Comparison of Pharmacological Properties between the Kappa Opioid Receptor Agonist Nalfurafine and 42B, Its 3-Dehydroxy Analogue: Disconnect between in Vitro Agonist Bias and in Vivo Pharmacological Effects.

Nalfurafine, a moderately selective kappa opioid receptor (KOR) agonist, is used in Japan for treatment of itch without causing dysphoria or psychotomimesis. Here we characterized the pharmacology of compound 42B, a 3-dehydroxy analogue of nalfurafine and compared with that of nalfurafine. Nalfurafine and 42B acted as full KOR agonists and partial µ opioid receptor (MOR) agonists, but 42B showed much lower potency for both receptors and lower KOR/MOR selectivity, different from previous reports. Molecular modeling revealed that water-mediated hydrogen-bond formation between 3-OH of nalfurafine and KOR accounted for its higher KOR potency than 42B. The higher potency of both at KOR over MOR may be due to hydrogen-bond formation between nonconserved Y7.35 of KOR and their carbonyl groups. Both showed modest G protein signaling biases. In mice, like nalfurafine, 42B produced antinociceptive and antiscratch effects and did not cause conditioned place aversion (CPA) in the effective dose ranges. Unlike nalfurafine, 42B caused motor incoordination and hypolocomotion. As both agonists showed G protein biases, yet produced different effects on locomotor activity and motor incoordination, the findings and those in the literature suggest caution in correlating in vitro biochemical data with in vivo behavior effects. The factors contributing to the disconnect, including pharmacodynamic and pharmacokinetic issues, are discussed. In addition, our results suggest that among the KOR-induced adverse behaviors, CPA can be separated from motor incoordination and hypolocomotion.

Quantitating Ligand Bias Using the Competitive Model of Ligand Activity.

G protein-coupled receptors (GPCRs) can interact with both G proteins and ß-arrestin proteins to propagate different signaling outputs. In some contexts, agonists may drive the receptor to preferentially engage one of these effectors over the other. Such "ligand bias" may present a means to impart pathway-selective signaling downstream of this class of receptors. In cases where physiological responses are mediated by diverse pathways, this could, in part, provide a means to refine GPCR therapeutics. Cell-based signaling assays are used to measure the potential for signaling bias in vitro, and these measures take into account potency, efficacy, and the overall capacity of the assay. However, narrow assay windows sometimes limit the confidence in estimating agonist activity, if a compound performs as a very weakly efficacious partial agonist. This lack of response in an assay hampers the ability to measure and compare potencies, and the degree of separation of an agonist's performance, between two assays. In this chapter, we describe in detail a method for the estimation of the relative activity of a partial agonist and provide a stepwise protocol for calculating bias when this case arises.

Analysis of Biased Agonism.

Agonists and most natural ligands bind to receptors in their inactive state and quickly induce an active receptor conformation that initiates cell signaling. The active receptor state initiates signaling because of its structural complementariness with coupling proteins that activate signaling pathways, such as G proteins and G protein-coupled receptor kinases. Agonist bias refers to the propensity of an agonist to direct receptor signaling through one pathway relative to another. Thus, if the agonist exhibits much higher affinity for active state 1 compared to active state 2, it will cause a robust activation of receptor coupling protein 1 but not 2, and ultimately, a preferential stimulation of signaling pathway 1. Biased agonists are potentially more selective therapeutic agents because there are numerous cases where the therapeutic and adverse effects of an agonist are mediated by distinct pathways involving G proteins and ß-arrestin. Given the mechanism for agonist bias, the most straightforward approach for quantifying bias involves the estimation of agonist affinity for the inactive receptor state and the active receptor states involved in signaling through different pathways. The approach provides quantitative estimates of the sensitivities of different signaling pathways, enabling one to determine to what extent the observed selectivity is caused by agonist or system bias. In addition, the approach is a powerful adjunct to in silico docking studies and can be applied to in vivo assays, structure-activity relationships, and the analysis of published agonist concentration-response curves.

PDE8 Is Expressed in Human Airway Smooth Muscle and Selectively Regulates cAMP Signaling by ß2-Adrenergic Receptors and Adenylyl Cyclase 6.

Two cAMP signaling compartments centered on adenylyl cyclase (AC) exist in human airway smooth muscle (HASM) cells, one containing ß2-adrenergic receptor AC6 and another containing E prostanoid receptor AC2. We hypothesized that different PDE isozymes selectively regulate cAMP signaling in each compartment. According to RNA-sequencing data, 18 of 24 PDE genes were expressed in primary HASM cells derived from age- and sex-matched donors with and without asthma. PDE8A was the third most abundant of the cAMP-degrading PDE genes, after PDE4A and PDE1A. Knockdown of PDE8A using shRNA evoked twofold greater cAMP responses to 1 µM forskolin in the presence of 3-isobutyl-1-methylxanthine. Overexpression of AC2 did not alter this response, but overexpression of AC6 increased cAMP responses an additional 80%. We examined cAMP dynamics in live HASM cells using a fluorescence sensor. PF-04957325, a PDE8-selective inhibitor, increased basal cAMP concentrations by itself, indicating a significant basal level of cAMP synthesis. In the presence of an AC inhibitor to reduce basal signaling, PF-04957325 accelerated cAMP production and increased the inhibition of cell proliferation induced by isoproterenol, but it had no effect on cAMP concentrations or cell proliferation regulated by prostaglandin E2. Lipid raft fractionation of HASM cells revealed PDE8A immunoreactivity in buoyant fractions containing caveolin-1 and AC5/6 immunoreactivity. Thus, PDE8 is expressed in lipid rafts of HASM cells, where it specifically regulates ß2-adrenergic receptor AC6 signaling without effects on signaling by the E prostanoid receptors 2/4-AC2 complex. In airway diseases such as asthma and chronic obstructive pulmonary disease, PDE8 may represent a novel therapeutic target to modulate HASM responsiveness and airway remodeling.

Estimation of the receptor-state affinity constants of ligands in functional studies using wild type and constitutively active mutant receptors: Implications for estimation of agonist bias.

We describe a method for estimating the affinities of ligands for active and inactive states of a G protein-coupled receptor (GPCR). Our protocol involves measuring agonist-induced signaling responses of a wild type GPCR and a constitutively active mutant of it under control conditions and after partial receptor inactivation or reduced receptor expression. Our subsequent analysis is based on the assumption that the activating mutation increases receptor isomerization into the active state without affecting the affinities of ligands for receptor states. A means of confirming this assumption is provided. Global nonlinear regression analysis yields estimates of 1) the active (Kact) and inactive (Kinact) receptor-state affinity constants, 2) the isomerization constant of the unoccupied receptor (Kq-obs), and 3) the sensitivity constant of the signaling pathway (KE-obs). The latter two parameters define the output response of the receptor, and hence, their ratio (Kq-obs/KE) is a useful measure of system bias. If the cellular system is reasonably stable and the Kq-obs and KE-obs values of the signaling pathway are known, the Kact and Kinact values of additional agonists can be estimated in subsequent experiments on cells expressing the wild type receptor. We validated our method through computer simulation, an analytical proof, and analysis of previously published data. Our approach provides 1) a more meaningful analysis of structure-activity relationships, 2) a means of validating in silico docking experiments on active and inactive receptor structures and 3) an absolute, in contrast to relative, measure of agonist bias.

Cooperativity Has Empirical and Ultimate Levels of Explanation.

Controversy over the meaning of pharmacological parameters often arises because of a lack of appreciation of different hierarchical levels of analysis. In a recent letter in Trends in Pharmacological Sciences, Zhang and Kavana [1] concluded that my two-state model for allosterism lacks cooperativity, even though Figures 5 and 6 in my review [2] illustrate examples of how the two-state model yields specific cooperativity values. Here, I explain how the two-state model (receptor-state analysis) gives rise to the cooperativity parameter (α) of the allosteric ternary complex model (receptor-population analysis).

Functional studies cast light on receptor states.

Contemporary analysis of the functional responses of G-protein-coupled receptors (GPCRs) usually addresses drug-receptor interactions from the perspective of the average behavior of the receptor population. This behavior is characterized in terms of observed affinity and efficacy. Efficacy is a measure of how well a drug activates the receptor population and observed affinity a measure of how potently a drug occupies the receptor population. The latter is quantified in terms of the dissociation constant of the ligand-receptor complex. At a deeper level of analysis, drug-receptor interactions are described in terms of ligand affinity constants for active and inactive receptor states. Unlike observed affinity and efficacy, estimates of receptor state affinity constants are unperturbed by G proteins, guanine nucleotides, or other signaling proteins that interact with the receptor. Recent advances in the analysis of the functional responses of GPCRs have enabled the estimation of receptor state affinity constants. These constants provide a more fundamental measure of drug-receptor interactions and are useful in analyzing structure-activity relationships and in quantifying allosterism, biased signaling, and receptor-subtype selectivity.

Intrinsic relative activities of κ opioid agonists in activating Gα proteins and internalizing receptor: Differences between human and mouse receptors.

Several investigators recently identified biased κ opioid receptor (KOP receptor) agonists. However, no comprehensive study of the functional selectivity of available KOP receptor agonists at the human and mouse KOP receptors (hKOP receptor and mKOP receptor, respectively) has been published. Here we examined the ability of over 20 KOP receptor agonists to activate G proteins and to internalize the receptor. Clonal neuro-2a mouse neuroblastoma (N2a) cells stably transfected with the hKOP receptor or mKOP receptor were used. We employed agonist-induced [(35)S]GTPγS binding and KOP receptor internalization as measures of activation of G protein and ß-arrestin pathways, respectively. The method of Ehlert and colleagues was used to quantify intrinsic relative activities at G protein activation (RAi-G) and receptor internalization (RAi-I) and the degree of functional selectivity between the two [Log RAi-G - logRAi-I, RAi-G/RAi-I and bias factor]. The parameter, RAi, represents a relative estimate of agonist affinity for the active receptor state that elicits a given response. The endogenous ligand dynorphin A (1-17) was designated as the balanced ligand with a bias factor of 1. Interestingly, we found that there were species differences in functional selectivity. The most striking differences were for 12-epi-salvinorin A, U69,593, and ICI-199,441. 12-Epi-salvinorin A was highly internalization-biased at the mKOP receptor, but apparently G protein-biased at hKOP receptor. U69,593 was much more internalization-biased at mKOP receptor than hKOP receptor. ICI199,441 showed internalization-biased at the mKOP receptor and G protein-biased at the hKOP receptor. Possible mechanisms for the observed species differences are discussed.

A novel method for analyzing extremely biased agonism at G protein-coupled receptors.

Seven transmembrane receptors were originally named and characterized based on their ability to couple to heterotrimeric G proteins. The assortment of coupling partners for G protein-coupled receptors has subsequently expanded to include other effectors (most notably the ßarrestins). This diversity of partners available to the receptor has prompted the pursuit of ligands that selectively activate only a subset of the available partners. A biased or functionally selective ligand may be able to distinguish between different active states of the receptor, and this would result in the preferential activation of one signaling cascade more than another. Although application of the "standard" operational model for analyzing ligand bias is useful and suitable in most cases, there are limitations that arise when the biased agonist fails to induce a significant response in one of the assays being compared. In this article, we describe a quantitative method for measuring ligand bias that is particularly useful for such cases of extreme bias. Using simulations and experimental evidence from several κ opioid receptor agonists, we illustrate a "competitive" model for quantitating the degree and direction of bias. By comparing the results obtained from the competitive model with the standard model, we demonstrate that the competitive model expands the potential for evaluating the bias of very partial agonists. We conclude the competitive model provides a useful mechanism for analyzing the bias of partial agonists that exhibit extreme bias.

A kinetic model of GPCRs: analysis of G protein activity, occupancy, coupling and receptor-state affinity constants.

CONTEXT: G protein-coupled receptors are vital macromolecules for a wide variety of physiological processes. Upon agonist binding, these receptors accelerate the exchange of GDP for GTP in G proteins coupled to them. The activated G protein interacts with effector proteins to implement downstream biological functions. OBJECTIVE: We present a kinetic, quaternary complex model, based on a system of coupled linear first-order differential equations, which accounts for the binding attributes of the ligand, receptor, G protein and two types of guanine nucleotide (GDP and GTP) as well as for GTPase activity. METHODS: We solved the model numerically to predict the extents of G protein activation, receptor occupancy by ligand and receptor coupling that result from varying the ligand concentration, presence of GDP and/or GTP, the ratio of G protein to receptor and the equilibrium constants governing receptor pre-coupling and constitutive activity. We also simulated responses downstream from G protein activation using a transducer function. RESULTS: Our model shows that agonist-induced G protein activation can occur with either a net decrease or increase in total receptor-G protein coupling. In addition, we demonstrate that affinity constants of the ligand for both the active and inactive states of the receptor can be derived to a close approximation from analysis of simulated responses downstream from receptor activation. DISCUSSION AND CONCLUSION: The latter result validates our prior methods for estimating the active state affinity constants of ligands, and our results on receptor coupling have relevance to studies investigating receptor-G protein interactions using fluorescence techniques.

International Union of Basic and Clinical Pharmacology. XC. multisite pharmacology: recommendations for the nomenclature of receptor allosterism and allosteric ligands.

Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties.

Estimation of ligand affinity constants for receptor states in functional studies involving the allosteric modulation of G protein-coupled receptors: implications for ligand bias.

INTRODUCTION: The affinity constants of a ligand for active and inactive states of a receptor ultimately determine its capacity to activate downstream signaling events. In this report, we describe a reverse-engineering strategy for estimating these microscopic constants. METHODS: Our approach involves analyzing responses measured downstream in the signaling pathway of a G protein-coupled receptor under conditions of allosteric modulation and reduced receptor expression or partial receptor inactivation. The analysis also yields estimates of the isomerization constant of the unoccupied receptor, the sensitivity constant of the signaling pathway, and the more empirical parameters of the receptor population including the observed affinities and efficacies of allosteric and orthosteric ligands - including inverse agonists - and the efficacy of the unoccupied receptor (i.e., constitutive activity). RESULTS AND DISCUSSION: We validate our approach with an analytical proof and by analysis of simulated data. We also use our method to analyze data from the literature. We show that the values of the microscopic constants of orthosteric and allosteric ligands are constant regardless of the allosteric interaction and the nature of the receptor-signaling pathway as long as the same active state mediates the response. Our analysis is useful for quantifying probe-dependent allosteric interactions and the selectivity of agonists for different signaling pathways. Knowing the isomerization constant and sensitivity constant of a signaling pathway in a given cell line or tissue preparation enables future investigators to estimate the affinity constants of agonists for receptor states simply through analysis of their concentration-response curves. Our approach also provides a means of validating in silico estimates of ligand affinity for crystal structures of active and inactive states of the receptor.

Effects of asparagine mutagenesis of conserved aspartic acids in helix 2 (D2.50) and 3 (D3.32) of M1-M4 muscarinic receptors on the irreversible binding of nitrogen mustard analogs of acetylcholine and McN-A-343.

We investigated how asparagine mutagenesis of conserved aspartic acids in helix 2 (D2.50) and 3 (D3.32) of M1-M4 muscarinic receptors alters the irreversible binding of acetylcholine mustard and BR384 (4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophenyl)carbamate), a nitrogen mustard derivative of McN-A-343 ([4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl] trimethylammonium chloride). The D2.50N mutation moderately increased the affinity of the aziridinium ions of acetylcholine mustard and BR384 for M2-M4 receptors and had little effect on the rate constant for receptor alkylation. The D3.32N mutation greatly reduced the rate constant for receptor alkylation by acetylcholine mustard but not by BR384, although the affinity of BR384 was reduced. The combination of both mutations (D2.50N/D3.32N) substantially reduced the rate constant for receptor alkylation by BR384 relative to that of wild type and mutant D2.50N and D3.32N receptors. The change in binding affinity caused by the mutations suggests that the D2.50N mutation alters the interaction of acetylcholine mustard with D3.32 of the M1 and M3 receptors but not that of the M4 receptor. BR384 exhibited the converse relationship. The simplest explanation is that acetylcholine mustard and BR384 alkylate at least two residues on M1-M4 receptors and that the D2.50N mutation alters the rate of alkylation of D3.32 relative to another residue, perhaps D2.50 itself.

What ligand-gated ion channels can tell us about the allosteric regulation of G protein-coupled receptors.

The GABA(A) receptor is the target for a number of important allosteric drugs used in medicine, including benzodiazepines and anesthetics. These modulators have variable effects on the potency and maximal response of macroscopic currents elicited by different GABA(A) receptor agonists, yet this modulation is consistent with a two-state model in which the allosteric ligand has invariant affinity constants for the active and inactive states. Analysis of the effects of an allosteric agonist, like etomidate, on the population current provides a means of estimating the gating constant of the unliganded GABA(A) receptor (∼10(-4)). In contrast, allosteric interactions at the M(2) muscarinic receptor are often inconsistent with a two-state model. Analyzing allosterism within the constraints of a two-state model, nonetheless, provides an unbiased measure of probe dependence as well as clues to the mechanism of allosteric modulation. The rather simple allosteric effect of affinity-only modulation is difficult to explain and suggests modulation of a peripheral orthosteric ligand-docking site on the M(2) muscarinic receptor.

Muscarinic agonists and antagonists: effects on gastrointestinal function.

Muscarinic agonists and antagonists are used to treat a handful of gastrointestinal (GI) conditions associated with impaired salivary secretion or altered motility of GI smooth muscle. With regard to exocrine secretion, the major muscarinic receptor expressed in salivary, gastric, and pancreatic glands is the M3 with a small contribution of the M1 receptor. In GI smooth muscle, the major muscarinic receptors expressed are the M2 and M3 with the M2 outnumbering the M3 by a ratio of at least four to one. The antagonism of both smooth muscle contraction and exocrine secretion is usually consistent with an M3 receptor mechanism despite the major presence of the M2 receptor in smooth muscle. These results are consistent with the conditional role of the M2 receptor in smooth muscle. That is, the contractile role of the M2 receptor depends on that of the M3 so that antagonism of the M3 receptor eliminates the response of the M2. The physiological roles of muscarinic receptors in the GI tract are consistent with their known signaling mechanisms. Some so-called tissue-selective M3 antagonists may owe their selectivity to a highly potent interaction with a nonmuscarinic receptor target.

Characterization of muscarinic receptors in the human bladder mucosa: direct quantification of subtypes using 4-DAMP mustard.

OBJECTIVE: To characterize pharmacologically relevant muscarinic receptors in the human bladder mucosa and detrusor muscle using radioligand binding assays with [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) and 4-DAMP mustard. METHODS: Muscarinic receptors in homogenates of bladder mucosa, detrusor muscle, and parotid gland were measured using the radioligand [3H]NMS. 4-DAMP mustard was used to inactivate M3 receptors irreversibly. RESULTS: Specific [3H]NMS binding in the homogenates of the mucosa and detrusor muscle was saturable and of high affinity as shown by dissociation constants (Kd) of 260 ±82 and 237 ±49 pM, respectively. Antimuscarinic agents (oxybutynin, propiverine, tolterodine, and darifenacin) and their active metabolites competed with [3H]NMS for the binding sites in the human mucosa in a concentration-dependent manner. These agents exhibited similar affinity in the detrusor muscle. The Bmax. values of [3H]NMS in the detrusor, bladder mucosa, and parotid gland were significantly decreased by pretreatment with 4-DAMP mustard (36%, 41% and 63%, respectively). CONCLUSION: The density and binding affinity profile of the muscarinic receptor population in the human bladder mucosa was shown to be similar to that of the detrusor muscle. The density of the M3 subtype in the mucosa was similar to that in the detrusor muscle but lower than that in the parotid gland.

Analysis of agonism and inverse agonism in functional assays with constitutive activity: estimation of orthosteric ligand affinity constants for active and inactive receptor states.

We describe a modification of receptor theory for the estimation of observed affinities (K(obs)) and relative efficacies of orthosteric ligands in functional assays that exhibit constitutive activity. Our theory includes parameters for the fractions of the occupied receptor population in the active (intrinsic efficacy, ε) and inactive (ε(i)) states and analogous parameters for the fractions of the free receptor population in the active (ε(sys)) and inactive (ε(i-sys)) states. The total stimulus represents the summation of the active states of the free and occupied receptor populations. A modified operational model is developed that expresses the response as a logistic function of the total stimulus. This function includes the standard parameters related to affinity and efficacy (K(obs) and τ) as well as a parameter proportional to the activity of the free receptor complex, τ(sys). Two related parameters are proportional to the fraction of the free (τ(i-sys)) and occupied (τ(i)) receptor populations in the inactive state. We show that the estimates of the affinity constants of orthosteric ligands for the active (K(b)) and inactive (K(a)) states of the receptor are equivalent to τK(obs)/τ(sys) and τ(i)K(obs)/τ(i-sys), respectively. We verify our method with computer simulation techniques and apply it to the analysis of M(2) and M(3) muscarinic receptors. Our method is applicable in the analysis of ligand bias in drug discovery programs.

Analysis of functional responses at G protein-coupled receptors: estimation of relative affinity constants for the inactive receptor state.

We describe a modification of receptor theory that enables the estimation of relative affinity constants for the inactive state of a G protein-coupled receptor. Our approach includes the traditional parameters of observed affinity (K(obs)) and efficacy (fraction of ligand-receptor complex in the active state, ε) and introduces the concept of the fraction of the ligand-receptor complex in the inactive state (intrinsic inactivity, ε(i)). The relationship between receptor activation and the ligand concentration is known as the stimulus, and the operational model expresses the response as a logistic function of the stimulus. The latter function includes K(obs) and the parameter τ, which is proportional to ε. We introduce the parameter τ(i), which is proportional to ε(i). We have previously shown that the product, K(obs)τ, of one agonist, expressed relative to that of another (intrinsic relative activity, RA(i)), is a relative measure of the affinity constant for the active state of the receptor. In this report, we show that the product, K(obs)τ(i), of one agonist, expressed relative to that of another (intrinsic relative inactivity, RI(i)), is a relative measure of the affinity constant for the inactive state of the receptor. We use computer simulation techniques to verify our analysis and apply our method to the analysis of published data on agonist activity at the M(3) muscarinic receptor. Our method should have widespread application in the analysis of agonist bias in drug discovery programs and in the estimation of a more fundamental relative measure of efficacy (RA(i)/RI(i)).

Quantifying agonist activity at G protein-coupled receptors.

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors. Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (K(b)) is much greater than that for the inactive state (K(a)). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (K(obs)), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (ε) (Figure 2). Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the K(obs) and relative efficacy of an agonist. In this report, we show how to modify this analysis to estimate the agonist K(b) value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate K(b) in absolute units of M(-1). Our method of analyzing agonist concentration-response curves consists of global nonlinear regression using the operational model. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of K(obs) and a parameter proportional to efficacy (τ). The estimate of τK(obs) of one agonist, divided by that of another, is a relative measure of K(b) (RA(i)). For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (τ(sys)). In this case, the K(b) value of an agonist is equivalent to τK(obs)/τ(sys). Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.

Mutagenesis of nucleophilic residues near the orthosteric binding pocket of M1 and M2 muscarinic receptors: effect on the binding of nitrogen mustard analogs of acetylcholine and McN-A-343.

Investigating how a test drug alters the reaction of a site-directed electrophile with a receptor is a powerful method for determining whether the drug acts competitively or allosterically, provided that the binding site of the electrophile is known. In this study, therefore, we mutated nucleophilic residues near and within the orthosteric pockets of M(1) and M(2) muscarinic receptors to identify where acetylcholine mustard and 4-[(2-bromoethyl)methyl-amino]-2-butynyl-N-(3-chlorophenyl)carbamate (BR384) bind covalently. BR384 is the nitrogen mustard analog of [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride (McN-A-343). Mutation of the highly conserved aspartic acid in M(1) (Asp105) and M(2) (Asp103) receptors to asparagine largely prevented receptor alkylation by acetylcholine mustard, although modest alkylation still occurred at M(2) D103N at high concentrations of the mustard. Receptor alkylation by BR384 was also greatly inhibited in the M(1) D105N mutant, but some alkylation still occurred at high concentrations of the compound. In contrast, BR384 rapidly alkylated the M(2) D103N mutant. Its affinity was reduced to one tenth, however. The alkylation of M(2) D103N by BR384 was competitively inhibited by N-methylscopolamine and allosterically inhibited by gallamine. Mutation of a variety of other nucleophilic residues, some in combination with D103N, had little effect on M(2) receptor alkylation by BR384. Our results suggest that BR384 alkylates at least one residue other than the conserved aspartic acid at the ligand-binding site of M(1) and M(2) receptors. This additional residue seems to be located within or near the orthosteric-binding pocket and is not part of the allosteric site for gallamine.