Effect of novel negative allosteric modulators of neuronal nicotinic receptors on cells expressing native and recombinant nicotinic receptors: implications for drug discovery

Allosteric modulation of nAChRs is considered to be one of the most promising approaches for drug design targeting nicotinic acetylcholine receptors (nAChRs). We have reported previously on the pharmacological activity of several compounds that seem to act noncompetitively to inhibit the activation of alpha3beta4(*) nAChRs. In this study, the effects of 51 structurally similar molecules on native and recombinant alpha3beta4 nAChRs are characterized. These 51 molecules inhibited adrenal neurosecretion activated via stimulation of native alpha3beta4(*) nAChR, with IC(50) values ranging from 0.4 to 13.0 microM. Using cells expressing recombinant alpha3beta4 nAChRs, these molecules inhibited calcium accumulation (a more direct assay to establish nAChR activity), with IC(50) values ranging from 0.7 to 38.2 microM. Radiolabeled nAChR binding studies to orthosteric sites showed no inhibitory activity on either native or recombinant nAChRs. Correlation analyses of the data from both functional assays suggested additional, non-nAChR activity of the molecules. To test this hypothesis, the effects of the drugs on neurosecretion stimulated through non-nAChR mechanisms were investigated; inhibitory effects ranged from no inhibition to 95% inhibition at concentrations of 10 microM. Correlation analyses of the functional data confirmed this hypothesis. Several of the molecules (24/51) increased agonist binding to native nAChRs, supporting allosteric interactions with nAChRs. Computational modeling and blind docking identified a binding site for our negative allosteric modulators near the orthosteric binding site of the receptor. In summary, this study identified several molecules for potential development as negative allosteric modulators and documented the importance of multiple screening assays for nAChR drug discovery.     

Allosteric modulators of rhodopsin-like G protein-coupled receptors: opportunities in drug development

Rhodopsin-like (class A) G protein-coupled receptors (GPCRs) are one of the most important classes of drug targets. The discovery that these GPCRs can be allosterically modulated by small drug molecules has opened up new opportunities in drug development. It will allow the drugability of "difficult targets", such as GPCRs activated by large (glyco)proteins, or by very polar or highly lipophilic physiological agonists. Receptor subtype selectivity should be more easily achievable with allosteric than with orthosteric ligands. Allosteric modulation will allow a broad spectrum of pharmacological effects largely expanding that of orthosteric ligands. Furthermore, allosteric modulators may show an improved safety profile as compared to orthosteric ligands. Only recently, the explicit search for allosteric modulators has been started for only a few rhodopsin-like GPCRs. The first negative allosteric modulators (allosteric antagonists) of chemokine receptors, maraviroc (CCR5 receptor), used in HIV therapy, and plerixafor (CXCR4 receptor) for stem cell mobilization, have been approved as drugs. The development of allosteric modulators for rhodopsin-like GPCRs as novel drugs is still at an early stage; it appears highly promising.     

Allosteric ligands for G protein‐coupled receptors: A novel strategy with attractive therapeutic opportunities

Allosteric receptor ligands bind to a recognition site that is distinct from the binding site of the endogenous messenger molecule. As a consequence, allosteric agents may attach to receptors that are already transmitter‐bound. Ternary complex formation opens an avenue to qualitatively new drug actions at G protein‐coupled receptors (GPCRs), in particular receptor subtype selective potentiation of endogenous transmitter action. Consequently, suitable exploitation of allosteric recognition sites as alternative molecular targets could pave the way to a drug discovery paradigm different from those aimed at mimicking or blocking the effects of endogenous (orthosteric) receptor activators. The number of allosteric ligands reported to modulate GPCR function is steadily increasing and some have already reached routine clinical use. This review aims at introducing into this fascinating field of drug discovery and at providing an overview about the achievements that have already been made. Various case examples will be discussed in the framework of GPCR classification (family A, B, and C receptors). In addition, the behavior at muscarinic receptors of hybrid derivatives incorporating both an allosteric and an orthosteric fragment in a common molecular skeleton will be illustrated. © 2009 Wiley Periodicals, Inc. Med Res Rev, 30, No. 3, 463–549, 2010     

The impact of orthosteric radioligand depletion on the quantification of allosteric modulator interactions

Radioligand binding assays remain a common method for quantifying the effects of allosteric modulators at G protein-coupled receptors. The allosteric ternary complex model (ATCM) is the simplest model applied to derive estimates of modulator affinity (K(B)) and cooperativity (alpha), which are necessary for understanding structure-activity relationships. However, the increasing drive toward assay miniaturization in modern drug discovery may lead to conditions where appreciable ligand depletion occurs in the assay. Theoretical simulations investigating the impact of orthosteric radioligand depletion on the estimation of ATCM parameters revealed the following. 1) For allosteric inhibitors, application of the standard ATCM to data obtained under depletion conditions leads to an underestimation of pK(B) and an overestimation of log alpha. 2) For allosteric enhancers, the opposite was noted, but not always; the nonlinear regression algorithm is more likely to struggle to converge to a satisfactory solution of (nondepletion) ATCM parameters in this situation. 3) Application of a novel ATCM that explicitly incorporates orthosteric ligand depletion will yield more reliable model estimates, provided the degree of depletion is not high (< approximately 50%). Subsequent experiments investigated the interaction between [3H]N-methylscopolamine and the allosteric enhancer, alcuronium, or inhibitor, gallamine, in the presence of increasing concentrations of M(2) muscarinic acetylcholine receptor and showed that application of an ATCM that explicitly incorporates radioligand depletion can indeed give more robust estimates of modulator affinity and cooperativity estimates than the standard model. These results have important implications for the quantification of allosteric modulator actions in binding-based discovery assays.     

Allosteric modulation of G protein-coupled receptors by amiloride and its derivatives. Perspectives for drug discovery?

The function of G protein-coupled receptors (GPCRs) can be modulated by compounds that bind to other sites than the endogenous orthosteric binding site, so-called allosteric sites. Structure elucidation of a number of GPCRs has revealed the presence of a sodium ion bound in a conserved allosteric site. The small molecule amiloride and analogs thereof have been proposed to bind in this same sodium ion site. Hence, this review seeks to summarize and reflect on the current knowledge of allosteric effects by amiloride and its analogs on GPCRs. Amiloride is known to modulate adenosine, adrenergic, dopamine, chemokine, muscarinic, serotonin, gonadotropin-releasing hormone, GABA_B, and taste receptors. Amiloride analogs with lipophilic substituents tend to be more potent modulators than amiloride itself. Adenosine, α-adrenergic and dopamine receptors are most strongly modulated by amiloride analogs. In addition, for a few GPCRs, more than one binding site for amiloride has been postulated. Interestingly, the nature of the allosteric effect of amiloride and derivatives varies considerably between GPCRs, with both negative and positive allosteric modulation occurring. Since the sodium ion binding site is strongly conserved among class A GPCRs it is to be expected that amiloride also binds to class A GPCRs not evaluated yet. Investigating this typical amiloride-GPCR interaction further may yield general insight in the allosteric mechanisms of GPCR ligand binding and function, and possibly provide new opportunities for drug discovery.     

Harnessing Allostery: A Novel Approach to Drug Discovery

Allostery is the most direct and efficient way for regulation of biological macromolecule function, ranging from the control of metabolic mechanisms to signal transduction pathways. Allosteric modulators target to allosteric sites, offering distinct advantages compared to orthosteric ligands that target to active sites, such as greater specificity, reduced side effects, and lower toxicity. Allosteric modulators have therefore drawn increasing attention as potential therapeutic drugs in the design and development of new drugs. In recent years, advancements in our understanding of the fundamental principles underlying allostery, coupled with the exploitation of powerful techniques and methods in the field of allostery, provide unprecedented opportunities to discover allosteric proteins, detect and characterize allosteric sites, design and develop novel efficient allosteric drugs, and recapitulate the universal features of allosteric proteins and allosteric modulators. In the present review, we summarize the recent advances in the repertoire of allostery, with a particular focus on the aforementioned allosteric compounds.     

Allosteric properties of G protein-coupled receptor oligomers

Allosteric regulation of ligand binding is a well-established mechanism regulating the function of G protein-coupled receptors (GPCR). Allosteric modulators have been considered so far as molecules binding to an allosteric site, distinct from that of the reference ligand (orthosteric site), and able to modulate the binding affinity at the orthosteric site and/or the signaling properties resulting from orthosteric site occupancy. Given that most GPCR are known to form dimers or higher order oligomers, we explored whether allosteric interactions could also occur between protomers within oligomeric arrays, thereby influencing binding and signaling receptor properties. Two main conclusions emerged from such studies. First, allosteric modulators can affect one receptor by binding to another receptor within a dimeric or oligomeric complex. Second, allosteric modulators might act on a given receptor by targeting the "orthosteric site" in another receptor of the complex. Allosteric regulation within di(oligo)mers thus implies that the pharmacological properties of a given receptor subtype can be influenced by the array of dimerization partners coexpressed in each particular cell type. Ligands could thus act as agonists or antagonists on 1 receptor, while modulating allosterically the function of a variety of other receptors to which they do not bind directly. Allosteric regulation across GPCR oligomeric interfaces is expected to greatly influence the practice of pharmacology. It will likely affect the design of drug discovery programs, which rely mostly on the overexpression of the receptor of interest in a cell line, thereby focusing on homo-oligomers and ignoring the potential effects of other partners.     

Synergistic regulation mechanism of iperoxo and LY2119620 for muscarinic acetylcholine M2 receptor

Muscarinic acetylcholine receptors are GPCRs that regulate the activity of a diverse array of central and peripheral functions in the human body, including the parasympathetic actions of acetylcholine. The M2 muscarinic receptor subtype plays a key role in modulating cardiac function and many important central processes. The orthosteric agonist and allosteric modulator can bind the pocket of M2. However, the detailed relationship between orthosteric agonist and allosteric modulator of M2 is still unclear. In this study, we intend to elucidate the residue-level regulation mechanism and pathway via a combined approach of dynamical correlation network and molecular dynamics simulation. Specifically computational residue-level fluctuation correlation data was analyzed to reveal detailed dynamics signatures in the regulation process. A hypothesis of “synergistic regulation” is proposed to reveal the cooperation affection between the orthosteric agonist and allosteric modulator, which is subsequently validated by perturbation and mutation analyses. Two possible synergistic regulation pathways of 2CU-I178-Y403-W400-F396-L114-Y440-Nb9 and IXO-V111-F396-L114-Y440-Nb9 were identified by the shortest path algorithm and were confirmed by the mutation of junction node. Furthermore, the efficiency of information transfer of bound M2 is significant higher than any single binding system. Our study shows that targeting the synergistic regulation pathways may better regulate the calcium channel of M2. The knowledge gained in this study may help develop drugs for diseases of the central nervous system and metabolic disorders.

Dynamics correlation network was used to reveal the synergistic regulation mechanism of iperoxo and LY2119620 for muscarinic acetylcholine M2 receptor.     

Application of a kinetic model to the apparently complex behavior of negative and positive allosteric modulators of muscarinic acetylcholine receptors

The binding of allosteric modulators to G protein-coupled receptors (GPCRs) is often described by an equilibrium allosteric ternary complex model (ATCM). This study evaluated the effects of three modulators on the binding of [(3)H]N-methylscopolamine ([(3)H]NMS) to the human M(2) muscarinic acetylcholine receptor (mAChR). The binding of each modulator was more complex than predicted by the ATCM; the inhibitors heptane-1,7-bis-(dimethyl-3-phthalimidopropyl)-ammonium bromide and gallamine yielded biphasic curves that were described empirically by a two-site binding model, whereas the enhancer alcuronium yielded a bell-shaped curve. Radioligand dissociation assays revealed that the modulators retarded [(3)H]NMS kinetics such that the system never attained equilibrium. Subsequent application of a kinetic ATCM accommodated and quantified all experimental observations. Our findings confirm and extend previous studies on the use of a kinetic ATCM for mAChR allosteric enhancers, but also highlight how complex curves displayed by allosteric inhibitors can be misinterpreted in terms of multisite orthosteric binding. It is possible that similar behavior of other allosteric modulators at GPCRs may reflect nonequilibrium binding artifacts rather than deviation from an ATCM.     

Allosteric Modulation: An Alternate Approach Targeting the Cannabinoid CB1 Receptor

The cannabinoid CB1 receptor is a G protein coupled receptor and plays an important role in many biological processes and physiological functions. A variety of CB1 receptor agonists and antagonists, including endocannabinoids, phytocannabinoids, and synthetic cannabinoids, have been discovered or developed over the past 20 years. In 2005, it was discovered that the CB1 receptor contains allosteric site(s) that can be recognized by small molecules or allosteric modulators. A number of CB1 receptor allosteric modulators, both positive and negative, have since been reported and importantly, they display pharmacological characteristics that are distinct from those of orthosteric agonists and antagonists. Given the psychoactive effects commonly associated with CB1 receptor agonists and antagonists/inverse agonists, allosteric modulation may offer an alternate approach to attain potential therapeutic benefits while avoiding inherent side effects of orthosteric ligands. This review details the complex pharmacological profiles of these allosteric modulators, their structure–activity relationships, and efforts in elucidating binding modes and mechanisms of actions of reported CB1 allosteric modulators. The ultimate development of CB1 receptor allosteric ligands could potentially lead to improved therapies for CB1‐mediated neurological disorders.     

Drug discovery and the human kinome: recent trends

A major new trend in drugs targeted at protein kinases is the discovery of allosteric modulators. These compounds differ from ATP-centric drugs in that they do not compete with ATP for binding to the catalytic domain, generally acting by inducing conformational changes to modulate activity. They could provide a number of advantages over more classical protein kinase drugs. For example, they are likely to be more selective, since they bind to unique regions of the kinase and may be useful in overcoming resistance that has developed to drugs that compete with ATP. They offer the ability of activating the kinases either by removing factors that inhibit kinase activity or by simply producing changes to the enzyme to foster catalytic activity. Furthermore, they provide more subtle modulation of kinase activity than simply blocking ATP access to inhibit activity. One hurdle to overcome in discovering these compounds is that allosteric modulators may need to inhibit protein-protein interactions; generally difficult to accomplish with small molecules. Despite the technical problems of identifying allosteric modulators, major gains have been made in identifying allosteric inhibitors and activators of the growth factor receptors as well as soluble tyrosine and serine/threonine kinases and some of these drugs are now in various stages of clinical trials. This review will focus on the discovery of novel allosteric modulators of protein kinases and drug discovery approaches that have been employed to identify such compounds.     

Determining the potency and molecular mechanism of action of insurmountable antagonists

Insurmountable antagonism (maximal response to the agonist depressed) can result from a temporal inequilibrium involving a slow offset orthosteric antagonist or be the result of an allosteric modulation of the receptor. The former mechanism is operative when the antagonist, agonist, and receptors cannot come to proper equilibrium during the time allotted for collection of agonist response (hemi-equilibrium conditions). Allosteric effects (changes in the conformation of the receptor through binding of the allosteric modulator to a separate site) can preclude the agonist-induced production of response, leading to depression of maximal responses. In these cases, the effects on receptor affinity can be observed as well. The first premise of this article is that system-independent estimates of insurmountable antagonist potency can be made with no prior knowledge of molecular mechanism through the use of pA(2) (-log molar concentration of antagonist producing a 2-fold shift of the concentration response curve) measurements The relationship between the pA(2) and antagonist pK(B) (-log equilibrium dissociation constant of the antagonist-receptor complex) is described; the former is an extremely close approximation of the latter in most cases. The second premise is that specially designed experiments are required to differentiate orthosteric versus allosteric mechanisms; simply fitting of data to orthosteric or allosteric theoretical models can lead to ambiguous results. A strategy to determine whether the observed antagonism is orthosteric (agonist and antagonist competing for the same binding site on the receptor) or allosteric in nature is described that involves the detection of the hallmarks of allosteric response, namely saturation and probe dependence of effect.     

Interactions of noncompetitive inhibitors with nicotinic receptors in the rat brain

This study was performed to determine how drugs that inhibit the function of peripheral nicotinic receptors (noncompetitive inhibitors), interact with nicotinic receptors in the brain. By using [3H]MCC (methylcarbamylcholine ) as a ligand for nicotinic receptors, competition studies at a fixed concentration of radioligand and saturation studies were performed with various noncompetitive inhibitors. [3H]MCC labeled high affinity nicotinic receptor sites in the rat brain at equilibrium. The sites appeared to represent desensitized nicotinic receptors, comprising a fraction of the total pool of these receptors. At micromolar concentrations, noncompetitive inhibitors interacted distinctively with [3H]MCC binding sites. Mecamylamine behaved as an allosteric inhibitor, as it decreased the apparent density of [3H]MCC binding sites. Tetracaine had mixed allosteric/competitive properties, reducing both the density and the affinity of binding. Chlorpromazine manifested a biphasic effect, increasing receptor density at concentrations of approximately 50 to 500 microM and reducing the affinity at higher concentrations. The results suggest that noncompetitive inhibitors bind to different, but interacting sites associated with desensitized nicotinic receptors in the brain, as well as to recognition sites for acetylcholine.     

Drugs interacting with G protein α subunits: selectivity and perspectives

Summary— Extracellular signal molecules as diverse as hormones, neurotransmitters and photons use a signal transduction pathway involving a receptor, a G protein and effectors. Compounds that interact directly with G proteins can mimic the receptor-G protein interaction or can block the activation of G proteins by receptors. Several binding sites exist on the G_α protein that may be exploited for the design of synthetic stimulatory or inhibitory ligands. The effector binding site is regulated by endogenous proteins and appears to be a target for selective exogenous ligands. The GTP binding site presents a large homology within the G protein families and therefore the nucleotide analogs might not be considered as a tool to discriminate between the G protein subclasses. In contrast, different experimental strategies have substantiated the specificity in the interaction between a receptor and a G protein, the receptor binding site of G proteins should be considered as potential drug targets. Drugs interfering with this site such as mastoparan and related peptides, GPAnt-2 and suramin, are lead compounds in the design of selective G protein antagonists. Benzalkonium chloride and methoctramine have agonist or antagonist properties, depending on G protein subtypes. Such compounds would be very useful to delineate the functions of G proteins and G protein-coupled receptors, to understand some side effects of drugs used in therapy and to develop new therapeutic agents.     

G protein coupling and signaling pathway activation by m1 muscarinic acetylcholine receptor orthosteric and allosteric agonists

The M(1) muscarinic acetylcholine (mACh) receptor is among a growing number of G protein-coupled receptors that are able to activate multiple signaling cascades. AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine) is an allosteric agonist that can selectively activate the M(1) mACh receptor in the absence of an orthosteric ligand. Allosteric agonists have the potential to stabilize unique receptor conformations, which may in turn cause differential activation of signal transduction pathways. In the present study, we have investigated the signaling pathways activated by AC-42, its analog 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone), and a range of orthosteric muscarinic agonists [oxotremorine-M (oxo-M), arecoline, and pilocarpine] in Chinese hamster ovary cells recombinantly expressing the human M(1) mACh receptor. Each agonist was able to activate Galpha(q/11)-dependent signaling, as demonstrated by an increase in guanosine 5'-O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding to Galpha(q/11) proteins and total [(3)H]inositol phosphate accumulation assays in intact cells. All three orthosteric agonists caused significant enhancements in [(35)S]GTPgammaS binding to Galpha(i1/2) subunits over basal; however, neither allosteric ligand produced a significant response. In contrast, both orthosteric and allosteric agonists are able to couple to the Galpha(s)/cAMP pathway, enhancing forskolin-stimulated cAMP accumulation. These data provide support for the concept that allosteric and orthosteric mACh receptor agonists both stabilize receptor conformations associated with Galpha(q/11)- and Galpha(s)-dependent signaling; however, AC-42 and 77-LH-28-1, unlike oxo-M, arecoline, and pilocarpine, do not seem to promote M(1) mACh receptor-Galpha(i1/2) coupling, suggesting that allosteric agonists have the potential to activate distinct subsets of downstream effectors.     

Pharmacological characterization of LY593093, an M1 muscarinic acetylcholine receptor-selective partial orthosteric agonist

Alzheimer's disease and schizophrenia are characterized by expression of psychotic, affective, and cognitive symptoms. Currently, there is a lack of adequate treatment for the cognitive symptoms associated with these diseases. Cholinergic signaling and, in particular, M1 muscarinic acetylcholine receptor (m1AChR) signaling have been implicated in the regulation of multiple cognitive domains. Thus, the M1AChR has been identified as a therapeutic drug target for diseases, such as schizophrenia and Alzheimer's disease, that exhibit marked cognitive dysfunction as part of their clinical manifestation. Unfortunately, the development of selective M1 agonist medications has not been successful, mostly because of the highly conserved orthosteric acetylcholine binding site among the five muscarinic receptor subtypes. More recent efforts have focused on the development of allosteric M1AChR modulators that target regions of the receptor distinct from the orthosteric site that are less conserved between family members. However, orthosteric and allosteric ligands may differentially modulate receptor function and ultimately downstream signaling pathways. Thus, the need for highly selective M1AChR orthosteric agonists still exists, not only as a potential therapeutic but also as a pharmacological tool to better understand the physiologic consequences of M1AChR orthosteric activation. Here, we describe the novel, potent and selective M1AChR orthosteric partial agonist LY593093 [N-[(1R,2R)-6-({(1E)-1-[(4-fluorobenzyl)(methyl)amino]ethylidene})amino)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]biphenyl-4-carboxamide]. This compound demonstrates modest to no activity at the other muscarinic receptor subtypes, stimulates Gα(q)-coupled signaling events as well as β-arrestin recruitment, and displays significant efficacy in in vivo models of cognition.     

An approach to drug abuse, intoxication and withdrawal

The symptomatic effects of drug abuse are a result of alterations in the functioning of the following neurotransmitters or their receptors: acetylcholine, dopamine, gamma-aminobutyric acid, norepinephrine, opioids and serotonin. Anticholinergic drugs antagonize acetylcholine receptors. Dissociative drugs affect all transmitter sites. Opiates act on both opioid and adrenergic receptor sites. Psychedelic drugs stimulate serotonin release, and sedative-hypnotic drugs potentiate the gamma-aminobutyric acid receptor. Specific signs and symptoms are associated with the neurotransmitters and receptors affected by each drug class. By recognizing symptomatic changes related to particular neurotransmitters and their receptors, family physicians can accurately determine the drug class and intervene appropriately to counteract drug-induced effects.     

Radioreceptor assay of anticholinergic drugs

The radioreceptor assay (RRA) of anticholinergic drugs in plasma and other biological fluids presents methodological difficulties. The specificity is not comparable with some pure chemical methods because drug metabolites which are active in binding to muscarinic receptors participate in the assay. There are also problems with serum dilution, with the lipophilicity of the radioligands and drugs, and with the protein binding of the drugs. However, the RRA of anticholinergic drugs is rapid and sensitive. In racemic drugs, only active stereoisomers show affinity for the muscarinic receptors. Results with RRA have been shown to correlate with anticholinergic effects, both desired and side effects. A careful familiarization with the method used for each individual drug analysis allows its useful clinical application.     

Progress in brain penetration evaluation in drug discovery and development

This review discusses strategies to optimize brain penetration from the perspective of drug discovery and development. Brain penetration kinetics can be described by the extent and time to reach brain equilibrium. The extent is defined as the ratio of free brain concentration to free plasma concentration at steady state. For all central nervous system (CNS) drug discovery programs, optimization of the extent of brain penetration should focus on designing and selecting compounds having low efflux transport at the blood-brain barrier (BBB). The time to reach brain equilibrium is determined by both BBB permeability and brain tissue binding. Rapid brain penetration can be achieved by increasing passive permeability and reducing brain tissue binding. Although many drug transporters have been identified at the BBB, the available literature demonstrates only the in vivo functional importance of P-glycoprotein (P-gp) in limiting brain penetration of its substrates. Drug-drug interactions mediated by P-gp at the BBB are possible due to inhibition or induction of P-gp. For newly identified drug transporters at the BBB, more research is needed to reveal their in vivo significance. We propose the following strategies for addressing drug transporters at the BBB. 1) Drug discovery screens should be used to eliminate good P-gp substrates for CNS targets. Special consideration could be given to moderate P-gp substrates as potential CNS drugs based on a high unmet medical need and the presence of a large safety margin. 2) Selection of P-gp substrates as drug candidates for non-CNS targets can reduce their CNS-mediated side effects.     

The pharmacology of McN-A-343

The unusual pharmacology of McN-A-343 was first described by Roszowski in 1961. The agonist appeared to be a selective stimulant of muscarinic receptors in sympathetic ganglia, now known to be the muscarinic M? receptor subtype. However, subsequent research demonstrated that McN-A-343 is a partial agonist with similar affinity at all five muscarinic acetylcholine receptor subtypes and its relative selectivity depends on a higher efficacy at the M? (and M?) subtypes. Being a partial agonist its action is also dependent on factors, such as receptor density and coupling efficacy between receptor activation and tissue response. Nevertheless, the relatively high efficacy at M? receptors led to its widespread use as an aid to distinguish responses mediated through M? receptors from those utilizing M? or M? muscarinic receptor subtypes, especially in the CNS. There is also evidence that it has an allosteric action at some receptor subtypes. Recently, it was demonstrated that McN-A-343 can bind to an allosteric site on the M? receptor as well as to the orthosteric site and has thus been termed a "bitopic agonist". This allosteric site differs from that occupied by allosteric modulators, such as gallamine. Comparison of comparable mutagenic changes in M? and M? receptors also suggests that McN-A-343 utilizes different regions of the two receptors for ERK1/2 activation. McN-A-343 has a number of non-muscarinic actions. These include activation of some types of nicotinic acetylcholine receptors, antagonism of serotonin 5-HT? and 5-HT? receptor subtypes, inhibition of the uptake mechanism and a local anesthetic action.