophobic residues are shown in green. Hydrophilic residues are shown in orange, and the phenylalanine from b4 is shown inblue: (A) topview instereomode (intracellular side)withexposedresidues rendered inwireframemode; (B) side viewof the channel

with the a3, b4, and a3 helices shown from left to right.Two of the b4 helices have been removed for clarity. Exposed residues arerendered in CPKmode.Labels show thenumberingof ringsaccording toTable III. See reference [20] fordetails.

Figure 4. Thehydrophobicpocket identifiednear theV/Fring inthe�3b4 subtypeof theneuronalnicotinicacetylcholine receptor;the figure renderedwithmolecularareaandonehelix hasbeen removed for clarity. [Color figure canbe viewed in the online issue,

which is availableat www.interscience.wiley.com.]


The mirror image relationship between the two enantiomers and their lack of conformational

mobility produce two unique orientations, which result in distinctly different interactionswith nearby

amino acidmoieties comprising the lumen. In the case ofDM(Fig. 5A), the bridgehead nitrogen atom

was oriented towards the hydroxyl moiety on the S residue (position 8) located on the a3 helix,

thereby increasing the probability of H-bond formation between the two moieties. With LM, the

bridgehead nitrogen atomwas pointing away from the two helices forming thea3 andb4 subunits dueto the steric considerations, thereby reducing the probability and strength of any H-bond interaction

(Fig. 5B). The calculated difference in methorphan–nAChR complexes, DDG(n) derived by

DGDM�DGLM, was�0.33 kcal/mol in favor of DM. This difference is in agreement with the value

obtained from van’t Hoff studies conducted using an immobilized a3b4 nAChR liquid

chromatographic stationary phase in which observed DDG� was �0.29 kcal/mol.41

Based upon the results from the docking experiments, the V/F cleft appears to play a key role in

the enantioselective binding of DM and LM to the a3b4 nAChR. This particular hydrophobic pocketis a unique feature of the a3b4 nAChR and should differ from the binding area produced by the V/V

Figure 5. The molecular model of dextromethorphan (A) and levomethorphan (B) docked at theV/F ring of the �3b4 neuronalnicotinic acetylcholine receptor. [Color figure canbe viewed in the online issue, which is availableat www.interscience.wiley.com.]


moieties present in the non-b4 containing nAChR subtypes. This hypothesis is consistent with the

observed differences in pharmacological responses. In general, receptors containing the b2 subunitdesensitize faster than those containing the b4 subunit.42 Papke and co-workers have also reported

that the recovery from non-competitive inhibition by mecamylamine appears to be a single

exponential process for receptors containing the b2 subunit and more complex for a3b4 nAChRs.16

In the case of the a3b4 nAChR, the recovery occurred in two phases, one of which took over 30 min.

3. Applying the a3b4 nAChR Model to In Silico Drug Discovery

Non-competitive inhibitors (NCIs) that bind in the central lumen of the nAChR are assumed to

sterically block the channel, thereby preventing ion flux.33However, the results of the docking studies

suggest that at least in the case of the a3b4 nAChR, non-competitive inhibition is due to an alternative

mechanism. The free energies of binding (DG� values) for DM and LMwith the a3b4 nAChR in the

desensitized state were �8.73 and �8.40 kcal/mol, respectively, and the DG� value calculated for

the mecamylamine, a prototypical NCI, was �6.41 kcal/mol.20 Since the agonist-induced gating

of the nAChR has been described as an organized and sequential movement of segments of the

protein,27,31 it is reasonable to assume that formation of the NCI–nAChR complex increases the

energy of activation required to produce the conformational changes required in the gating process.

Thus, the binding of a NCI does not physically block the ion channel, but instead acts as a wedge,

essentially freezing the nAChR in a closed conformation.43

Since previous studies have associated the length of inhibitory activity of an NCI with the

stability of the NCI–nAChR complex,22,41 the molecular model of the a3b4 nAChR can be used to

design compounds with extended therapeutic activity. In addition, the results from the study of the

enantioselective interaction of DM and LM with the a3b4 nAChR demonstrate that for this class of

compounds, the increased stability of the DM relative to the LM is due to more efficient hydrogen

bonding interactions between the ammoniummoiety on themorphinan ring and the hydroxyl moiety

on the S molecule at position 8.20

Based upon these observations, one route to a more effective NCI of the a3b4 nAChR would be

to alter the molecular structure of DM in order to enhance hydrogen bond formation. This could be

accomplished by transforming the methyl group attached to the bridgehead amine into a longer

moiety with hydrogen bond acceptors. Three possible structures were designed and docked into

the a3b4 nAChR model, and the predicted DG values were determined (Table III).44 The results

of the docking simulations were consistent with the proposed approach. Compound DM-01 was

synthesized, and was an active inhibitor of nicotine-induced 86Rbþ efflux in a cell line that expressed

the a3b4 nAChR with an IC50 value of 22 mM.44

The internal surface of the lumen domain is highly electron rich,20 a feature that appears to be

responsible for the cation selectivity as well as for strong interactions with positively charged NCIs.

This aspect of the NCI–nAChR interaction can also be used in drug design to affect pharmacological

properties. An increase in the total negative charge on a molecule should reduce the stability of the

compound–nAChR complex and the pharmacological effect of the compound, while the opposite

effect should be produced by increasing the total positive charge on the molecule.

The effects of changes in molecular polarity are illustrated by the changes in the IC50 values and

dissociation rate constants (kd) of DM produced by N-demethylation, O-demethylation, and N,O-

didemethylation (Table IV, Fig. 6). The kd values were obtained using NLC on a column containing

immobilizedmembranes obtained from a cell line that expressed thea3b4 nAChR.20 In this study, thekd values of the O-demethylated compounds, dextrorphan and (þ)-3-hydroxymorphinan, were

twofold higher than those of the mexthoxy-containing analogs, DM and (þ)-3-methoxymorphinan.

These results were attributed to the fact that under the experimental conditions, the phenoxymoieties

of the dextrorphan and (þ)-3-hydroxymorphinan were partially ionized, and the additional negative

charge, relative to DM, destabilized the dextrorphan and (þ)-3-hydroxymorphinan nAChR


complexes relative to the complex formed by DM. In the case of (þ)-3-methoxymorphinan, N-

demethylation slightly increased the net positive charge on the molecule relative to DM but did not

alter the relative stabilities of the DM–nAChR and (þ)-3-methoxymorphinan–nAChR complexes.

The chromatographic results were reflected in the IC50 values obtainedwith the cell lines used to

create the affinity column.21 In these studies, O-demethylation increased the IC50 values, thereby

reducing the effectiveness of dextrorphan and (þ)-3-hydroxymorphinan, relative to DM, while the

N-demethylation of DM did not change the observed IC50 (see Appendix and Table IV). There was

Table III. Molecules Designed to Inhibit a3b4-nAChR Followed by DG� Values Obtained in

Docking Simulations (Reference DG of Dextromethorphan ¼ �8.73 kcal/mol)

Structure Compound ∆ G o

dextromethorphan

N

O

H -8.73 kcal/mol

DM-01 N

O

H

O

-9.09 kcal/mol

DM-02 N

O

H

O

-9.35 kcal/mol

DM-03 N

O

H

O

-10.31 kcal/mol

Table IV. Chromatographically Determined Dissociation Rates (kd) for the

Complexes between the a3b4 nAChR and Dextromethorphan and Three of its

Metabolites

Compound kd

(sec -1 ) IC 50

( µ µ M) Percent recovery

(7 min) Dextromethorphan 1.0 10.1 51

Dextrophan 2.3 29.6 (+)-3-Hydroxymorphinan 2.0 59.7 102 (+)-3-Methoxymorphinan 1.0 10.3

Determined using a liquid chromatography stationary phase containing immobilized cellular membranes

obtained fromthe KXa3b4R2 cell line, and functional inhibition (IC50 values) andpercent inhibition 7minafter

washoutdeterminedusingtheKXa3b4R2cell line.


also no significant difference between the IC50 value of verapamil and itsN-demethylatedmetabolite,

norverapamil, or between dilitiazem and N-desmethyldilitiazem (see Appendix).

In addition, O-demethylation of DM reduced the length of the pharmacological effect.22 At

7 min, DM continued to have a significant effect on nAChR activity (only 50% of the initial activity

had been recovered), while there was no residual effect of (þ)-3-hydroxymorphinan (Table IV).

The results of these studies demonstrate that the model of the central lumen of the a3b4 nAChRand docking simulations can be used to manipulate molecular structures to increase or decrease

interactions with this receptor. If an NCI is the desired compound, then themolecule can be altered to

increase either the hydrophobic or hydrogen bonding interactions, or both. If non-competitive

inhibition of the nAChR is an unwanted effect, then the electronegativity of the compound can be

increased in order to decrease the interactions with the nAChR.

B. The Ethidium-Binding Site

In 1987, Herz and co-workers identified a single, high-affinity, non-competitive-binding site for the

quaternary amine ethidium on the a(2)bgd nAChR isolated from the Torpedo californica.45 This site

has been described as a polyamine-binding site46 and studies using fluorescence titration and

photoaffinity-labeling have indicated that ethidium binds in the outer vestibule of the central lumen

about 46 A above the transmembrane portion of the receptor30,47 (Fig. 2).

Structure–activity relationship (SAR) studies have been used to identify the structural elements

related to NCI activity at the nAChR.46 In these studies, a series of compounds were synthesized

based upon the polyamine philanthotoxin structure, a family of NCIs isolated from wasp venoms,

which act as NCIs. The results of the study indicate that high affinity binding at the ethidium site is

associated with hydrophobic head groups and polyamine side chains.

While the ethidium-binding site has been extensively studied, only a few marketed

pharmaceuticals have been identified as binding at this site. The most important of these drugs are

chlorpromazine and clozapine.48 It is also likely, but unproven, that the neuromuscular blocking

agent atracurium binds at this site. It is of interest to note that at low concentrations, atracurium is an

agonist, while at higher concentration, it displays both competitive and non-competitive inhibition

with the a7 and a4b2 nAChRs, and only non-competitive inhibition with the a3b4 nAChR.49

N

O

CH3

CH3

H

NCH3

HO

H

N

OCH3

H

N

HO

H

dextromethorphan

(+)-3-methoxymorphinan

dextrorphan

(+)-3-hydroxymorphinan

Figure 6. Structuresofdextromethorphanand its demethylatedmetabolites.


Laudanosine, a metabolite of atracurium, also displays agonist, competitive antagonist, and NCI

properties,49 but most probably binds in the central lumen of the nAChR.49,50

C. The Quinacrine-Binding Site

The primary-binding site for the antimalarial agent quinacrine has been identified in the non-annular

lipid domain on the border between the nAChR and themembrane, and not in the luminal domain30,51

(Fig. 2). Using a series of spin-labeled state isomers and quinacrine fluorescence quenching, it

appears that the site is �7–12 A below the aqueous–lipid interface.30

At the present time, quinacrine is the only marketed pharmaceutical that has been identified as

binding at the quinacrine-binding site.30 This is most probably due to the experimental difficulties

associated with the determination of binding to this site. However, displacement chromatography on

an a3b4 nAChR column and cellular inhibition studies employing the cell line used to create

the chromatographic column demonstrated that the morphine derivatives buprenorphine and

naltrindole were NCIs of the a3b4 nAChR, with IC50 values of 13 and 28 mM, respectively, and that

these compounds bound at the quinacrine site on this receptor [unpublished data]. Thus, the

chromatographic approach appears to be a simple and direct method to determine whether a

compound binds at the quinacrine site. This technique is being applied to the study of a number of

known nAChR NCIs and the results may increase the importance of the quinacrine-binding site as a

target for drug discovery and a source of unexpected side effects.

5 . S Y N T H E S I S A N D C H A R A C T E R I Z A T I O N O F T H En A C h R S T A T I O N A R Y P H A S E

A. Liquid Chromatography Stationary Phases Containing Immobilized Proteins

Affinity chromatography is awell characterized and studied technique that has been primarily used as

a method to isolate and purify proteins, c.f.52 The standard experimental approach is to immobilize a

known ligand or an antibody on a chromatographic support and to use the resulting column to

chromatographically isolate target proteins from cellular lysates or incubates. However, Chaiken53

and Carr54 demonstrated that cytosolic proteins and enzymes could also be immobilized and used to

study ligand–protein interactions.

In 1996, Lundahl et al.55 reported the development of a chromatographic column containing a

transmembrane protein, the GLUT1 glucose transporter. This column had been created through the

entrapment of red blood cell membranes in proteoliposomes. Frontal affinity chromatography

techniques were used to demonstrate that the immobilized GLUT1 actively bound substrates and

inhibitors and that this technique could be used to determine binding affinities, Kd values. This

approach has been recently reviewed.56

Building on the work by Lundahl and co-workers, we have developed a series of liquid

chromatographic stationary phases containing immobilized cellular membrane fragments obtained

from cell lines expressing a target receptor, ion channel, or transporter. These phases have been based

upon the immobilized artificial membrane (IAM) stationary phase developed by Pidgeon et al.57 and

have included membranes containing opioid G-protein coupled receptors (GPCRs),58 b2-adrenergicGPCR,59 nAChRs,43,60 the drug transporter P-glycoprotein (Pgp),61,62 and the human organic cation

transporter-1 (hOCT1).63

The technology is not limited to a particular protein or family of proteins or a particular cell line.

For example, nAChRs were obtained from transfected HEK-29343 and SH-EP1 cell lines

[unpublished data], the organic cation transporter from a MDCK cell line,63 and the Pgp transporter

from a LCC-6 cell line.61,62 The binding capacities of the resulting columns depend upon quantity of

the immobilized functional protein, which is determined by the level of expression of the target


protein and the amount of the solubilized target protein. For example, the amount of cells required to

produce functional nAChR columns ranged from 1� 106 (a3b4 subtype)43 to 30� 106 (a7 subtype)[unpublished data].

In addition to cellular membranes, solubilized tissues can also be used to produce functional

affinity columns. For example, solubilized brain tissue was used to create a stationary phase that

contained co-immobilized nAChRs, g-amino-butyric acid receptors, and N-methyl-D-aspartate

receptors.64

B. Immobilization of nAChRs

Avariety ofmethods have been used to immobilize nAChR-containingmembranes. In one approach,

membranes from the Torpedo marmorata were immobilized on the surface of a gold Biacore chip

using phospholipids composed of a hydrophilic polyethyleneglycol spacer and a terminal thiol

moiety in the lipid headgroup.65 The lipid bilayerwas anchored to the gold surface of the chip through

sulfur–gold bonds. The resulting complex was used in surface plasmon resonance experiments to

quantify the binding of ligands and antibodies to the nAChR.

Cellular membranes containing the a3b4 nAChR have also been sterically entrapped in

Superdex beads60 and membranes from the Torpedo californica have been immobilized in sol–gel-

derived silicamaterials.66However, themost extensive studies on immobilized nAChRcolumns have

been conducted using IAM-immobilized membranes and columns have been created from cell lines

expressing the a3b2 nAChR, a3b4 nAChR, a4b2 nAChR, and a4b4 nAChR.43

C. Characterization of the Immobilized nAChR Stationary Phase

The initial characterization of the immobilized nAChR stationary phases has been accomplished

using frontal affinity chromatography. This technique is used to characterize the binding of small

molecules to the immobilized membrane-bound target (the nAChR) and to determine binding

affinities (Kd) and the number of active binding sites on the column (Bmax). A key requirement of this

approach is the existence of a compound which is known to bind to the target, the marker ligand. In

frontal affinity chromatography, the marker ligand is placed in the mobile phase and passed through

the column. The frontal regions are composed of the relatively flat initial portion of the

chromatographic traces, which represent the non-specific and specific binding of the marker to

the cellular membranes and the target. The saturation of the target by the marker produces a vertical

rise in the chromatographic trace, which plateaus when the target is saturated.

Typical chromatograms of 60 pM [3H]-labeled epibatidine ([3H]-EB) obtained using frontal

chromatography techniques on an a3b4 nAChR stationary are presented in Figure 7.60 The

chromatographic traces produced by 60 pM (Fig. 7, Curve A) and 450 pM [3H]-EB (Fig. 7, Curve B)

illustrate the effect on concentration on retention and demonstrate that the frontal chromatograms

were produced by a specific binding interaction.

Once it has been established that the immobilized target specifically binds themarker ligand, it is

possible to calculate Kd values of the marker or other ligands for the target receptor using

displacement chromatography. In the displacement studies, increasing concentrations of the marker

or other test ligands are added to the mobile phase and the effects on the retention volumes, measured

at the midpoint of the breakthrough curves, are determined. This is illustrated by the effects on the

chromatographic traces produced by 60 pM [3H]-EB produced by the addition of 60 nM nicotine

(Fig. 7, Curve C), and 1,000 nM nicotine (Fig. 7, Curve D).

The relationship between displacer concentration and retention volume can be established using

Equation 1 and can be used to determine the Kd value of the displacer and the number of active

binding sites, Bmax;55

½Nic�ðV� VminÞ ¼ Bmax½Nic� ðKdNic þ ½Nic�Þ�1 ð1Þ


where Vis retention volume of Nic; Vmin, the retention volume of Nic when the specific interaction is

completely suppressed (this value can be determined by running [3H]-EB with a high concentration

of displacer). From the plot of [Nic] (V�Vmin) versus [Nic], dissociation constant values, Kd, forNic

can be obtained. The data is normally analyzed using non-linear regression employing a standard

program such as Prism 4 software (Graph Pad Software, Inc., San Diego, CA) running on a personal

computer.

The Kd and Bmax values obtained for known nicotinic ligands obtained using columns produced

from cell lines expressing the a3b2 nAChR, the a3b4 nAChR, the a4b2 nAChR, or the a4b4 nAChRsubtypes are presented in Table V.43 The results demonstrate that frontal affinity chromatography can

not only be used to calculate Kd values on a specific target, but can also be used to distinguish binding

affinities between closely related subtypes of the same target.

The Kd values calculated using the chromatographic method were generally lower than the

corresponding values determined from the membrane-binding studies.43 However, while the Kd

values differed, linear regression analysis showed that the two data sets correlated with an

r2 ¼ 0.7952 (P < 0.0001), indicating that the observed differences were quantitative not qualitative.

The Bmax values are also reported in Table V. The calculated Bmax values represent the total

number of active binding sites available on the column and are directly related to the binding capacity

of the column. Since the level of expression of the nAChR subtypes varies between the cell lines and

since the number of cells used in the experiments ranged from1� 106 to 3� 106, the number of active

binding sites on each column is not equal. In fact, the Bmax values can vary between columns of the

same subtype.

0

25

50

75

100

3020100

Rentention volume (ml)

[Epi

batid

ine ]

(% o

f pl

atea

u)

ACBD

Figure 7. The elutionprofilesof [3H]-epibatidineon�3b4NR-IAM stationaryphase, where A ¼ 60 pM,B ¼ 450 pM,C ¼ 60 pM

[3H]-epibatidine in the presence of 60 nM (�)-nicotine; D ¼ 60 pM [

3H]-epibatidine in the presence of 1,000 nM (�)-nicotine.

Reprinted fromZhangetal.60Copyright1998, with permission fromElsevier.

Table V. Binding Affinities (nM) and Bmax Values Calculated by Frontal Affinity

Chromatography Using the Immobilized Nicotinic Receptor Columns

αα3β2 α3β4 α4β2 α4β4 Epibatidine 0.086 (0.57) 0.001 (0.035) 0.042 (0.16) 0.005 (0.061)

Nicotine 16.4 (10) 80 (440) 2.18 (47) 0.387 (40)

Cytisine 0.237 (2.1) 6.09 (1.5) 7.6 (220) 2.43 (37)

Bmax 13.5 fmol 24.3 fmol 572 fmol 4.76 fmol

Compared tobindingaffinitiescalculatedby filtrationassays inparentheses, seereference [43] fordetails.


Most cellular or membrane binding studies utilizing the nAChR are carried out using the ‘‘high

affinity’’ or desensitized state of the receptor, which is produced by exposure of the receptor to an

agonist. It was of interest to determine if the immobilized nAChRs were in the resting or desensitized

states and if exposure to an agonist would produce a conformational shift from one state to the other.

A study was performed comparing the retentions of luminal NCIs on freshly prepared nAChR

columns and after the columns were exposed to EB.43 The results demonstrated that the membranes

obtained from the expressed cell lines contained nAChRs that were predominantly in the resting state

and that exposure to EB shifted the population to the higher affinity desensitized state. Thus, the

immobilization process did not affect the conformational mobility of the membrane bound nAChRs,

although the transition from desensitized to resting statewas significantly slower for the immobilized

receptors relative to the intact cells.

6 . N O N - L I N E A R C H R O M A T O G R A P H Y

The shape of a chromatographic peak is the function of the specific and non-specific interactions

between the solute and the stationary phase. In particular, the kinetics involved in the formation and

dissolution of the solute-stationary phase complex, that is, the association and dissociation rate

constants, ka and kd, respectively. When the stationary phase contains an immobilized protein, the

dissociation of a ligand–protein complex is usually slower than the rate of complex formation

producing non-Gaussian peaks with tailing.

The degree of deviation from a Gaussian distribution is a function of applied ligand

concentration and the kinetics of ligand–receptor interactions occurring during the chromatographic

process. An example of the effect of solute concentration on peak shape is presented in Figure 8,19 in

which increasing concentrations of the NCI mecamylamine were chromatographed on an a3b4nAChR column.

Concentration-dependent asymmetry can be used with NLC techniques to characterize the

separation processes occurring on the column. The first formulation of non-linear conditions in

chromatography was derived by Thomas67 and this approached was labeled ‘‘quantitative affinity

chromatography’’ in a review by Jaulmes and Vidal-Madjar.68 In 1987, Wade et al.54 developed the

Impulse Input Solution for the mass balance equation. This approach is based upon the observation

0 5 10 15 20 25 30 35 40time [min]

1

2

5

10

20

50

100

200

500

1000

Figure 8. The effect of increasing concentraions of mecamylamine, from 1 to 1,000 mM, on the chromatographic profiles of

mecamylamine.Forexperimental details, see reference [19].


that when adsorption/desorption rates are slow, band broadening is insensitive to a moderate degree

of column overload. In contrast to numerical integration methods, this approach uses the analytical

solution, which can be applied directly to fit experimental peak profiles. The Impulse Input Equation

has been included in PeakFit v4.11 forWindows Software (SPSS, Inc., Chicago, IL) and can be easily

applied to NLC studies.

The mathematical approach used in the Impulse Input Solution for the NLC is described by

Equation 2 (PeakFit User’s Manual, p. 8–25):

y ¼ a0

a31� exp � a3

a2

� �� ffiffiffia1x

pI1

2ffiffiffiffiffia1x

p

a2

� �exp �x�a1

a2

� �

1� T a1a2; xa2

� �1� exp � a3

a2

� �h i24

35 ð2Þ

where

y—Intensity of signal,

x—Reduced retention time,

Tðu; vÞ ¼ expð�vÞZu

0

expð�tÞI0 2ffiffiffiffivt

p� dt

The T function acts as a ‘‘switching’’ function, which produce the skew in the peak profile when the

column is overloaded.

I0( ) and I1( ) are Modified Bessel functions,

a0—Area parameter,

a1—Center parameter, reveal to true thermodynamic capacity factor,

a2—Width parameter,

a3—Distortion parameter.

Once the NLC parameters have been established, they can be further processed for the

calculation of the k 0, Ka, ka, and kd values. These parameters were calculated using the following

relationships (PeakFit User’s Manual, p. 8–26):

k 0 ¼ a1—Real thermodynamic capacity factor, kd ¼ 1a2t0

—Solute desorption constant rate (t0 is the

dead time of a column),Ka ¼ a3C0—Equilibrium constant for adsorption (C0 is a concentration of

solute injected multiplied by a width of the injection pulse (as a fraction of column dead volume)),

ka ¼ kd ¼ Ka—Solute adsorption constant rate.

The application of NLC to the characterization of the interactions between NCIs and the

immobilizeda3b4 nAChRwas established using theNCIs ketamine, bupropion,mecamylamine, and

DM.19 The k 0(NLC), Ka, kd, and ka values calculated using the NLC parameters are presented in

TableVI. Once a column has been characterized usingNLC techniques, the concentration-dependent

studies do not have to be repeated for each text compound and a single concentration can be used to

establish the relative Ka, kd, and ka values.

A. Using NLC to Identify and Characterize NCI

1. Direct Determination of IC50 Values

It is commonly accepted that affinity chromatography can be used to derive binding affinities, which

can be correlated to data obtained using standard techniques such as membrane binding,

ultrafiltration, and equilibrium dialysis.69,70 It is also assumed that this technique is not applicable

to the direct determination of functional activities, such as IC50 or EC50 values. This may be partially


explained by the fact that the binding affinity of the drug towards the receptor is not necessarily

directly related with the efficacy of this drug acting on the pharmacological target.

However, the relationship between binding affinities and functional properties has been

established for competitive inhibitors of enzymes by Cheng and Prusoff.71 In this approach, the

binding affinity of the inhibitor (Ki), the functional strength of the inhibitor (IC50 value), and the EC50

of a specific marker agonist are related in the following manner:

Ki ¼I50

1þ SKm

� � ð3Þ

where I50 is the IC50 value, S is the concentration of the substrate (ormarker ligand), Km is the affinity

of the substrate (or maker ligand) for the target. This approach has also been used with receptors to

relate binding affinities and antagonist activity (Ki and IC50) and agonist activity (Kd and EC50).

Recently, we have demonstrated that the Cheng–Prusoff relationship can also be used with

affinity chromatography techniques to directly assess relativeEC50 values.72 These studies useda3b4

nAChR and a4b2 nAChR columns and single frontal displacement chromatography experiments.

The displacement of [3H]-EB by each of nine test compounds and two internal controls, nicotine and

carbachol, was calculated and expressed as Dml. Nicotine was used as an internal control for

compounds with agonist activity, and carbachol was used as an internal control for compounds with

veryweak agonistic activity. Since the concentration of themarker ligandwas a 1,000-fold lower than

the displacer, Equation 3 reduced to Affinity (Kd), expressed asDml, is equivalent to activity (EC50).

The test compounds were ranked using the Dml values relative to the Dml of nicotine and carbachol

(Table VII), and the corresponding EC50 values were confirmed using the nicotine-stimulated 86Rbþ

efflux assay and the cell line expressing the a3b4 nAChR.

Unfortunately, the Cheng–Prusoff relationship cannot be used with NCIs, and it was necessary

to develop an alternative method for the online estimation of relative antagonistic activities,

expressed as IC50 values. This was accomplished using NLC, chemometric and multivariate

analyses.21 In this approach, NLC retention values, k 0, were obtained for 29 compounds on an a3b4nAChR, and the corresponding molecular descriptors EHOMO and SYZ were generated (Table VIII).

EHOMO is the energy of the highest occupied molecular orbital and SYZ is the surface area of the

molecular projection onto the YZ plane. The two data sets were related using Equation 4, which had

been previously described.20

log k ¼ 5:255 ð�0:942Þ þ 0:491 ð�0:092ÞEHOMO þ 0:012 ð�0:005ÞSYZr ¼ 0:984; s ¼ 0:168; F ¼ 27:929; n ¼ 17

ð4Þ

The IC50 values for used in the study were also determined using nicotine-induced 86Rbþ efflux from

KXa3b4R2 cells (Table VIII). Linear regression analyses were used to compare IC50 values with log

k, EHOMO, and SYZ. No statistically significant relationships were observed (all P-values >0.05).

Table VI. Non-Linear Chromatography (NLC) Parameters Designated as Retention Factor (k 0NLC),

EquilibriumConstants for Adsorption (Ka), Association Rate Constant (ka), andDissociation Rate Constant (kd)

k’Compound NLC Ka (µµM-1 k) a (µM-1 sec-1) kd (sec-1)4.8 19.7 4.2 17.0 mecamylamine 6.9 15.0 2.3 12.0 Ketamine 5.8 10.5 1.8 14.0 bupropion

82.0 dextrometnorphan 0.8 16.7 20.1 Calculatedfor the interactionofmecamylamine, ketamine,bupropion,anddextromethorphanwithanimmobilizeda3b4nAChRstationaryphase.Seereference [19] fordetails.


However, a visual examination of a 3-D scatterplot of the chromatographic and molecular

parameters suggested that theNCIs could be subdivided into three separate clusters (Fig. 9). This was

confirmed usingmultidimensional cluster analysis, which is designed to specifically identify patterns

within multivariate datasets. The object was to sort cases into groups, or clusters, such that the degree

of association is strong betweenmembers of the same cluster andweak betweenmembers of different

clusters. Each cluster thus describes the class to which its members belong, and this description may

be abstracted through use from the particular to the general class.

The analysis identified three distinct clusters, which represents a qualitative grouping of

functional properties in which Cluster 1 contained compounds with IC50 values 1.9–3.0 mM, Cluster

2 contained compounds with IC50 values ranging from 7.0 to 139.7 mM, and Cluster 3 contained

compounds with IC50 values ranging from 1.0 to 6.6 mM21 (Table VIII, Fig. 9A).

The initial 19 test compounds contained 2 calcium channel blockers, verapamil and diltiazem,

and norverapamil, a primary metabolite of verapamil, and the data demonstrated that these

compoundswere efficient NCIs of the a3b4 nAChR (Table VIII). In order to further explore this class

of drugs and to validate the model, four additional calcium channel blockers (compounds 25, 27, 28,

29), five metabolites of diltiazem (compounds 20–24), and one additional metabolite of verapamil

(compound 26) were chromatographed on the a3b4 nAChR. The log k and molecular descriptors

were calculated for each compound (Table VIII) and used in the cluster analysis. The cluster analysis

placed compounds 20, 21, 23, 25, 27, and 28 in Cluster 1 and compounds 22, 23, 26, and 29 in Cluster

2 (Fig. 9B). The IC50 values for the compounds were then determined and 8 of the 10 compounds had

been placed in the correct cluster (Table VIII).

The molecular descriptors identified in the previous non-linear chromatographic study are

consistent with this mechanism, where EHOMO reflects the ability of the compound to transfer a

proton to the carboxylate moiety and SYZ is associated with the entrance of the compound into a

defined steric environment.20 The thermodynamic retention factor, k, reflects the equilibrium

between the initial binding of the compound to the nAChR, ka, and the stability of the final NCI–

nAChR complex, kd. All of these steps contribute to the observed IC50 value. Thus, it is not surprising

that linear regression analyses between only one of these factors and the IC50 values did not work,

while cluster analysis using all the three variables was able to identify populations with qualitatively

similar IC50 values.

Table VII. Comparison between theAgonist Activity of ConstrainedNicotine

and Anabasine Derivatives (TKS 1–9), Expressed as the EC50 Value

Sample EC50 [µµM] ∆ mL α3β4 ∆ mL α4β2

3H-EB (28.3 ± 1.6) x 10-3 -- ---- TKS-9 26.4 ± 1.8 0.34 1.60 TKS-6 20.8 ± 2.9 0.28 0.28

0.26 0.28 > 300 TKS-8 TKS-7 13.8 ± 3.0 0.20 0.24 Nicotine 19.8 ± 1.6 0.26 0.16 TKS-2 18.2 ± 3.4 -0.05 0 Carbachol >1000 -0.06 -0.04

-0.07 -0.04 >1000 TKS-4 0.17 -0.04 >1000 TKS-1 0.13 -0.12 >1000 TKS-3 0.18 -0.32 >1000 TKS-5

Determinedusingacell line expressingthea3b4neuronalnicotinicacetylcholinereceptor (a3b4nAChR)and their effect on the retentionof epibatidine (EB) in single displacement experiment, expressedasDml,calculatedasbreakthroughvolume of EBaloneminus thebreakthroughvolumeof EBafter theaddition

of the test ligand, on the immobilized a3b4 nAChR and a4b2 nAChR liquid chromatography stationary

phases.Seereference [72] fordetails.


The results of the study demonstrate that the experimental approach can be used to directly relate

chromatographic retention on an nAChR column to IC50 values obtained using the same cell line used

to construct the column. In addition, it took less than aweek to obtain the chromatographic data using

a single nAChR column, which was stable for>6months, and an automated LC–MS system. This is

in contrast to the 6weeks required to complete the functional studies for 18 compounds. These results

suggest that the chromatographic approachmay be useful in development of lead drug candidates that

act as NCIs of the nAChR and in the assessment of the probability of unexpected clinical toxicities

arising from off-target binding to the nAChR.

2. Direct Determination of NCI–nAChR Dissociation Rates Constants

The dissociation of a ligand–receptor complex plays a key role in the pharmacological effect of the

ligand. Thus, the ability to rapidly determine dissociation rate constants, kds, is an important addition

to drug discovery programs. Currently, stopped-flow analysis is the predominant method used to

determine pharmacologically relevant kds. While this technique is well known and accepted, it can

Table VIII. IC50 Values, Logarithm of Thermodynamic Retention Factors (log k)

and Molecular Descriptors (EHOMO and SYZ) and Assigned Clusters

logk Compound E HOMO S YZ Cluster IC 50

[ µ M] Methadone 1 1.9 1 66.6 -9.20 1.65 Diltiazem 2 2.3 1 62.3 -8.66 1.64 Norverapamil 3 2.6 1 69.1 -9.12 1.99 Verapamil 4 3.0 1 69.4 -9.04 1.98 Dextromethorphan 5 10.1 2 51.1 -8.72 1.79 Levomethorphan 6 10.9 2 52.7 -8.74 1.55 Dextrorphan 7 29.6 2 50.9 -8.77 1.43 (+)-3-hydroxy-mophinan 8 10.3 2 49.8 -8.78 1.75 (+)-3-methoxy-morphinan 9 54.2 2 49.3 -8.83 1.42 Clozapine 10 28.0 2 44.5 -7.69 2.19 Laudanosine 11 139.7 2 50.1 -8.54 1.36 Phencyclidine 12 7.0 2 51.6 -9.05 1.38 Adamantadine 13 3.4 3 35.4 -9.71 0.95 Bupropion 14 1.4 3 34.2 -9.51 1.11 Ketamine 15 1.4 3 42.5 -9.49 0.92 Mecamylamine 16 1.0 3 40.2 -9.22 1.04 Memantine 17 6.6 3 42.3 -9.71 1.22 Methamphetamine 18 401.2 3 27.8 -9.39 0.92 MK-801 19 26.6 3 43.0 -9.11 1.28 N-demethyl-diltiazem 20 4.2 1 66.5 -8.65 1.61 deacetyl-diltiazem 21 30.4 1 62.4 -8.58 1.60 N-demethyl-deacetyl- 22 diltiazem

77.6 2 57.4 -8.58 1.61

O-demethyl-deacetyl- 23 diltiazem

73.2 1 61.8 -8.41 1.45

N,O-didemethyl-deacetyl- 24 diltiazem

63.1 2 58.1 -8.65 1.48

galapamil 25 6.4 1 66.3 -9.05 1.88 D-620 26 (verapamil metabolite)

48.9 2 48.5 -9.35 1.25

nicardipine 27 2.5 1 65.2 -8.84 2.33 amlodipine 28 5.8 1 62.9 -8.72 2.00 nifedipine 29 24.7 2 58.4 -8.63 1.27

Determinedusing3-Dclusteranalysisofthe logk,EHOMO,andSYZvalues.Seereference [21] fordetails.


also be technically challenging and lengthy. Surface plasmon resonance techniques have also been

used to determine kd values of a variety of receptors including nAChRs. In the case of the nAChRs, the

study involved the immobilization of a characterized nAChR ligand, a bungarotoxin, the

solubilization of the nAChRs contained in Torpedo membranes, and their reconstitution in lipids

before screening.73

The binding kinetics of agonists to membrane-bound ligand gated ion channels, such as the

nAChR, have also been studied using electrophysiological approaches.74,75 However, the patch-

clamp techniques used in these studies are technically difficult and require multiple experiments.

Thus, this approach is also not easily adapted to the rapid screening of compound libraries.

As discussed above {see Section 4.A.3}, NLC techniques can be used to assess the kd values

associated with the binding of NCI to immobilized nAChRs. In particular, the chromatographically

determined kd values for DM (1.01 s�1) and LM (1.55 s�1) were compared to the percent recovery of

nicotine-stimulated [86Rbþ] efflux in a KXa3b4 cell line at 7 min post-NCI exposure, DM 38%, LM

63%.41 This comparison suggested that chromatographically determined kd values could be used to

assess the length of the inhibitory effect of NCIs on the pharmacological function of the nAChR. This

hypothesis was confirmed by a comparison of the chromatographically determined kd values of 12

NCIs of the a3b4 nAChR with the corresponding percent recovery of nicotine-stimulated [86Rbþ]

efflux at 7 min post-NCI exposure in intact KXa3b4 cells.22

In the study, the chromatograms from the NLC studies were analyzed using Equation 3 to

determine the kd values for each test compound as were the corresponding percent recoveries

(Table IX). The data sets were compared using Microsoft Excel and a non-linear correlation was

Figure 9. The results from the cluster analysis using the log k, EHOMO and SYZ parameters; where (A) are the results from the first

19 compounds; (B) are the results of the application of the cluster analysis to the test cohort of 10 calcium channel blockers and

theirmetabolites.Seereference [21] fordetails. [Color figurecanbeviewedintheonline issue,which isavailableatwww.interscience.

wiley.com.]


observed with an r2 value of 0.7458 (P ¼ 0.0048) (Fig. 10). The data was also tested for statistical

significance using a Spearman correlation and the two-tailed P-value was <0.001. This established

that NLC can be used to determine kd values which can be related to pharmacological activity.

However, it is reasonable to question whether at 7 min the difference between 100% recovery of

Figure 9. (Continued )

Table IX. Dissociation Rate Constants (kd) Determined

Using Non-Linear on an Immobilized a3b4 nAChR

Column and Percent Recovery of Nicotine-Induced

[86Rbþ] Efflux in KXa3b4 Cells 7 min after Exposure

to the Compounds

kName of compound d (s-1 7min % recovery )(n = 4)

78.1 ± 2.4 4.4 Nifedipine 28.8 ± 2.9 0.5 Nicardapine 92.4 ± 5.3 3.5 Dizocilpine(MK-801) 92.9 ± 4.1 5.0 Mecamylamine 88.4 ± 2.7 2.2 Laudanosine 37.9 ± 7.3 0.7 Verapamil

48.9 ± 19.9 0.7 Methoxyverapamil 50.9 ± 6.6 1.0 Dextromethorphan

100.5 ± 3.8 2.0 3-Hydroxymorphinan 52.1 ± 2.4 1.7 Diltiazem 67.4 ± 3.4 1.9 Desacetyl Diltiazem 68.1 ± 3.3 1.8 N-demethyl Diltiazem

Seereference [22] fordetails.


initial activity (3-hydroxymorphinan) and 88% (laudanosine) or 78% (nifedipine) has any

pharmacological relevance.

The results of the study indicated that the chromatographically determined kd values should be

used to establish qualitative relationships, rather than as quantitativemeasures. This assumption was

tested using the Fisher Exact Test to determine the predictive value of kd. When the question was

‘‘does a kd � 1 s�1 predict a percent recovery � 50%?,’’ a significant correlationwas observedwith a

two-sided P-value of 0.0101; when the question was ‘‘does a kd < 2 s�1 predict a percent recovery

<75%?,’’ a very significant correlation was observed with a two-sided P-value of 0.0013.

As previously discussed above {Section 4.A.3}, the data from this study also demonstrate that

the chromatographicallymeasured kd values could be used to identify functional groups that play key

roles in determining the relative length of the inhibitory effect. For example, in the case of DM and 3-

hydroxymorphinian, the O-demethylated metabolite of DM (see Fig. 6), the conversion of a phenolic

ether to a phenol increased the kd from 1.0 to 2.0 s�1, which was reflected in a corresponding increase

in the percent recovery at 7 min from 50 to 100% (Table IX).

The data from this studywere also consistentwith the previous observation that an increase in the

negative electrostatic potential of a compound will decrease the length of the inhibitory effect, and

vice versa, see Section 4.A.3, and also demonstrated that the chromatographic approach can identify

changes inmolecular structure that do not have an effect on the kd values, and, therefore on the length

of the inhibitory activity. This was illustrated by diltiazem and verapamil. With diltiazem, O-

deacetylation orN-demethylation produced essentially the same kd values and percent recovery at the

7-min time point (Table IX). The addition of a methoxy group to one of the phenyl rings of verapamil

to form methoxyverapamil also resulted in no significant change in the observed kd value or percent

recovery at 7 min (Table IX). These results are also consistent with the description of the NCI-a3b4nAChR-binding mechanism,20 as the transformations do not significantly alter the electronic

characteristics of the compounds.

The effect of an increase in the net positive charge of amolecule on kd and percent recovery at the

7-min time point was illustrated by nifedipine and nicardipine, where the primary difference between

the twomolecules is the addition of an aminomoiety to the latter compound. This change produced an

�eightfold reduction in the observed kd value of nicardipine relative to nifedipine, 0.5 and 4.4 s�1,

respectively, and a�60% reduction in the percent recovery at the 7-min time point, 28.9 and 78.1%,

respectively (Table IX).

R2 = 0.7458

0

20

40

60

80

100

120

6.05.04.03.02.01.00.0

kd

% r

eco

very

at

7 m

in

Figure 10. Thecorrelationobservedbetweenthedissociationrate constants (kd) determined for the interactionofthe compoundsused in this study with immobilized a3b4 nAChRs usingnon-linearchromatographyand thepercent recoveryofnicotine-induced[86Rb

þ] efflux in KXa3b4 cells at 7minafterexposure to the compoundsused in the study.


7 . C O N C L U S I O N S

In the discussion section of the manuscript reporting their early studies with an immobilized human

red cell glucose transporter, Lundahl and co-workers stated that ‘‘the immobilization procedure for

the red blood cells can probably be modified for immobilization of membranes from other

sources . . . ’’55 The authors further state that ‘‘the remarkable stability of the immobilized systems

may allow determination of equilibrium constants for various interactions and facilitates the study of

the effects of ionic strength, pH, temperature, lipid composition, and other parameters and conditions

on the affinities.’’55

The data presented in this review have demonstrated that Lundahl’s predictions were correct.

The immobilization of membrane fragments has been extended to include a variety of

transmembrane receptors obtained from different cell lines as well as tissues. The immobilized

receptors have been remarkably stable, for example, the nAChR columns are usually functional for

over 6 months, versatile, and applicable to routine determinations of binding affinities, binding sites,

and competitive binding interactions. Indeed, it has been demonstrated that the data obtained using

the quantitative affinity chromatographic approach is equivalent to data obtained using standard

binding techniques as well as functional techniques. An example of the latter case is the fact that there

was no significant difference in the identification of Pgp transporter substrates between chroma-

tographic method using an immobilized Pgp and transport studies using Caco-2 cell monolayers.76

What sets the chromatographic approach apart from other technologies, such as plate-based and

whole cell-basedmethods, is the ability to rapidly identify and characterize allosteric interactions. As

the interest allosteric modifiers increases, quantitative affinity chromatography could play an

important role in the development of therapeutic agents.

A C K N O W L E D G M E N T S

This work was supported by funds from the Intramural Research Program of the National Institute on

Aging of the National Institutes of Health.

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APPENDIX Marketed Drugs and Their Metabolites Identified as Non-Competitive Inhibitors of

Neuronal Nicotinic Acetylcholine Receptors with IC50 Values <100 mM

SiteSubtype Compound IC50 (µµM) Refadiphenine α3β4, α4β2, α4β4 A 1.8, 3.7, 6.3 U adamantadine α7, α4β2, α3β4 CL 6.5 (α7) Bamlodipine α3β4 C 5.8 U atracurium α7, α4β2, α3β4 D 35.6, 42.0, 3.9 U benzyltetrahydroisoquinolines α7, α4β2, α3β4 E See reference U buprenorphine α3β4 F 12.6 Q bupropion α3β4

α3β4, α4β2, α4β4 1.4 CL 1.8, 12, 14

GH

(2S,3S)-hydroxybupropiona α3β4, α4β2, α4β4 H 10, 3.3, 30.0 CL (2R,3R)-hydroxybupropiona α3β4, α4β2, α4β4 H 6.5, 31.0, 41.0 CL butorphanol α3β4 F 19.4 CL cembranoids α3β4, α4β2 I,J 2.2 – 33.1 U clomipramine α4β2 K 1.3 clozapine α3β4 C 28.0 U cocaine α4β2, α3β2

α3β4 CL5.5, 30.5 26.0 (K)I

LM

corticosterone α3β4 N 94.0 S dexamethasone α3β4 N 45.0 S dextromethorphan α3β4 8.9 CL

10.1OP

Dextrophana α3β4 P 29.6 CL (+)-3-hydroxymorphinanb α3β4 P 59.7 CL (+)-3-methoxymorphinanb α3β4 P 10.3 CL diltiazem α7, α4β2

α3β41.0, 3.8 U 2.3

QC

N-demethyl-dilitiazemc α3β4 C 2.3 U deacetyl-dilitiazemc α3β4 C 4.2 U N-demethyl-deacetyl-dilitiazemc α3β4 C 30.4 U O-demethyl-deacetyl-dilitiazemc α3β4 C 77.6 U N,O-didemethyl-deacetyl-dilitiazemc α3β4 C 73.2 U dimethisoquin α3β4 A 63.1 U (+)-dizocilpine (MK-801) α3β4

α3β457.0 (K)CL I

26.6MO

estradiol α3β4 N 43.0 S etomidate α4β2 R 33.0 CL fluoxetine α7, α3β4 S 11.0, 2.5 U galapamil α4β4 C 6.4 U halothane α4β2 R 27.0 U ibogaine α3β4 G 1.1 CL isofurane α3β4 R 56.0 CL ketamine α4β2, α3β4

α3β472.0, 9.5 CL 1.4

TC

laudanosine α7, α4β2, α4β3 D 18.3, 9.4, 38.4 U levomethorphan α3β4 P 10.9 CL levorphanol α3β4 P 39.9 CL lidocaine α3β4 A 63.0 U mecamylamine α3β4 O 1.0 CL memantine α3β4 C 6.6 CL

(Continued )


APPENDIX (Continued )

methadone α3β4 U 1.9 CL 18-methoxycoronaridine α3β4 V 0.75 U naltrindole α3β4 F 27.5 Q nefazodone α7, α3β4 S 33.0, 8.3 U nicardipine α3β4 C 2.5 U nifedipine α3β4 C 24.7 U paroxetine α7, α4β2 S 23.0, 4.9 U phencyclidine α3β4

α3β45.9 CL 7.0

GO

proadifen α3β4, α4β2, α4β4 A 0.6, 2.0, 1.5 U progesterone α3β4 N 11.0 S procaine α3β4 A 87.0 U propofal α3β4 P 5.4 CL propoxyphene α3β4 U 2.7 CL sertaline α7, α3β4 S 21, 3.1 U sevoflurane α4β2 R 98.0 U tetracaine α3β4, α4β2, α4β4 A 8.3, 27.0, 30.0 U tetrandrine α7, α4β2 W 2.7, 1.1 M thiopental α3β4 R 56.7 CL venlafaxine α3β4 S 12.0 U verapamil α7

α3β440.7 U 3.0

QC

Norverapamild α3β4 C 2.6 U D-620d α3β4 C 48.9 U

SiteSubtype Compound IC50 (µM) Ref

Wheresite refers toabindingsiteonthereceptor towhichthecompoundhasbeenshowntobind, andthesite is labeledasthecentral

lumen (CL), ethidium(E), quinacrine (Q), steroid (S), andUindicatesthat thesitehasnotbeenidentified.aBupropionmetabolite.

bDextromethorphanmetabolite.

cDiltiazemmetabolite.dVerapamilmetabolite.

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A. Gentry CL, Lukas RJ. Local anesthetics non-competitively inhibit function of four distinctnicotinic acetylcholine receptor subtypes. J Pharmacol Exp Ther 2001;299:1038–1048.

B. Matsubayashi H, SwansonKL,Albuquerque EX.Amantadine inhibits nicotinic acetylcholinereceptor function in hippocampal neurons. J Pharmacol Exp Ther 1997;281:834–844

C. Jozwiak K, Moaddel R, Yamaguchi R, Ravichandran S, Collins JR, Wainer IW. Qualitativeassessment of IC 50 values of inhibitors of the neuronal nicotinic acetylcholine receptor usinga single chromatographic experiment and multivariate cluster analysis. J Chromatography2005;819:169–174.

D. Chiodini F, Charpantier E, Muller D, Tassonyi E, Fuchs-Buder T, Bertrand D. Blockade andactivation of the human neuronal nicotinic acetylcholine receptors by atracurium andlaudanosine. Anesthesiology 2001;94:643–651.

E. Exley R, Iturriaga-Vasquez P, Lukas RJ, Sher E, Cassels BK, Bermudez I. Evaluation ofbenzyltetrahydroisoquinolines as ligands for neuronal nicotinic acetylcholine receptors. Brit JPharmacol 2005;146:15–24.

F. Jozwiak K, Ravichandran s, Collins JR,Wainer IW. Interaction of non-competitive inhibitorswith an immobilized a3b4 nicotinic acetylcholine receptor investigated by affinitychromatography, quantitative-structure activity relationship analysis, andmolecular docking.J Med Chem 2004;47:4008–4021.

G. Fryer JD, Lukas RJ. Non-competitive functional inhibition at diverse, human nicotinicacetylcholine receptor subtypes by bupropion, phencyclidine, and ibogaine. J Pharmacol ExpTher 1999;288:88–92.


Dr.RuinMoaddel received his Ph.D. from Northeastern University in 1999 and was a postdoctoral fellow in the

Department of Pharmacology at Georgetown University. He then joined the Bioanalytical and Drug Discovery

Section of the Gerontology Research Center, National Institute on Aging in 2001 as a Senior Scientist. His

research interests are in protein immobilizations and their application in Drug Discovery.

Dr. Krzysztof Jozwiak graduated from Medical University of Lublin, Poland in 2000 and was a postdoctoral

fellow in the Gerontology Research Center, NIA in Baltimore, MD under the supervision of Irving W. Wainer from

2000–2003. He is currently an Associate Professor at the Faculty of Pharmacy, Medical University of Lublin,

Poland. His main research interests include novel experimental approaches in characterization of ligand–

receptor interactions and molecular modeling applications in medicinal chemistry.

Professor IrvingW.Wainer received his Ph.D. degree in chemistry from Cornell University in 1970 from Prof.

Jerrold Meinwald, and did postdoctoral studies in molecular biology (University of Oregon) and clinical

pharmacology (Thomas Jefferson Medical School). Professor Wainer has published more than 300 scientific

papers and 9 books and 23 book chapters. He was founding editor of the journal Chirality (1988–1994) and

Senior Editor of the Journal of Chromatography B: Biomedical Sciences and Applications (1993–2003). In his

current position at the Laboratory for Clinical Investigation at the National Institute on Aging/National

Institutes of Health, Professor Wainer’s research program includes the development of new tools for the

identification and development of new drug substances based upon immobilized receptors, drug transporters and

enzymes placed in a flow system.

H. DamajMI, Carroll FI, Eaton JB,NavarroHA,BloughBE,Mirza S, Lukas RJ,Martin BR. Enantioselectiveeffects of hydroxy metabolites of bupropion on behavior and on function of monoamine transporters andnicotinic receptors. Mol Pharmacol 2004;66:675–682.

I. Fryer JD, Lukas RJ. Non-competitive functional inhibition at diverse, human nicotinic acetylcholinereceptor subtypes by bupropion phencyclidine, and ibogaine. J Pharmacol Exp Ther 1999;288:88–92.

J. EatonMJ, Ospina CA, Rodriguez AD, Eterovic VA. Differential inhibition of nicotine-and acetylcholine-evoked currents through a4b2 neuronal nicotinic receptors by tobacco cembranoids in Xenopus oocytes.Neurosci Lett 2004;366:97–102.

K. Lopez-Valdes HE, Garcia-Colunga J, Miledi R. Effects of clomipramine on neuronal nicotinicacetylcholine receptors. Eur J Pharmacol 2002;444:13–19.

L. DamajMI, Slemmer JE, Carroll FI,Martin BR. Pharmacological characterization of nicotine’s interactionwith cocaine and cocaine anaologs. J Pharmacol Exp Ther 1999;289:1229–1236.

M. Krivoshein AV, Hess GP.Mechanism-based approach to the successful prevention of cocaine inhibition ofthe neuronal (a3b4) nicotinic acetylcholine receptor. Biochem 2004;43:481–489.

N. Ke L, Lukas RJ. Effects of steroid exposure on ligand binding and functional activities of diverse nicotinicacetylcholine receptor subtypes. J Neurochem 1996;67:1100–1112.

O. Hernandez SC, Bertolino M, Xiao Y, Pringle KE, Caruso FS, Kellar KJ. Dextromethorphan and itsmetabolite dextrorphan block a3b4 neuronal nicotinic receptors. J Pharmacol Exp Ther 2000;293:962–967.

P. Jozwiak K, Hernandez SC, Kellar KJ, Wainer IW. Enantioselective interactions of dextromethorphan andlevomethorphan with the a3b4-nicotinic acetylcholine receptor: Comparison of chromatographic andfunctional data. J Chromatography B 2003;797:373–379.

Q. Houlihan LM, Slater EY, Beadle DJ, Lukas RJ, Bermudez I. Effects of diltiazem on human nicotinicacetylcholine and GABA receptors. Neuro Pharmacol 2000;39:2533–2542.

R. Tassonyi E, Charpantier E,Muller D, Dumont L, Bertrand D. The role of nicotinic acetylcholine receptorsin the mechanisms of anesthesia. Brain Res 2002;57:133–150.

S. Fryer JD, Lukas RJ. Antidepressants non-competitively inhibit nicotinic acetylcholine receptor function. JNeurochem 1999;72:1117–1124.

T. Yamakura T, Chavez-Noriega LE, Harris RA. Subunit-dependent inhibition of human neuronal nicotinicacetylcholine receptors and other ligand-gated ion channels by dissociative anesthetics ketamine anddizocilpine 2000;92:1144–1153.

U. Xiao Y, Smith RD, Caruso FS, Kellar KJ. Blockade of rat a3b4 nicotinic receptor function by methadone,its metabolites, and structural analogs. J Pharmacol Exp Ther 2001;299:366–371.

V. Glick SD, Maisonneuve IM, Kitchen BA. Modulation of nicotine self-administration in rats bycombination therapywith agents blocking a3b4 nicotinic receptors. Eur J Pharmacol. 2002;448:185–191.

W. Slater Y, Houlihan LM, Cassels BK, Lukas RJ, Bermudez I. Effects of the plant alkaloid tetrandrine onhuman nicotinic acetylcholine receptors. Eur J Pharmacol 2002;450:213–221.


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Allosteric modifiers of neuronal nicotinic acetylcholine receptors: New methods, new opportunities

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