The muscarinic acetylcholine receptors (mAChRs) are members of the G protein-coupled receptor (GPCR) superfamily and mediate the majority of the actions of acetylcholine (ACh), in both the central nervous system (CNS) and the periphery (Wess et al., 2003). Within the CNS, the M₁ and M₄ mAChRs show a higher level of expression than the M₂, M₃, and M₅ subtypes and have been implicated in the regulation of cognition, sensory processing, motor coordination, and attention (Wess et al., 2007). However, these mAChRs are also characterized by a very high degree of sequence conservation within the orthosteric binding site (i.e., the site on the receptor that binds its endogenous agonist) (Wess et al., 2003; Gregory et al., 2007). As a consequence, it has traditionally been very difficult to discover orthosteric agonists or antagonists, either as therapeutic agents or pharmacological tools, that selectively target M₁ or M₄ receptors in the CNS without also interacting with other receptor subtypes, especially M₂ and M₃ receptors that mediate many peripheral cholinergic side-effects (Wess et al., 2007).

One promising approach to overcome some of the difficulties associated with mAChR drug targeting is to focus on allosteric binding sites, which are topographically distinct from the orthosteric site and thus less conserved between subtypes (Christopoulos, 2002). Allosteric modulators are ligands that bind to these sites and regulate either the affinity and/or efficacy of a classic ligand interacting with the orthosteric site (Christopoulos and Kenakin, 2002; May et al., 2007a). There are now numerous examples of GPCR allosteric modulators with greater selectivity for their targets than orthosteric agonists or antagonists (May et al., 2007b). With respect to the mAChRs, we and others (Felder et al., 2004; Chan et al., 2008; Shirey et al., 2008) have recently disclosed a new class of allosteric potentiator, exemplified by LY2033298 (Fig. 1) and related compounds, which selectively enhance the actions of ACh at the M₄ mAChR to the relative exclusion of other mAChR subtypes. Moreover, LY2033298 displays in vivo efficacy in preclinical rodent models predictive of antipsychotic drug effects, suggesting that selective activation or potentiation of M₄ mAChRs may represent a novel approach to treating schizophrenia (Chan et al., 2008).

An alternative approach to selectively targeting a given GPCR to the exclusion of others is to generate a mutant receptor that does not respond to its endogenous ligand but gains responsiveness to otherwise inert biological compounds (Bishop et al., 2000; Scearce-Levie et al., 2001). If the resulting mutant GPCR retains the fidelity of the native receptor with respect to its signaling properties while being tailored to respond only to an exogenous ligand (or ligands) specific for the mutant, then one gains a powerful tool. In particular, the mutant GPCR can be introduced into a chosen cellular environment, and the resulting biological effects observed in the presence of the exogenous ligand must reflect solely the activation of the chosen receptor and no other. Moreover, this can be performed in native tissues without the possibility of unwanted activation of the mutant GPCR by the endogenous agonist. Using random mutagenesis coupled with directed molecular evolution in yeast, we recently generated such a mutant GPCR, termed a “designer receptor exclusively activated by a designer drug” (DREADD), for each of the mAChR subtypes (Armbruster et al., 2007). We identified a novel mutation of two conserved orthosteric-site residues (Y¹¹³C and A²⁰³G in the M₄ mAChR) that yielded a profound loss in potency and efficacy of ACh for activating classic second messenger pathways, but a robust gain in responsiveness to clozapine-N-oxide (CNO), a metabolite of the atypical antipsychotic agent clozapine that is otherwise biologically inert at the wild-type (WT) mAChRs. Subsequent expression of an M₄ DREADD into primary hippocampal neurons, followed by exposure to CNO, demonstrated its ability to induce neuronal silencing in a native environment, a property associated with ACh actions at the WT M₄ mAChR (Armbruster et al., 2007). This finding, supported by additional experiments examining different signaling pathways of other mAChR DREADDs (Armbruster et al., 2007), suggests that the DREADDs retain the signaling properties of native mAChRs despite losing the ability to recognize ACh, an important requirement if DREADD-based data are to be used to provide valid physiological insights.

Given that DREADDs represent a new type of reverse-engineered GPCR with significant potential as biological tools, we sought to perform a detailed characterization of the molecular pharmacology of a diverse range of orthosteric and allosteric ligands at the M₄ DREADD to gain further insight into the modes of activation and ligand interactions at this receptor. In addition to the classic orthosteric agonists ACh and pilocarpine, we used 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride (McN-A-343), a partial agonist that has been suggested to recognize both an allosteric and the orthosteric site on the M₂ mAChR (Birdsall et al., 1983; Waelbroeck, 1994; May et al., 2007a); clozapine, CNO, and N-desmethylclozapine (NDMC), a biologically active metabolite of clozapine and a functionally selective mAChR agonist (Sur et al., 2003; Davies et al., 2005); and our novel M₄-selective allosteric potentiator, LY2033298 (Fig. 1). We present evidence for the existence of a topographically distinct allosteric site on the M₄ DREADD that retains its ability to interact with orthosteric ligands but displays markedly different types of cooperative effects depending on the nature of the ligand and the receptor.

Materials and Methods

Materials. Chinese hamster ovary (CHO) Flp-In cells and Hygromycin B were purchased from Invitrogen (Carlsbad, CA). Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum (FBS) were from Gibco (Gaithersburg, MD) and JRH Biosciences (Lenexa, KS), respectively. The AlphaScreen SureFire phospho-ERK1/2 reagents were kindly donated by Dr. Michael Crouch (TGR Biosciences, South Australia, Australia), whereas the AlphaScreen streptavidin donor beads and anti-IgG (protein A) acceptor beads used for phosphorylated ERK1/2 (pERK1/2) detection, [³H]quinuclidinyl benzilate ([³H]QNB; specific activity, 52 Ci/mmol) and [³H]N-methylscopolamine ([³H]NMS; specific activity, 72 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). NDMC and CNO were purchased from Tocris Bioscience (Bristol, UK) and BIOMOL Research Laboratories (Plymouth Meeting, PA), respectively. LY2033298 was synthesized in-house at Eli Lilly (Indianapolis, IN). All other chemicals were from Sigma Chemical Co. (St. Louis, MO).

cDNA Constructs and Generation of Stable Cell Lines. cDNA encoding the M₄ DREADD was generated in pcDNA3.1+ as described previously (Armbruster et al., 2007). cDNA encoding the WT M₄ mAChR was obtained from Missouri University of Science and Technology (http://www.cdna.org) and was provided in pcDNA3.1+. Sequences encoding the M₄ DREADD and WT M₄ mAChR were amplified by PCR and cloned into the Gateway entry vectors pDONR201 and pENTR/D-TOPO, respectively, using the BP Clonase enzyme mix and pENTR directional TOPO cloning kit, respectively, according to the manufacturer's instructions (Invitrogen). Both constructs were subsequently transferred into the Gateway destination vector pEF5/frt/v5/dest using the LR Clonase enzyme mix (Invitrogen), and the constructs were used to transfect Flp-In CHO cells (Invitrogen) as described previously (May et al., 2007a). Cells were selected using 400 μg/ml hygromycin B to generate cell lines stably expressing each receptor construct and, after selection, were maintained in high glucose DMEM containing 10% FBS, 16 mM HEPES, and 200 μg/ml hygromycin B.

Membrane Preparations. Cells were grown until approximately 90% confluence and harvested using 2 mM EDTA in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, and 1.5 mM KH₂PO₄). Cells were pelleted by centrifugation for 10 min at 1200g, and the pellets were resuspended in 30 ml of buffer containing 20 mM HEPES and 10 mM EDTA at pH 7.4. All subsequent steps were performed at 4°C. The cell suspension was homogenized using a Polytron homogenizer (PT 1200 CL; Kinematica, Basel, Switzerland), with two 10-s bursts separated by cooling on ice. The cell homogenate was centrifuged for 5 min at 1700g, and the supernatant was transferred to new tubes and further centrifuged (90 min, 38,000g) in a Sorval centrifuge. The pellet was resuspended in 10 ml of buffer (20 mM HEPES and 0.1 mM EDTA, pH 7.4) and briefly homogenized to ensure uniform consistency. Membranes were aliquoted and stored at -80°C. The protein concentration was determined by the method of Bradford using bovine serum albumin as a standard (Bradford, 1976).

Radioligand Binding Assays. Saturation and competition binding assays were performed using 15 and 75 μg of membrane expressing the M₄ WT and DREADD receptors, respectively. For saturation binding assays, membranes were incubated with the orthosteric antagonists [³H]QNB or [³H]NMS in HEPES buffer (20 mM HEPES, 100 mM NaCl, and 10 mM MgCl, pH 7.4) at 37°C for 1 h before termination of the assay by rapid filtration onto GF/B grade filter paper (Whatman, Maidstone, UK) using a Brandel harvester, followed by three 2-ml washes with ice-cold NaCl (0.9%). Nonspecific binding was defined in the presence of 100 μM atropine and radioactivity was determined by liquid scintillation counting. For inhibition binding assays, membranes were incubated in HEPES buffer containing 100 μM GppNHp and increasing concentrations of the cold ligand for 3 h at 37°C in the presence of a [³H]QNB concentration equal to its equilibrium dissociation constant at each receptor (0.09 and 6 nM for the WT M₄ mAChR and M₄ DREADD, respectively) as determined from saturation binding experiments. For interaction studies, competition of [³H]QNB binding by ACh, clozapine, or CNO was performed in the presence of 1 and 10 μM LY2033298, the allosteric modulator. For all experiments, nonspecific binding was defined by 100 μM atropine, and the effects of vehicle were determined. The reaction was terminated and radioactivity counted as described previously.

Extracellular Signal Regulated Kinase 1/2 Phosphorylation Assays. Initial ERK1/2 phosphorylation time course experiments were performed to determine the time at which ERK1/2 phosphorylation was maximal after stimulation by each agonist. Cells were seeded into transparent 96-well plates at 4 × 10⁴ cells per well and grown overnight or until confluent. Cells were then washed twice with phosphate-buffered saline and incubated in serum-free DMEM at 37°C for at least 4 h to allow FBS-stimulated pERK1/2 levels to subside. Cells were stimulated with agonist using a staggered addition approach. For subsequent agonist-stimulated concentration-response experiments, cells were incubated at 37°C with each agonist for the time required to generate a maximal pERK1/2 response (5 min for ACh, LY2033298, clozapine, and CNO and 8 min for pilocarpine, McN-A-343, and NDMC). For functional interaction studies, cells were incubated at 37°C with varying concentrations of agonist in the absence and presence of different concentrations of a second compound, which was added simultaneously with the agonist. For all experiments, 10% FBS was used as a positive control, and vehicle controls were also performed. The reaction was terminated by removal of drugs and lysis of cells with 100 μl of SureFire lysis buffer (as provided by the manufacturer). The lysates were agitated for 1 to 2 min and were diluted at a ratio of 4:1 (v/v) lysate/Surefire activation buffer in a total volume of 50 μl. Under low light conditions a 1:240 (v/v) dilution of AlphaScreen beads: Surefire reaction buffer was prepared and this was mixed with the activated lysate mixture in a ratio of 6:5 (v/v), respectively, in a 384-well opaque Optiplate. Plates were incubated in the dark at 37°C for 1.5 h before the fluorescence signal was measured using a Fusion-α plate reader (PerkinElmer Life and Analytical Sciences) using standard AlphaScreen settings.

Data analysis. All data were analyzed using Prism 5.01 (GraphPad Software, San Diego, CA). For radioligand saturation binding data, nonspecific and total binding data were fitted to the following equation.

where Y is radioligand binding, B_max is the total receptor density, [A] is the radioligand concentration, K_A is the equilibrium dissociation constant of the radioligand, and NS is the fraction of nonspecific radioligand binding.

For radioligand inhibition binding experiments, specific binding of each orthosteric ligand was fitted to both a one-site (eq. 2) and a two-site (eq. 3) binding equation, and an F test was used to determine which equation better fitted the data:

where Top and Bottom are the maximal and minimal asymptotes of the curve, respectively, log[B] is the concentration of inhibitor, logIC₅₀ is the logarithm of the concentration of inhibitor that reduces half the maximal radioligand binding for each binding site, and fraction_1 is the proportion of high-affinity binding sites. IC₅₀ values were converted to K_B values (inhibitor equilibrium dissociation constant) using the Cheng and Prusoff (1973) equation.

For some experiments, as indicated under Results, the shifts of the competitor versus [³H]QNB competition binding curve obtained in the absence or presence of 1 and 10 μM LY2033298 were fitted to the following form of a simple allosteric ternary complex model (Ehlert, 1988):

where [I] denotes the concentration of competitor, IC₅₀ denotes its potency in the absence of modulator, [B] denotes the concentration of LY2033298, K_B denotes the modulator equilibrium dissociation constant, and α denotes the “binding” cooperativity factor, which is a measure of the magnitude and direction of the allosteric effect the modulator exerts on the affinity of ligand I. Values of α > 1 denote positive cooperativity, values < 1 (but greater than 0) denote negative cooperativity, values = 1 denote neutral cooperativity, and values approaching zero denote inhibition that is indistinguishable from competitive (orthosteric) antagonism. This analysis also assumes that the interaction between the modulator and the radiolabel is neutrally cooperative (α = 1), as was the case for the current study.

Concentration-response data generated from the ERK1/2 phosphorylation assays were normalized to the response generated by 10% FBS and fitted to the following three-parameter logistic equation:

where E is response, E_max and Bottom are the top and bottom asymptotes of the curve, respectively, [A] is the agonist concentration, and pEC₅₀ is the negative logarithm of the agonist concentration that gives a response halfway between E_max and Bottom.

Functional experiments measuring the interaction between ACh and clozapine at the WT M₄ mAChR were globally fitted to the following logistic equation of agonist-antagonist interaction (Motulsky and Christopoulos, 2004):

where s represents the Schild slope for the antagonist, and pA₂ represents the negative logarithm of the concentration of antagonist that shifts the agonist EC₅₀ by a factor of 2; all other parameters are as defined in eq. 5.

For some of the functional interaction experiments, datasets were also fitted to the following operational model for the interaction between an agonist and allosteric modulator (Price et al., 2005; Leach et al., 2007):

where E denotes the effect, A denotes the agonist, K_A denotes its equilibrium dissociation constant, B denotes the allosteric modulator, β denotes an empirical proportionality constant (“activation” cooperativity factor) that quantifies the change in stimulus imparted to the receptor by the agonist as a result of the presence of allosteric modulator, E_m denotes the maximum possible effect, τ_A is an operational measure of agonist (A) efficacy, and n is a “transducer” slope factor that governs the shape of the stimulus-response function. [A] and α are as defined above.

All parametric measures of potency, affinity, operational efficacy, and cooperativity were estimated as logarithms (Christopoulos, 1998). Statistical comparisons between parameters were performed using Student's t test or F test, where appropriate, with p < 0.05 taken as indicating significance.

Results

The M₄ DREADD Displays Reduced Affinity for Prototypical Orthosteric Antagonists. Initial radioligand saturation binding studies used both the benzilate [³H]QNB and the tropate [³H]NMS as prototypical orthosteric mAChR antagonists. [³H]QNB bound with high affinity to the WT M₄ mAChR (pK_A 10.1 ± 0.1; n = 3), and detected a receptor expression level of 1.6 ± 0.2 pmol/mg of membrane protein. Y¹¹³C and A²⁰³G, the mutations responsible for generating the M₄ DREADD, led to a profound and significant (p < 0.05) decrease in the binding affinity of [³H]QNB (pK_A 8.2 ± 0.1; n = 3), as well as reduction in receptor expression (0.6 ± 0.1 pmol/mg), in keeping with our previous findings (Armbruster et al., 2007). [³H]NMS labeled a lower number of M₄ binding sites than [³H]QNB for both the WT (1.1 ± 0.2 pmol/mg; n = 3) and the DREADD (0.09 ± 0.04 pmol/mg; n = 3), possibly because of its hydrophilic nature, which does not allow it to access membrane-sequestered receptors. Similar to [³H]QNB, however, [³H]NMS also bound to the WT M₄ mAChR with much higher affinity (pK_A, 9.6 ± 0.1) than at the DREADD (pK_A, 7.3 ± 0.3).

The Nature of the Interacting Ligand Dictates Its Differential Affinity for the M₄ DREADD. Because [³H]QNB had higher affinity for both the WT and the DREADD than [³H]NMS, subsequent inhibition binding assays were performed using the benzilate as a probe of the orthosteric M₄ mAChR pocket. As shown in Fig. 2, A and B, the classic orthosteric agonists ACh and pilocarpine displayed a reduced potency for inhibiting [³H]QNB binding at the M₄ DREADD relative to the WT. Even though all experiments were performed in the presence of 100 μM GppNHp to promote receptor-G protein uncoupling, the binding of ACh at the WT could still be resolved into two apparent affinity states but reverted to a single low-affinity state at the DREADD (Table 1). Given that multiphasic agonist binding curves are common for high-affinity agonists and generally related to receptor-G protein coupling propensity (Kenakin, 1997), our finding suggests that the DREADD mutation may be affecting receptor-G protein coupling or conformational activation, in addition to direct effects on orthosteric ligand affinity.

It is noteworthy that when these experiments were repeated with McN-A-343 (Fig. 2C) or LY2033298 (Fig. 2D), a different profile of behaviors was noted. In particular, the binding of McN-A-343 was insensitive to the DREADD mutation (Table 1), a finding that is consistent with the hypothesis that this agonist uses a different mode of binding when interacting with mAChRs. Furthermore, LY203398 did not seem to interact at all with [³H]QNB over the concentration ranges tested, suggesting that the interaction between the modulator and this orthosteric antagonist is characterized by neutral cooperativity (α = 1) at both the WT M₄ receptor and the DREADD; we could not use higher concentrations of the modulator because of solubility limits and nonspecific effects (not shown). In contrast, and in agreement with our initial study on the mAChR DREADDs (Armbruster et al., 2007), clozapine, CNO, and NDMC bound with significantly higher affinity to the M₄ DREADD than to the WT M₄ mAChR (Fig. 3, Table 1).

Allosteric Modulation by LY2033298 of Ligand Affinity Is Modified on the M₄ DREADD. To gain additional insight into the nature of allosteric interactions between the orthosteric and allosteric binding sites on the WT M₄ mAChR and the DREADD, additional competition binding studies were performed using ACh, clozapine, or CNO in the absence or presence of LY2033298. As shown in Fig. 4A, LY2033298 caused a robust and significant enhancement in the potency of ACh to inhibit [³H]QNB binding that was virtually lost at the M₄ DREADD (Fig. 4B). This finding clearly indicates that the positive cooperativity between ACh and LY2033298 is sensitive to the conformation of the orthosteric pocket, converting to neutral cooperativity at the M₄ DREADD. Interestingly, in these experiments the biphasic nature of ACh binding was retained at the DREADD, although the high affinity state represented a small fraction of the overall binding (Fig. 4B; Supplementary Table) in contrast to the WT, where the fraction of high affinity sites progressively increased with increasing modulator concentrations (Supplemental Table). Despite these complexities in the binding isotherms of ACh, and because the cooperativity between the allosteric modulator and the radioligand was neutral in all instances, we applied a simple allosteric ternary complex model to the LY2033298-mediated translocation of the ACh curves (eq. 4) to derive an approximation of the affinity of LY2033298 and its cooperativity with ACh at the WT receptor. This analysis yielded a value of pK_B = 5.43 ± 0.09 (n = 3) and Logα = 1.78 ± 0.06 (α = 60), which were in good agreement with our previous study of this interaction using [³H]NMS as the orthosteric probe (Chan et al., 2008).

When these experiments were repeated with either clozapine (Fig. 4, C and D) or CNO (Figs. 4, E and F) instead of ACh, very small effects were noted in the presence of LY2033298, with only a modest enhancement of inhibitor potency (Supplemental Table) at the highest concentration used (10 μM). The shifts were too small to allow application of eq. 4 unless we assumed apK_B of 5.43 for LY2033298 at the WT receptor, in which case we could estimate a value of Logα = 0.65 ± 0.05 (α = 4.4) for clozapine and 0.55 ± 0.05 (α = 3.5) for CNO (n = 3). This weak interaction is either unaltered or slightly reduced at the M₄ DREADD (Figs. 4, D and F).

LY2033298 Is an Allosteric Agonist of M₄ mAChR-Mediated ERK1/2 Phosphorylation, and Its Efficacy Is Sensitive to the DREADD Mutations. Agonist-stimulated ERK1/2 phosphorylation was used as a measure of receptor activity to investigate the effects of the Y¹¹³C and A²⁰³G mutations on the function of the M₄ mAChR. In addition to the reduction in agonist affinity noted in our competition binding studies, both ACh and pilocarpine displayed a profound loss in the ability to stimulate ERK1/2 phosphorylation, as evidenced by a large reduction in potency (nearly 10,000-fold) and maximal effect for ACh and complete abolition of response to pilocarpine (Fig. 5, A and B; Table 2), in agreement with our prior findings (Armbruster et al., 2007). It is noteworthy that McN-A-343, which was a more efficacious partial agonist than pilocarpine at the WT M₄ mAChR (Fig. 5C), also lost its ability to activate the M₄ DREADD despite having an unaltered affinity for the mutant receptor (Table 1). This finding suggests that the activating conformation McN-A-343 engenders is as sensitive to the DREADD mutations as those promoted by ACh and pilocarpine. To our surprise, we also discovered a novel property of LY2033298: the ability to act as a robust agonist of WT M₄ mAChR-mediated ERK1/2 phosphorylation in the absence of orthosteric ligand (Fig. 5D; Table 2). This is in contrast to our prior study, in which LY2033298 was either quiescent in the absence of orthosteric agonist for mediating intracellular calcium mobilization or only very weakly stimulated guanine nucleotide exchange on activated G proteins (Chan et al., 2008). Moreover, we found that the agonistic properties of LY2033298 were completely lost at the M₄ DREADD (Fig. 5D; Table 2), suggesting that LY2033298 has either a markedly reduced affinity for the allosteric site on the M₄ DREADD or, as with McN-A-343, that the activating conformation it engenders requires Tyr¹¹³ and Ala²⁰³ to be intact for transmission to the intracellular environment. In contrast, clozapine and its metabolites were converted from either inactive (clozapine, CNO) or very low efficacy (NDMC) ligands at the WT M₄ mAChR to efficacious and potent agonists at the M₄ DREADD (Fig. 6; Table 2).

Functional Interaction Studies Unmask Allosteric Effects on Agonist Efficacy and Reveal that LY2033298 Retains the Ability to Recognize an Allosteric Site on the M₄ DREADD. To investigate the potential for allosteric effects on agonist signaling efficacy, functional interaction studies between ligands were performed at both the WT M₄ mAChR and the M₄ DREADD. In the first instance, we investigated the effects of coaddition of ACh with LY2033298 at the WT M₄ mAChR. As shown in Fig. 7A, the major effect observed was an elevation in ERK1/2 phosphorylation owing to the intrinsic efficacy of LY2033298. No significant potentiation was noted in ACh potency at the highest concentration of LY2033298 used (1 μM), even though this concentration was sufficient to enhance ACh binding (Fig. 4A). A possible explanation for this discrepancy is a synergistic desensitization of the two ligands, which would offset any affinity-based gains in ACh potency by decreasing its efficacy; the reduction in ACh maximum response in the presence of 1 μM LY2033298 is also in agreement with this hypothesis. Alternatively, and given the variability in the data, it is possible that this observation simply reflects the difficulty in accurately determining ACh concentration-response curves as a result of the reduced system response window in the presence of high levels of receptor activation caused by LY2033298. In contrast, the interaction between clozapine and ACh at the WT M₄ mAChR exhibited behavior consistent with a simple competitive interaction (Fig. 7B). Application of eq. 6 to the data yielded a pA₂ estimate of 7.88 ± 0.08 (n = 3-6) for clozapine, which is in good agreement with other studies (http://pdsp.med.unc.edu) but is significantly higher (p < 0.05) than the corresponding estimate obtained from the radioligand binding assays (6.6; Table 1); this discrepancy may be due to differences in the assay conditions for clozapine between the binding and functional experiments.

Because LY2033298 demonstrated agonistic properties at the WT M₄ mAChR, we were able to investigate its interactions with clozapine and CNO in a functional format for comparison with the modest (slightly positive) interaction noted in the binding interaction assays. As shown in Fig. 7, C and D, increasing concentrations of either clozapine or CNO resulted in a progressive reduction in the maximum agonist effect of LY2033298, clearly indicating a noncompetitive interaction. This finding suggests that the clozapine-like compounds exert a negative allosteric effect (negative activation cooperativity) on the intrinsic efficacy of LY2033298, despite their slightly positive binding cooperativity. We attempted to quantify this negative activation cooperativity by fitting an operational model of allosterism (eq. 7) to the data in Fig. 7, C and D. Because of the large number of model parameters, we fixed the estimates of pK_A (agonist affinity) and Logα (cooperativity) to the corresponding values for LY2033298 determined in the interaction binding studies. The remaining model parameters were estimated by nonlinear regression, and the results are shown in Table 3, where a number of features can be noted. First, the functional estimate of the affinity (pK_B) of clozapine as an allosteric antagonist of LY2033298 is in excellent agreement with the pA₂ value obtained for clozapine's competitive interaction with ACh under the same functional assay conditions; this is consistent with the fact that clozapine should be mediating both these effects (orthosteric competition with ACh; allosteric modulation with LY2033298) from the same binding site. Second, a pK_B estimate for CNO as an allosteric modulator of LY2033298 efficacy was also obtained. Third, the estimates of logβ for both ligands were highly negative; with respect to CNO, the nonlinear regression trended toward a boundary value, indicating that the parameter was practically indistinguishable from β = 0 and could thus not be estimated accurately. This is consistent with the notion that the efficacy modulation is so highly negative that LY2033298 is incapable of generating a stimulus on a receptor occupied by clozapine-like compounds.

Finally, we performed interaction studies on the M₄ DREADD. The most striking result was obtained when LY2033298 was coadministered with ACh. Figure 8A shows that, despite losing agonistic properties, LY2033298 was able to “rescue” ACh potency and efficacy at the otherwise functionally inactive M₄ DREADD, providing conclusive evidence for the presence of an allosteric site on this mutant receptor that retained the capacity to modulate orthosteric functionality. Furthermore, application of our operational model of allosterism to the data set yielded the parameters shown in Table 3, where it can be seen that the functional estimate of ACh affinity is in good agreement with the corresponding value from the radioligand binding assays (Table 1). Furthermore, and of importance, we also obtained an estimate of the affinity of LY2033298 for the DREADD, which we were unable to do from the binding studies. The LY2033298 pK_B value of 5.39 for the DREADD is very similar to the value determined for the WT M₄ mAChR (5.43), indicating that the affinity of LY2033298 for the allosteric site is not affected by the Y¹¹³C and A²⁰³G mutations in the orthosteric pocket. However, the cooperativity between ACh and LY2033298 changed dramatically on the DREADD. Because the radioligand binding studies indicated a neutral cooperativity at the level of binding affinity between the two at the DREADD (Fig. 4B; Logα = 0), the functional rescue by LY2033298 of ACh signaling must be totally due to positive activation cooperativity. In our model, this is quantified by the parameter logβ, which indicated a greater than 100-fold enhancement in the efficacy of ACh by LY2033298. In contrast, LY2033298 did not appreciably affect the potency or maximum response of either clozapine or CNO as agonists at the DREADD receptor (Fig. 8, B and C), indicating neutral cooperativity with these DREADD agonists.

Discussion

This study provides new evidence for conformational flexibility in the function of the M₄ mAChR. By using structurally diverse orthosteric ligands such as ACh and the clozapine-like molecules on a reverse-engineered M₄ DREADD, we identified differential effects on binding and signaling that suggest multiple modes of receptor engagement. Furthermore, through analysis of the actions of a novel allosteric agonist/modulator, LY2033298, we provide evidence for the retention of an allosteric site on the M₄ DREADD that has altered modes of cooperativity with the orthosteric site compared with the WT but can still restore functionality to an otherwise unresponsive receptor. Given that allosteric ligands are increasingly being discovered for many other GPCRs, these findings are likely to have relevance beyond the mAChR family.

Radioligand binding assays found that the M₄ DREADD had reduced affinity for orthosteric antagonists, such as [³H]-QNB and [³H]NMS, as well as for classic orthosteric agonists such as ACh and pilocarpine. In contrast, clozapine and its two metabolites, CNO and NDMC, showed an increase in binding affinity. These findings are the first to demonstrate that the divergent effects of the DREADD mutations on the function of ACh-like compounds, on the one hand, and clozapine-like compounds, on the other (Armbruster et al., 2007), are due in part to changes in binding affinity. Additional insights were gained from investigation of mAChR ligands believed to act allosterically. McN-A-343 is a partial agonist suggested either to act solely through an allosteric site (Birdsall et al., 1983) or to recognize both orthosteric and allosteric sites on the M₂ mAChR (Waelbroeck, 1994; May et al., 2007a). The fact that its affinity was unaltered at the M₄ DREADD (Fig. 2C, Table 1) is consistent with a nonclassical mode of binding compared with ACh, but further experiments are required to conclusively address the nature of the interaction between McN-A-343 and the M₄ mAChR. In contrast, the novel modulator LY2033298 did not seem to interact with the orthosteric antagonist [³H]QNB at either the WT M₄ mAChR or the DREADD. Given that positive cooperativity could be demonstrated between this compound and ACh in binding (α = 60) at the WT M₄ mAChR (Fig. 4A) and signaling (β = 155) at the DREADD (Fig. 8A; Table 3), the lack of interaction with [³H]QNB is indicative of neutral binding cooperativity (α = 1) at both receptors.

In addition to the varied pattern of effects noted for the DREADD mutations on ligand affinities, different behaviors were observed on ligand efficacies in functional assays of ERK1/2 phosphorylation. As anticipated, ACh and pilocarpine had markedly reduced potencies and maximal responses at the DREADD relative to the WT M₄ mAChR. In addition, the clozapine-like compounds displayed the opposite properties at the DREADD, namely a gain of functional potency and efficacy. Most surprising, however, were the findings with LY2033298, which displayed agonistic properties at the WT receptor; we had not detected appreciable levels of agonism in our initial characterization of this compound using intracellular calcium mobilization or guanine nucleotide exchange as functional readouts (Chan et al., 2008). This result may be due to differences in receptor-effector coupling between the cell lines used in our studies (CHO FlpIn versus CHO-K1) or due to LY2033298 trafficking receptor stimulus to the ERK1/2 pathway as opposed to classic pathways such as calcium mobilization or nucleotide exchange. Further studies are currently under way to address this issue. However, the agonist activity of both LY2033298 and McN-A-343 was lost upon mutation of Y¹¹³C and A²⁰³G, and this was not due to a loss in binding affinity at the M₄ DREADD. Therefore, there are two important implications of these findings. First, because Tyr¹¹³ and Ala²⁰³ are located in the orthosteric binding pocket, these residues could directly affect the binding of prototypical orthosteric compounds, such as ACh, pilocarpine, and [³H]QNB, but have a minimal effect on the binding of ligands such as LY2033298 and McN-A-343. The fact that clozapine and its metabolites gain affinity at the DREADD suggests that they adopt a different orientation within the orthosteric site relative to ACh, such that the mutation of Tyr¹¹³ and Ala²⁰³ leads to a gain in the free energy of binding for clozapine-like molecules. The second major implication is that, in addition to their involvement in ligand binding, Tyr¹¹³ and Ala²⁰³ must be involved in a conformational switch that links receptor occupancy to activation. Moreover, this conformational switch is not restricted to prototypical orthosteric ligands but is also part of the network that atypical agonists such as LY2033298 and McN-A-343 use to transmit their signal via the receptor to the cell.

Subsequent experiments focused on the interactive properties between orthosteric and allosteric sites on the M₄ mAChR to determine whether the magnitude and direction of allosteric modulation was also affected by the DREADD mutation. The ability of LY2033298 to bind to a topographically distinct site as an allosteric modulator was readily apparent in radioligand binding assays of the WT M₄ mAChR, where LY2033298 caused a concentration-dependent leftward translocation of the ACh/[³H]QNB competition binding curve (Fig. 4A). Application of an allosteric model to the data yielded affinity and cooperativity estimates for the interaction that were in general agreement with our previous study (Chan et al., 2008), although we acknowledge that the nature of the interaction at the molecular level is more complex because the modulator, in this instance, seemed to affect the proportion of high-affinity states for ACh in addition to its affinity. In contrast, the cooperativity was only weakly positive with clozapine and CNO, highlighting the fact that allosteric interactions can be highly probe-dependent (Leach et al., 2007). Additional evidence for an allosteric mechanism of LY2033298 action at the WT M₄ mAChR was obtained when it was tested against clozapine and CNO in functional assays of ERK1/2 phosphorylation, where increasing concentrations of either clozapine or CNO markedly reduced the efficacy of LY2033298; in our operational model of allosterism, this is reflected in values of the activation cooperativity parameter, β, trending toward zero (Table 3), even though the binding cooperativity parameter, α, is (albeit only slightly) greater than 1. Differential effects on binding cooperativity, on the one hand, and activation cooperativity, on the other, have previously been noted for other allosteric modulators of GPCRs (Christopoulos, 2002) and highlight the need to study allosteric modulator actions in a variety of different assay formats.

When interaction experiments were repeated on the DREADD, it became readily apparent that the mutations had a profound effect on the cooperativity between the orthosteric and allosteric sites. The most striking finding was that despite both ACh and LY2033298 losing their agonistic effects at the M₄ DREADD, the combination of the two led to a significant functional rescue of ACh potency and efficacy (Fig. 8A). This finding clearly demonstrates that LY2033298 is still able to bind to the M₄ DREADD (with similar affinity as for the WT; Table 3) and act as an allosteric modulator of the orthosteric site, even though it can no longer act as an allosteric agonist. In addition, the nature of the allosteric interaction on the DREADD seems totally driven by positive activation cooperativity, because radioligand binding studies revealed neutral binding cooperativity between ACh and LY2033298 on the DREADD in contrast to the positive cooperativity observed at the WT receptor (Fig. 4B). Perhaps most importantly, this finding has also identified another novel approach to exploiting DREADDs; although we have no reason to assume that activation of the DREADD by an exogenous ligand (e.g., CNO) does not faithfully recapitulate the signaling properties of the native mAChR, our ability to now engender responsiveness to the native agonist by using an allosteric modulator such as LY2033298 provides a new opportunity to directly address this assumption.

Because the allosteric interaction between LY2033298 and the clozapine-like compounds at the WT M₄ mAChR was different from that between the modulator and ACh, it was perhaps not so surprising that the DREADD mutation led to a different clozapine/CNO-LY2033298 interaction compared with ACh. In this case, however, the interaction between either clozapine or CNO and LY2033298 was characterized by a neutral activation cooperativity; i.e., the effect of the DREADD mutation seemed to be a functional “uncoupling” of the allosteric site and the orthosteric site when the latter is used by clozapine-like molecules. This is further evidence that the transmission of cooperative effects between orthosteric and allosteric sites on the M₄ mAChR relies on the complementarity between specific molecules and their attachment points, as well as conformational cross-talk between the two sites. Figure 9 summarizes the main elements of our findings for ACh and CNO in schematic form, although it should be noted that this is not intended to represent a model for the underlying molecular mechanisms governing the observed behaviors. Indeed, the observed affinity modulation by LY2033298 of ACh at the WT M₄, as well as the efficacy modulation at the DREADD, may reflect a common molecular mechanism at both receptors, whereby LY2033298 allosterically promotes an active receptor state that would manifest as an increase in apparent affinity for a highly efficacious agonist (ACh at the M₄ WT) but an increase in efficacy for a low efficacy agonist (ACh at the M₄ DREADD; see Ehlert and Griffin, 2008).

In conclusion, this study has used a novel pharmacological probe, LY2033298, and a novel biological tool, the M₄ DREADD, to gain new insights into the molecular pharmacology of the M₄ mAChR. We have found that Tyr¹¹³ and Ala²⁰³ are not only involved in the binding and activation mechanism of orthosteric mAChR ligands but also participate in the activation and cooperative mechanisms of allosteric ligands. The finding that LY2033298 can rescue function of an endogenous ligand at a mutationally impaired receptor also has significant relevance for the potential development of allosteric modulators for diseases in which receptor mutations cause decreased orthosteric activity; if the allosteric site remains intact, it can be used as an alternative drug target. Improved understanding of the structure-function relationships of both orthosteric and allosteric GPCR sites may thus be used for the development of more selective therapeutics for a number of disorders.