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     

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.     

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.     

Use of Allosteric Targets in the Discovery of Safer Drugs

The need for drugs with fewer side effects cannot be overemphasized. Today, most drugs modify the actions of enzymes, receptors, transporters and other molecules by directly binding to their active (orthosteric) sites. However, orthosteric site configuration is similar in several proteins performing related functions and this leads to a lower specificity of a drug for the desired protein. Consequently, such drugs may have adverse side effects. A new basis of drug discovery is emerging based on the binding of the drug molecules to sites away (allosteric) from the orthosteric sites. It is possible to find allosteric sites which are unique and hence more specific as targets for drug discovery. Of many available examples, two are highlighted here. The first is caloxins - a new class of highly specific inhibitors of plasma membrane Ca²⁺ pumps. The second concerns the modulation of receptors for the neurotransmitter acetylcholine, which binds to 12 types of receptors. Exploitation of allosteric sites has led to the discovery of drugs which can selectively modulate the activation of only 1 (M1 muscarinic) out of the 12 different types of acetylcholine receptors. These drugs are being tested for schizophrenia treatment. It is anticipated that the drug discovery exploiting allosteric sites will lead to more effective therapeutic agents with fewer side effects.Key Words: Adverse effects, Allosterism, Drug specificity, Drug targets, Orthosteric sites, Pharmacology, Side effects     

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.     

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 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.     

Positive and negative allosteric modulation of metabotropic glutamate receptors: emerging therapeutic potential

Metabotropic glutamate receptors (mGluRs) modulate neuronal activity in the central and peripheral nervous systems, and since their discovery have attracted considerable attention as putative therapeutic targets for a range of neurological and psychiatric disorders. A number of competitive agonists and antagonists acting at the N-terminal glutamate binding site have been identified, the majority of which are conformationally constrained or substituted amino acid analogues. These ligands have greatly facilitated investigation of the physiological and pathological roles of the receptor family. However, their utility and therapeutic potential has been restricted by relatively poor bioavailability and central nervous system (CNS) penetration, as well as limited chemical tractability and, generally, a lack of selectivity for individual mGluRs. Recently, a number of non-competitive mGluR ligands have been identified which bind within the receptor transmembrane heptahelical domain. These include both positive and negative allosteric modulators. Positive allosteric modulators do not exhibit intrinsic agonism but facilitate agonist-mediated receptor activity. Negative allosteric modulators include both non-competitive antagonists and inverse agonists. Allosteric modulation offers the potential for improved selectivity, particularly for individual receptors within the mGluR family, and enhanced chemical tractability relative to competitive agonists/antagonists. In addition, positive allosteric modulation provides a distinct, and perhaps superior, profile to receptor agonism, offering the potential for facilitation of physiologically appropriate receptor activation with reduced liability for receptor desensitisation and/or tolerance. Thus, the emerging field of positive and negative allosteric modulation of the mGluR family offers considerable promise for the development of novel therapeutics.     

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.     

An atlas and functional analysis of G-protein coupled receptors in human islets of Langerhans

G-protein coupled receptors (GPCRs) regulate hormone secretion from islets of Langerhans, and recently developed therapies for type-2 diabetes target islet GLP-1 receptors. However, the total number of GPCRs expressed by human islets, as well as their function and interactions with drugs, is poorly understood. In this review we have constructed an atlas of all GPCRs expressed by human islets: the ‘islet GPCRome’. We have used this atlas to describe how islet GPCRs interact with their endogenous ligands, regulate islet hormone secretion, and interact with drugs known to target GPCRs, with a focus on drug/receptor interactions that may affect insulin secretion. The islet GPCRome consists of 293 GPCRs, a majority of which have unknown effects on insulin, glucagon and somatostatin secretion. The islet GPCRs are activated by 271 different endogenous ligands, at least 131 of which are present in islet cells. A large signalling redundancy was also found, with 119 ligands activating more than one islet receptor. Islet GPCRs are also the targets of a large number of clinically used drugs, and based on their coupling characteristics and effects on receptor signalling we identified 107 drugs predicted to stimulate and 184 drugs predicted to inhibit insulin secretion. The islet GPCRome highlights knowledge gaps in the current understanding of islet GPCR function, and identifies GPCR/ligand/drug interactions that might affect insulin secretion, which are important for understanding the metabolic side effects of drugs. This approach may aid in the design of new safer therapeutic agents with fewer detrimental effects on islet hormone secretion.     

A new approach for studying GPCR dimers: drug-induced inactivation and reactivation to reveal GPCR dimer function in vitro, in primary culture, and in vivo

GPCRs are a major family of homologous proteins and are key mediators of the effects of numerous endogenous neurotransmitters, hormones, cytokines, therapeutic drugs, and drugs-of-abuse. Despite the enormous amount of research on the pharmacological and biochemical properties of GPCRs, the question as to whether they exist as monomers, dimers, or higher order structures in the body is unanswered. The GPCR dimer field has been dominated by techniques involving recombinant cell lines expressing mutant receptors, often involving the solubilization of the receptors. These techniques cannot be applied in vivo or even to primary cell cultures. This review will focus on a novel approach to exploring the functional properties of homodimers. Studies of the 5-HT₇ and 5-HT_2A serotonin receptors have revealed that binding of a pseudo-irreversible antagonist (“inactivator”) to one of the orthosteric sites of a homodimer abolishes all receptor activity, and subsequent binding of a competitive antagonist to the orthosteric site of the second protomer releases the inactivator, allowing the receptor to return to an active state. This approach demonstrates allosteric crosstalk between protomers of native GPCR homodimers, indicating that GPCRs do exist and function as homodimers in both recombinant cells and rat primary astrocytes. This technique can be applied universally using intact recombinant or primary cells in culture, membrane homogenate preparations and, potentially, in vivo. The data obtained using the 5-HT₇ and 5-HT_2A receptors are strongly supportive of a GPCR homodimer structure, with little evidence of monomer involvement in the function of these receptors.     

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.     

G protein-coupled receptors: a count of 1001 conformations

G protein-coupled receptors (GPCRs) were initially regarded to adopt an inactive and an active conformation and to activate a single type of G protein. Studies with recombinant cell systems have led to a more complex picture. First, GPCRs can activate distinct G protein species. Second, GPCR multistate models have been invoked to explain their complex behaviour in the presence of agonists, antagonists and other binding partners. The occurrence of intermediate receptor conformational states during GPCR activation and antagonist binding is suggested by fluorescence measurements and studies with constitutively active receptor mutants and insurmountable antagonists. Different agonists may trigger distinct effector pathways through a single receptor by dictating its preference for certain G proteins (i.e. 'agonist trafficking'). Structural modification and exogenous and endogenous (e.g. other cellular proteins, lipids) allosteric modulators also affect ligand-GPCR interaction and receptor activation. These new developments in GPCR research could lead to the development of more selective therapeutic drugs.     

Regulation of M2 muscarinic acetylcholine receptor expression and signaling by prolonged exposure to allosteric modulators

The effects of prolonged exposure of M(2) muscarinic acetylcholine receptors (mAChRs), stably expressed in Chinese hamster ovary cells, to the allosteric modulators gallamine, alcuronium, and heptane-1,7-bis (dimethyl-3'-phthalimidopropyl)-ammonium bromide (C(7)/3'-phth) were compared with the effects of the agonist carbachol (CCh) and antagonists atropine and N-methylscopolamine (NMS). Intact cell saturation binding assays using [(3)H]NMS found that pretreatment of the cells for 24 h with CCh caused a significant down-regulation of receptor number, whereas atropine, NMS, and all three allosteric modulators caused receptor up-regulation. Functional assays using a cytosensor microphysiometer to measure whole-cell metabolic rate found no acute effects of gallamine on receptor signaling, whereas atropine seemed to behave as an inverse agonist. Pretreatment of the cells with gallamine (20 microM) or atropine (20 nM) resulted in a significant enhancement of the maximal effect evoked by CCh. In contrast, CCh (100 microM) pretreatment resulted in a significant reduction in maximal receptor signaling capacity. Time-course experiments revealed that the effects of atropine and gallamine on receptor up-regulation are only visualized after at least 12-h ligand exposure, compared with the more rapid effects of CCh, which achieve steady-state down-regulation within 90 min. Additional experiments monitoring CCh-mediated M(2) mAChR internalization in the presence of gallamine revealed that part of the mechanism underlying the effects of the modulator on receptor expression may involve a change in receptor internalization properties. These findings suggest that, like orthosteric ligands, G protein-coupled receptor allosteric modulators also are able to mediate long-term effects on receptor regulation.     

The G protein-coupled receptors: pharmacogenetics and disease

Genetic variation in G-protein coupled receptors (GPCRs) is associated with a wide spectrum of disease phenotypes and predispositions that are of special significance because they are the targets of therapeutic agents. Each variant provides an opportunity to understand receptor function that complements a plethora of available in vitro data elucidating the pharmacology of the GPCRs. For example, discrete portions of the proximal tail of the dopamine D1 receptor have been discovered, in vitro, that may be involved in desensitization, recycling and trafficking. Similar in vitro strategies have been used to elucidate naturally occurring GPCR mutations. Inactive, over-active or constitutively active receptors have been identified by changes in ligand binding, G-protein coupling, receptor desensitization and receptor recycling. Selected examples reviewed include those disorders resulting from mutations in rhodopsin, thyrotropin, luteinizing hormone, vasopressin and angiotensin receptors. By comparison, the recurrent pharmacogenetic variants are more likely to result in an altered predisposition to complex disease in the population. These common variants may affect receptor sequence without intrinsic phenotype change or spontaneous induction of disease and yet result in significant alteration in drug efficacy. These pharmacogenetic phenomena will be reviewed with respect to a limited sampling of GPCR systems including the orexin/hypocretin system, the beta2 adrenergic receptors, the cysteinyl leukotriene receptors and the calcium-sensing receptor. These developments will be discussed with respect to strategies for drug discovery that take into account the potential for the development of drugs targeted at mutated and wild-type proteins.     

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.     

A glance at G-protein-coupled receptors for lipid mediators: a growing receptor family with remarkably diverse ligands

A plethora of lipid-like molecules known to act as intracellular second messengers are now recognized to signal cells through plasma membrane 7 transmembrane G-protein-coupled receptors (GPCRs). This has been the result of a decade-long genetic hunt for novel sequences encoding 7 transmembrane receptor proteins and the efforts to pair novel sequences with biologically active substances of (partly) unknown molecular mechanism of action. Identification of novel GPCR ligand pairs represents the first step to shed more light into the mode of action of novel cellular signaling molecules in human health and disease and might represent a fruitful source for the development of new drugs, judged on the successful history of GPCR as drug targets. Since 2000, more than 16 reports became available on lipid mediators--as diverse as lysophospholipids, arachidonic acid metabolites, short-, medium-, and long-chain fatty acids as well as steroid-like molecules--exerting their effects as extracellular mediators via rhodopsin-like family GPCRs. These reports have opened new avenues for research in human lipid receptor physiology and pharmacology. Here, the current knowledge on the recently deorphanized lipid receptors, including their isolation, expression pattern, function, and possible physiological or pathological roles will be reviewed.     

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.     

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.     

Toward selective drug development for the human 5-hydroxytryptamine 1E receptor: a comparison of 5-hydroxytryptamine 1E and 1F receptor structure-affinity relationships

The 5-hydroxytryptamine (5-HT) 1E receptor is highly expressed in the human frontal cortex and hippocampus, and this distribution suggests the function of 5-HT(1E) receptors might be linked to memory. To test this hypothesis, behavioral experiments are needed. Because rats and mice lack a 5-HT(1E) receptor gene, knockout strategies cannot be used to elucidate this receptor's functions. Thus, selective pharmacological tools must be developed. The tryptamine-related agonist BRL54443 [5-hydroxy-3-(1-methylpiperidin-4-yl)-1H-indole] is one of the few agents that binds 5-HT(1E) receptors with high affinity and some selectively; unfortunately, it binds equally well to 5-HT(1F) receptors (K(i) ≈ 1 nM). The differences between tryptamine binding requirements of these two receptor populations have never been extensively explored; this must be done to guide the design of analogs with greater selectivity for 5-HT(1E) receptors versus 5-HT(1F) receptors. Previously, we determined the receptor binding affinities of a large series of tryptamine analogs at the 5-HT(1E) receptor; we now examine the affinities of this same series of compounds at 5-HT(1F) receptors. The affinities of these compounds at 5-HT(1E) and 5-HT(1F) receptors were found to be highly correlated (r = 0.81). All high-affinity compounds were full agonists at both receptor populations. We identified 5-N-butyryloxy-N,N-dimethyltryptamine as a novel 5-HT(1F) receptor agonist with >60-fold selectivity versus 5-HT(1E) receptors. There is significant overlap between 5-HT(1E) and 5-HT(1F) receptor orthosteric binding properties; thus, identification of 5-HT(1E)-selective orthosteric ligands will be difficult. The insights generated from this study will inform future drug development and molecular modeling studies for both 5-HT(1E) and 5-HT(1F) receptors.