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Research ArticleCellular and Molecular

Engineered Context-Sensitive Agonism: Tissue-Selective Drug Signaling through a G Protein-Coupled Receptor

Wiebke K. Seemann, Daniela Wenzel, Ramona Schrage, Justine Etscheid, Theresa Bödefeld, Anna Bartol, Mareille Warnken, Philipp Sasse, Jessica Klöckner, Ulrike Holzgrabe, Marco DeAmici, Eberhard Schlicker, Kurt Racké, Evi Kostenis, Rainer Meyer, Bernd K. Fleischmann and Klaus Mohr
Journal of Pharmacology and Experimental Therapeutics February 2017, 360 (2) 289-299; DOI: https://doi.org/10.1124/jpet.116.237149

Abstract

Drug discovery strives for selective ligands to achieve targeted modulation of tissue function. Here we introduce engineered context-sensitive agonism as a postreceptor mechanism for tissue-selective drug action through a G protein-coupled receptor. Acetylcholine M2-receptor activation is known to mediate, among other actions, potentially dangerous slowing of the heart rate. This unwanted side effect is one of the main reasons that limit clinical application of muscarinic agonists. Herein we show that dualsteric (orthosteric/allosteric) agonists induce less cardiac depression ex vivo and in vivo than conventional full agonists. Exploration of the underlying mechanism in living cells employing cellular dynamic mass redistribution identified context-sensitive agonism of these dualsteric agonists. They translate elevation of intracellular cAMP into a switch from full to partial agonism. Designed context-sensitive agonism opens an avenue toward postreceptor pharmacologic selectivity, which even works in target tissues operated by the same subtype of pharmacologic receptor.

Introduction

Differences in structure between receptor subtypes allow for the design of subtype-selective drugs. In the present study we shift focus from differences in receptor structure toward differences in subsequent intracellular signal propagation to engender tissue-selectivity of drug action. This concept is also addressed as “cell-based functional selectivity” (Kenakin, 2007) or “phenotypic pharmacology” (Nelson and Challiss, 2007) and is based on the stoichiometry and sensitivity of the cellular components driving the cellular response. It should not be confounded with “receptor-based selectivity” or “ligand-induced bias” (Kenakin, 2007). In contrast to conventional binding selectivity, cell state-dependent signaling will generate selectivity even between tissues endowed with the same receptor subtype. For this principle to work it is crucial to chemically encode agonist sensitivity at a level that discriminates between different states of cellular activity. Such “chemically engineered” context-dependent signaling will be introduced here. As a paradigm we will elucidate why activation of the M2 muscarinic acetylcholine receptor can be exploited to suppress pain (Matera et al., 2014), even though this subtype would in principle also mediate dangerous slowing of the heart rate.

Remarkably, even permanently charged muscarinic ligands unable to penetrate the blood–brain barrier are powerful analgesics in animal experiments, and such peripheral compounds lack unwanted central tremorgenic activity (Barocelli et al., 2001; Matera et al., 2014). Therefore, permanently charged M2-selective agonists should be useful for the treatment of pain disorders.

In mammalian hearts, M2 receptor-signaling is preferentially mediated via activation of inhibitory G proteins (Gi/o), whereas counteracting adrenergic β-receptors activate stimulatory G protein (Gs) signaling (for reviews, see Brodde and Michel, 1999; Haga, 2013). This functional antagonism converges on the level of intracellular cAMP. Therefore, we hypothesized less effective cholinergic signaling by dualsteric agonists to occur in tissues under conditions of elevated cAMP. This would allow the organism to maintain sufficient blood pressure in spite of muscarinic receptor activation.

To probe the feasibility of this concept, we applied a novel type of agonist engineered to transduce receptor activation with down-tuned efficacy (Bock et al., 2012). These “dualsteric” (bitopic orthosteric/allosteric) ligands activate the receptor protein from the orthosteric transmitter binding site and simultaneously bind to the receptor’s allosteric vestibule, thereby hindering the receptor from adopting the fully active state (Supplemental Fig. 1; for review, see Mohr et al., 2013). The term “dualsteric” illustrates that ligand binding is present at two sites on one and the same receptor protein. For comparison, we included a conventional full agonist (oxotremorine M) and an archetypal weak partial agonist (pilocarpine).

Action of these tools was investigated in different M2 receptor expression systems. First, we quantified ligand-induced G protein activation in [35S]GTPγS binding experiments performed with membrane homogenates of Chinese hamster ovary cells stably transfected with the human M2 receptor (CHO-hM2). To illustrate the influence of the cellular context on compound-induced receptor signaling, we, second, applied murine hippocampal brain slices to detect ligand-induced inhibition of acetylcholine release via presynaptic inhibitory M2 receptors. Third, we analyzed the negative chronotropic response in spontaneously beating murine embryonic cardiomyocytes (eCM) after agonist application. The orthosteric agonist oxotremorine M (OxoM), the dualsteric agonist iper-6-phth (I-6-p) and the partial agonist pilocarpine (Pilo) did not fully inhibit acetylcholine release from cholinergic nerve endings of the hippocampus. In contrast to this, all compounds induced full intrinsic efficacy in eCM, that is, complete silencing of spontaneous beating. The intensity of drug action is well known to depend on the level of receptor expression (Kenakin, 2007; Rajagopal et al., 2011). However, the functional status of the receptor protein may be also critical. Here, we show that the intrinsic efficacy of the dualsteric compound for slowing spontaneously beating of eCM declines with elevated intracellular cAMP. Additionally, left ventricular catheter measurements in anesthetized mice disclose strongly improved cardiac tolerability of the applied dualsteric agonist compared with the conventional orthosteric agonist OxoM.

To validate the concept of context-dependent efficacy, M2 receptor signaling induced by dualsteric compounds was analyzed in recombinant and native M2 receptor expression systems, that is, CHO-hM2 and human lung fibroblasts (MRC-5), respectively. Taken together, detailed molecular pharmacology and physiology studies with dualsteric ligands pinpoint a novel approach for pharmacologic selectivity: engineered context-sensitive signaling of G protein-coupled receptors (GPCRs).

Materials and Methods

Test Compounds

Oxotremorine M iodide (OxoM), pilocarpine hydrochloride (Pilo), atropine sulfate, N-methylscopolamine bromide, isoprenaline hydrochloride (ISO), metoprolol tartrate, forskolin (FSK), isobutylmethylxanthine (IBMX), and hemicholinium were obtained from Sigma-Aldrich Chemie (Steinheim, Germany). [3H]N-methylscopolamine bromide ([3H]NMS) and [3H]choline were purchased from PerkinElmer Life and Analytical Sciences (Homburg, Germany).

The dualsteric agonists were kindly provided from Prof. DeAmici and Prof. Holzgrabe. Synthesis of the dualsteric agonists iper-6-phth [I-6-p (Antony et al., 2009)] and iper-8-phth [I-8-p (Bock et al., 2012)] is described elsewhere.

Cell Culture

We used Flp-In-Chinese Hamster Ovary cells (R75807; Invitrogen, Carlsbad, CA) stably expressing the human M2 receptor (CHO-hM2) and the human lung fibroblast cell line MRC-5 (CCL-171; American Type Culture Collection [ATCC], Manassas, VA). Cell lines were used in experiments until reaching passage 50 and 10 for CHO-hM2 and MRC-5, respectively. MRC-5 and CHO cells were negatively tested for mycoplasma contamination. Both cell lines were cultured as described previously elsewhere (Schrage et al., 2013).

Animals

Wild-type mice (C57BL/6 and CD1 strain) of both sexes were used in the described experiments. Mice (Charles River, Sulzfeld, Germany) were housed in isolated ventilated cages at 24°C with a 12-hour light/dark cycle. Animals taken for organ and embryo isolation were killed by cervical dislocation. Animal experiments were approved by the responsible federal state authority (Landesamt fuer Natur-, Umwelt- und Verbraucherschutz Nordrhein-Westfalen).

[35S]GTPγS Accumulation

Preparation of membrane homogenates (CHO-hM2) and [35S]GTPγS binding experiments were conducted as described previously elsewhere (Schrage et al., 2013). In short, homogenates of membranes from CHO-hM2 wild-type cells (40 μg/ml) were incubated with 0.07 nM [35S]GTPγS and agonist-induced [35S]GTPγS incorporation was measured after 1 hour. Experiments were performed in quadruplicate. A sample size of n ≥ 3 was elected on the basis of earlier publications (Antony et al., 2009; Schröder et al., 2010; Schrage et al., 2013).

Inhibition of Acetylcholine Release

Superfusion studies on hippocampal brain slices of female 8-week-old CD1 mice were performed as described elsewhere (Schulte et al., 2012). Briefly, slices (0.3 mm thick, diameter 2 mm) were preincubated with [3H]choline (0.1 µM) for 30 minutes. Tissues were superfused at 37°C for 110 minutes with oxygenated physiologic salt solution containing 3.25 mM calcium and 10 µM hemicholinium to block high-affinity choline uptake. Agonist was added from 60 minutes of superfusion onward. Two periods of electrical field stimulation for 2 minutes (3 Hz, 200 mA, 2 milliseconds) were applied after 40 minutes (S1) and 90 minutes (S2) of superfusion. Fractional rates of tritium overflow without (S1) and with agonist (S2) were calculated, and the agonist-induced inhibition of transmitter release was finally expressed as the S2/S1 ratio. OxoM and I-6-p were tested on brain slices obtained from three and seven independent isolations, respectively. Pilo was tested in a supramaximal concentration of 1 mM on two brain slices obtained from the same isolation.

Beating Frequency of Cardiomyocytes

Atrial cardiomyocytes were enzymatically isolated by collagenase treatment according to Fleischmann et al. (2004) from murine embryonic midstage hearts (mouse strain CD1; sex of the used embryo “unknown”). After isolation on day 13.5–16.5 from female pregnant mice, cells were plated on sterile gelatin-coated glass coverslips and kept in the incubator (37°C, 5% CO2) for 48 hours. On the day of experiment, coverslips were transferred into a temperature-controlled recording device (37°C) and superfused with increasing agonist-containing buffer solution (140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM HEPES and 10 mM glucose; pH 7.4 with NaOH). As cellular readout, the negative chronotropic effect in spontaneously beating cardiomyocytes was measured by videomicroscopy as described previously elsewhere (Dobrowolski et al., 2008). Movies of beating cardiomyocytes were recorded with a Sony XCD-X710 camera at 30 frames/second, and beating areas were analyzed offline with a custom-written software (Labview 7.1 and IMAQ; National Instruments, Austin, TX). The test compound effect was determined as the average frequency over the last 30 seconds in steady-state and was normalized to baseline frequency. Each agonist was tested at least in three independent experiments.

Cardiomyocyte Sarcomere Shortening

Ventricular cardiomyocytes were prepared from male adult mouse hearts (mouse strain C57BL/6, age 20–25 weeks) as described previously elsewhere (Tiemann et al., 2003) and kept in oxygenated Tyrode’s solution (135 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 2 mM HEPES; 9 mM glucose, bovine serum albumin 1 mg/ml, and trypsin inhibitor 0.017 mg/ml; pH 7.4 with NaOH) at room temperature until use. Cells were allowed to attach to laminin-coated microscope slides and stimulated externally with 40 V for 0.4 milliseconds. Sarcomere shortening was measured under a pacing frequency of 2 Hz at 36°C in absence and presence of 1 µM ISO. Subsequently, isolated cardiomyocytes were superfused with ISO-containing muscarinic agonist solutions.

Agonists were tested in a concentration of 10 µM and shortening recordings evaluated by calculating the mean of five shortenings in the steady state. Sarcomere shortening was recorded using a video imaging system and SarcLen software (IonOptix, Milton, MA). The effect of the tested agonists was normalized to the effect of ISO and plotted in a bar diagram. OxoM and I-6-p were tested in cardiomyocytes of at least four different isolations.

Left Ventricular Catheter Measurements

Measurements were performed as described previously elsewhere (Wenzel et al., 2012). Briefly, female mice (age 8–12 weeks) of the CD1 strain were anesthetized by inhalation of 1% isoflurane and 0.6 l/min O2. The right carotid artery was exposed, and a small catheter (1F, Millar) was inserted through an incision and placed into the left ventricle. OxoM, I-6-p (0.4 μmol/kg each) or metoprolol (3 mg/kg) were injected into the left jugular vein. Left ventricular pressure was recorded continuously via the Millar Aria 1 system connected to a PowerLab A/D converter (AD Instruments, Spechbach, Germany).

For analysis, maximal systolic pressures and the heart rate before and 2 minutes after injection were compared. Based on earlier publications (Barocelli et al., 2001; El Beheiry et al., 2011), a sample size of n ≥ 5 was elected. Experiments were performed without randomization; no blinding was performed.

Dynamic Mass Redistribution

To detect GPCR signaling on whole-cell level in CHO-hM2 and MRC-5, we used label-free biosensor technology based on dynamic mass redistribution (DMR) (Schröder et al., 2010). The principle of DMR was described elsewhere in detail for CHO (Schröder et al., 2011) and MRC-5 cells (Lamyel et al., 2011). Experiments were conducted in Hank’s balanced salt solution (HBSS, 14025; Invitrogen) buffer supplemented with 20 mM HEPES (pH 7.0) at 28°C. DMR responses of CHO-hM2 and MRC-5 were quantified using the agonist-induced maximal response between 0 and 1800 seconds. In CHO-hM2, agonists were additionally tested in presence of 10 µM FSK. Independent experiments were performed in triplicate. As reported previously for CHO cells (Schröder et al., 2011), the concentration–effect curves of MRC-5 were almost independent from the time point of DMR reading (Supplemental Fig. 5). Independent experiments were conducted at least three times in triplicate or quadruplicate according to earlier publications (Schröder et al., 2010; Bock et al., 2012; Schrage et al., 2013).

Radioligand Binding Assays

[3H]NMS Dissociation Assay.

Two-point [3H]NMS dissociation experiments with membrane homogenates expressing muscarinic receptors were conducted in 5 mM NaKPi buffer (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4) at 23°C as described previously elsewhere (Huang et al., 2005). Preparation of membrane homogenates (CHO and MRC-5) and cardiac tissue homogenates (murine ventricular tissue from adult C57BL/6 mice, age and sex “unknown”) was conducted according to Schrage et al. (2013) and Keller et al. (2015), respectively.

[3H]NMS] Competition Assay.

Whole-cells experiments using CHO-hM2 and MRC-5 cells were conducted in HBSS (14025; Invitrogen) buffer supplemented with 20 mM HEPES (pH 7.0) at 28°C as described previously for MRC-5 (Schrage et al., 2013).

Radioligand binding experiments were conducted at least three times in duplicate. The sample size was selected based on earlier publications (Bock et al., 2012; Schrage et al., 2013).

cAMP Determination

The intracellular cAMP of CHO-hM2 and MRC-5 cells was determined using the HTRF-cAMP dynamic kit (Cisbio, Bagnols-sur-Cèze Cedex, France) following the manufacturer’s instructions with the following modification: experiments were performed in absence of IBMX to avoid interference with basal cAMP. Experiments were conducted in HBSS (14025; Invitrogen) buffer supplemented with 20 mM HEPES (pH 7.0), as the DMR experiments. Fluorescence was quantified on a Mithras LB 940 reader (Berthold Technologies, Bad Wildbad, Germany). A cAMP standard curve with the final concentrations of 0, 0.17, 0.69, 2.78, 11.1, 44.5, 178, and 712 nM cAMP was tested to define the measurement window of the assay (Supplemental Fig. 3A). Then, cellular cAMP was related to the protein content of the cells determined according to Lowry using human serum albumin as a standard (Lowry et al., 1951). Experiments were performed in triplicate using cells in suspension.

Statistics and Data Analysis

Data are shown as mean values ± S.E.M. for n independent observations. F tests were performed to check the variance between statistically tested groups; no difference was detected. Based on the assumption of normal data distribution, comparison of means was performed using Student’s two-side t tests (paired and unpaired) or one-way analysis of variance. Analysis of variance was followed by Dunnett’s or Tukey’s post-test, as appropriate. P < 0.05 was considered statistically significant.

All data were analyzed using Prism 6.0 (GraphPad Software, San Diego, CA). Functional and binding data were analyzed using a four-parameter logistic function yielding the inflection point (IC50), the upper and lower plateau, and the slope factor of the curves. In case of functional experiments, the inflection point represents the potency (EC50) and the upper plateau describes the maximal inducible effect (Emax). If the observed slope factors did not differ significantly from unity (F test, P > 0.05), the slope factor was constrained to 1.

In case of [3H]NMS competition binding experiments, the IC50 values were converted to apparent binding constants (KA) using the Cheng-Prusoff correction (Cheng and Prusoff, 1973). Receptor density (Bmax) was calculated from IC50 values according to DeBlasi et al. (1989).

Additionally, mean values from DMR experiments with CHO-hM2 and MRC-5 cells were analyzed by the operational model of agonism (Black and Leff, 1983) to estimate the coupling efficiency as described earlier (Rajagopal et al., 2011; Schrage et al., 2013) with KA fixed to the agonist’s dissociation binding constant estimated from whole-cell radioligand binding experiments (Supplemental Table 2):Embedded Image(1)In eq. 1, E is the response, Emax is the maximal response of the system (determined by the full agonist OxoM), τ is the coupling efficacy between the agonist/receptor complex and its downstream signaling partners, KA is the agonist’s dissociation binding constant, and [A] is the concentration of agonist.

The maximum effect of an agonist predicted from the coupling efficiency was calculated according to eq. 2 (Black et al., 2010) and plotted as a theoretical curve in Fig. 5 (lower panel) with Emax of the system fixed to 1.

Embedded Image(2)

Results

Cellular Context Determines the Intensity of Muscarinic M2 Receptor Signaling.

Ligands are traditionally subdivided in full agonists and partial agonists or antagonists depending on their ability to elicit a receptor-mediated physiologic or pharmacologic response (Stephenson, 1956). It is important to note that the amount of detectable receptor activation is dependent on the system and assay (Langmead and Christopoulos, 2013). In systems with high receptor density and/or assays with significant signal amplification, full and partial agonists can induce the same maximum effect (Rajagopal et al., 2011; Schrage et al., 2016). Therefore, we determined the intrinsic efficacy of muscarinic agonists first in an assay with low signal amplification using receptor homogenates of CHO-hM2 cells.

[35S]GTPγS-accumulation is a functional readout to detect inhibitory G protein activation that is close to the receptor and directly linked to the agonist-induced receptor activation (Fig. 1A) (Milligan, 2003). The orthosteric activator OxoM is known to be as effective as acetylcholine and represents a full agonist (Schrage et al., 2013). Pilo is known to induce less G protein activation via the M2 receptor than OxoM (Van Gelderen et al., 1996) and was employed as a “partial agonist.” In contrast to OxoM, which elicited maximal G protein activation, Pilo and the prototypal dualsteric agonist I-6-p induced only effects of 60% ± 3% and 75% ± 3%, respectively (Fig. 1B). The potency for receptor-mediated [35S]GTPγS incorporation was similar for OxoM and I-6-p, whereas Pilo was found to be less effective (Fig. 1B; pEC50: 8.07 ± 0.19 for OxoM, 7.49 ± 0.26 for I-6-p, and 5.80 ± 0.12 for Pilo). However, the quantification of agonist-induced G protein activation in a particular cellular system (in this case CHO-hM2) does not allow for deriving pharmacologic parameters of agonist behavior in a different cellular context.

Fig. 1.
Fig. 1.

System dependence of agonist-induced M2 receptor activation. Shown are simplified illustrations of the tested systems (upper panels) and the corresponding concentration–effect curves of OxoM, Pilo and I-6-p (lower panels). The green triangles represent muscarinic agonists, and the red-labeled terms are the detected readouts of the corresponding assays. All data are mean ± S.E.M. of n independent experimental days. (A, D) G protein activation was quantified in [35S]GTPγS binding experiments. (B, E) Inhibition of acetylcholine release was detected in adult murine hippocampal brain slices. Pilo (1 mM) was tested on two brain slices obtained from the same preparation (0.79, 0.81). (C) Modified from Schulte et al., 2012. (E, F) Reduction of spontaneous beating was measured in isolated embryonic cardiomyocytes isolated from murine atria.

To analyze the effect of muscarinic agonists in native cellular systems with dominant M2 receptor expression, we used murine hippocampal brain slices and murine eCM to determine inhibitory muscarinic autoreceptor function and the muscarinic-mediated negative chronotropic effect, respectively. In the central nervous system, autoinhibition of acetylcholine release is primarily mediated via activation of presynaptic Gi/o-coupled muscarinic acetylcholine receptors which inhibit voltage-sensitive calcium channels that are involved in the regulation of neutrotransmitter release (Fig. 1C) (Caulfield, 1993; Shapiro et al., 1999). The M2 receptor subtype is known to be the dominant muscarinic autoreceptor in the hippocampus (Zhang et al., 2002). As expected, all agonists failed to fully inhibit acetylcholine release in the hippocampal brain slices (Fig. 1D); even iperoxo, an agonist with supraphysiologic efficacy (Schrage et al., 2013), did not fully inhibit acetylcholine release (Etscheid et al., 2014). Therefore, the effect of OxoM was considered as the maximum inducible effect in this system.

Compared with OxoM, the dualsteric ligand I-6-p was clearly less effective and behaved as a weak partial agonist (Fig. 1D). Due to the already low effect of I-6-p, Pilo was only tested in the concentration of 1 mM and was as effective as I-6-p. In comparison with [35S]GTPγS-accumulation experiments, potencies were shifted to higher concentrations but were again in the same range for OxoM and I-6-p (pEC50: 6.58 ± 0.27 and 4.79 ± 0.80 for OxoM and I-6-p, respectively).

Because muscarinic acetylcholine receptor activation is also known to mediate dangerous slowing of the heart rate (Fig. 1E), we next investigated the effect in cardiomyocytes. For this, we measured the agonist-induced negative chronotropic effect on spontaneous beating eCM by videomicroscopy. This assay is an indirect detection method for M2 receptor activation (Fleischmann et al., 2004). Therefore, eCM were superfused with increasing concentrations of test compound. Cumulative administration of either the orthosteric activator OxoM, the partial agonist Pilo, or the dualsteric agonist I-6-p caused concentration-dependent lowering of spontaneous beating rates (Fig. 1F). OxoM and I-6-p possessed similar potencies (pEC50: 6.88 ± 0.10 and 6.62 ± 0.10 for OxoM and I-6-p, respectively) and showed full intrinsic efficacy. Even the less potent agonist Pilo (pEC50: 5.21 ± 0.12) caused a complete arrest of spontaneous beating. Upon washout with agonist-free solution, all tested cells resumed spontaneous beating (OxoM: 94% ± 3% [n = 33], I-6-p: 80% ± 3% [n = 34], Pilo: 64% ± 4% [n = 36]).

In sum, the distinct efficacy profiles of the full agonist OxoM, the partial agonist Pilo and the dualsteric agonist I-6-p in three different expression systems (i.e., membrane homogenates of CHO-hM2 cells, hippocampal brain slices, and eCM) nicely demonstrate the critical role of the cellular context with respect to full and partial agonism of ligands.

Dualsteric Muscarinic Agonist Action Is Sensitive to Intracellular cAMP in Cardiomyocytes.

In cardiomyocytes, activation of M2 receptors leads to inhibition of the adenylyl cyclase via activation of Gi/o proteins and hence inhibits formation of the second messenger cAMP (Brodde and Michel, 1999). Therefore, it is imaginable that the intensity of cellular readout depends on the basal intracellular cAMP level. To check whether dualsteric agonist action is reduced in cases of increased intracellular cAMP in eCM, I-6-p-induced decrease of eCM beating was tested under basal conditions and under sympathetic stimulation. In Figure 2 real-time recordings are shown in the upper panels (Fig. 2, A and B), and single data points are plotted in the lower panels (Fig. 2, C and D).

Fig. 2.
Fig. 2.

Chronotropic response to muscarinic agonists in spontaneously beating murine embryonic atrial cardiomyocytes (eCM). Increasing concentrations of the muscarinic agonists were applied cumulatively and normalized to baseline frequency. All experiments were performed on at least three independent days; a minimum of three data points was conducted per concentration. (A, B) Representative real-time recordings of spontaneously beating cells and their response to I-6-p under basal conditions (A) and in presence of sympathetic stimulation (B). The dotted lines represent cardiac arrest (0 bpm). The washout period is marked with gray arrows. (C, D) Increasing concentrations of I-6-p were applied under basal conditions (C) and in presence of 100 nM ISO and 100 µM IBMX (D). The dotted lines represent the mean basal frequency: 140 ± 4 bpm (n = 34) and 139 ± 7 bpm (n = 15) in case of C and D, respectively. The washout effect of each tested cell is marked in gray. The response of each individual cell to ISO and IBMX is visualized by a connecting line in D.

To mimic sympathetic stimulation, eCM were pretreated with the β-adrenergic agonist ISO in combination with the phosphodiesterase inhibitor IBMX. The combination of ISO (100 nM) and IBMX (100 µM) provides maximal stimulation of the system (Ji et al., 1999; Malan et al., 2004). Cardiomyocytes responded with an increase of spontaneous beating rate by at least 15% (15 out of 20 cells; responding cells were plotted in Fig. 2D and their individual response visualized via a connecting line). In comparison with baseline frequency, ISO induced a significant increase in beating frequency (basal: 139 ± 7 beats per minute [bpm] versus ISO/IBMX: 197 ± 11 bpm; mean values ± S.E.M., P < 0.0001, paired t test). In the presence of ISO and IBMX, I-6-p failed to achieve full intrinsic efficacy even at concentrations of 10 µM (Fig. 2D). In contrast, OxoM 10 µM could still completely suppress spontaneous beating. We checked our findings of dualsteric agonist action also in adult ventricular cardiomyocytes by measuring sarcomere shortening as an inotropic response. In the presence of ISO, I-6-p induced significantly less negative inotropy in these cells than the orthosteric agonist OxoM (Supplemental Fig. 2).

Taken together, these findings are in line with the hypothesis that a given adrenergic tone provides better protection against negative chronotropy and inotropy induced by the dualsteric muscarinic agonist I-6-p compared with the purely orthosteric agonist OxoM.

The Dualsteric Muscarinic Agonist I-6-p Engenders Superior Cardiovascular Tolerability In Vivo.

Systemic administration of muscarinic agonists in mammals is known to cause a pronounced reduction of heart rate and mean arterial pressure (Barocelli et al., 2001; Takakura et al., 2003; Fisher et al., 2004). To check the cardiodepressive action of the orthosteric agonist OxoM and the dualsteric agonist I-6-p in vivo, we performed left ventricular catheter measurements in anesthetized mice. Because of their permanent charge, neither compound was likely to pass the blood–brain barrier, which rules out central cardiodepressive effects.

Both agonists were administered intravenously by bolus injection into the left jugular vein. OxoM was administered at a dose of 100 µg/kg to induce a maximal effect (Barocelli et al., 2001). Based on the mean body weight and blood volume of mice (i.e., 30 g and 4 ml, respectively), this will result in an initial blood concentration of 3 µM. The dualsteric agonist I-6-p was applied at the same concentration as OxoM as both compounds were equipotent in eCM (Fig. 1F).

Real-time recordings of left ventricular systolic pressure (LVSP) (Fig. 3, A and C) and heart rate (HR) (Fig. 3, B and D) revealed a pronounced suppression of cardiovascular function in the case of OxoM, whereas I-6-p was well tolerated. In particular, OxoM was lethal in 60% of the animals, whereas all mice of the I-6-p-group survived. Statistical analysis of cardiac function showed that, starting from similar baseline levels of LVSP and HR, OxoM induced a significantly stronger cardiovascular depression in mice compared with the dualsteric agonist I-6-p (Fig. 3, E versus F; LVSP, reduction to 54% ± 6% [OxoM] versus 90% ± 3% [I-6-p], P < 0.001; HR, reduction to 40% ± 5% [OxoM] versus 86% ± 2% [I-6-p], P < 0.001).

Fig. 3.
Fig. 3.

Effects of OxoM and I-6-p on blood pressure and heart rate in anesthetized mice. (A, B) Original traces of left ventricular systolic blood pressure (LVSP) and heart rate (HR) recorded in response to OxoM (0.4 µmol/kg). (C, D) Original traces of LVSP and HR recorded in response to I-6-p (0.4 µmol/kg). (E, F) Statistical analysis of changes in LVSP and HR, respectively, in response to either metoprolol (3 mg/kg), OxoM, or I-6-p. P < 0.05 was considered statistically significant according to Student’s t test (***P < 0.001). The paired t test was used to check the effect of metoprolol and the unpaired t test to compare the interindividual effects of both test compounds: OxoM versus I-6-p. Indicated data are mean ± S.E.M. of n independent experiments.

Due to the small effect of I-6-p, a second dose was administered 10 minutes after the first one. The second application induced no further effect, thus indicating that the applied concentration of I-6-p was maximally active (LVSP, reduction by 10% ± 3% [first application] versus reduction by 1% ± 2% [second application]; HR, reduction by 14% ± 2% [first application] versus reduction by 2% ± 1% [second application]).

To check the basal β-adrenergic tone of anesthetized mice in our experiments, we applied the β-adrenoceptor blocker metoprolol by intravenous-injection (3 mg/kg according to El Beheiry et al., 2011). Relative to baseline, treatment with metoprolol resulted in a LVSP of 90% ± 4% (P > 0.05, paired t test) and a HR of 81% ± 2% (P < 0.01, paired t test) (Fig. 3, E and F), indicating a significant endogenous sympathetic tone in our experimental conditions. Therefore, we hypothesized that I-6-p showed better in vivo cardiovascular tolerability in mice because of a higher sensitivity to the endogenous β-sympathetic tone.

Intracellular cAMP Mediates Loss of Sensitivity to Dualsteric Muscarinic Agonists.

Experiments on isolated cardiomyocytes and in anesthetized mice suggested that interventions to increase intracellular cAMP weaken cholinergic Gi/o-signaling in the case of the dualsteric ligand I-6-p. For the analysis and quantification of signaling efficiency, we took advantage of CHO cells as recombinant host to recapitulate this signaling paradigm by recombinant expression of the human muscarinic M2 receptor subtype (CHO-hM2, i.e., hM2 “overexpression” system). Cells were studied in the absence and presence of FSK, a direct activator of adenylyl cyclases (Seamon et al., 1981) that increased intracellular cAMP in a concentration-dependent manner (Supplemental Fig. 3, B and C).

After FSK-pretreatment (10 µM), which increased intracellular cAMP to the level in human lung fibroblasts (MRC-5) (Supplemental Fig. 3C and Supplemental Table 1), we checked for potential effects of high intracellular cAMP levels on compound efficacy. MRC-5 were additionally applied, which express the M2 subtype endogenously (Matthiesen et al., 2006; Schrage et al., 2013). This was additionally verified by orthosteric [3H]NMS dissociation experiments employing the allosteric inhibitor action of W84 (Supplemental Fig. 4).

To quantify muscarinic agonist action on whole-cell level in all three different cellular systems (i.e., CHO-hM2, CHO-hM2 + FSK, and MRC-5), we used the label-free optical biosensor Epic and measured agonist-induced DMR in living cells (Schröder et al., 2010, 2011); this is outlined in Fig. 4. To gain deeper insight into how the cellular context influences agonist efficiency, we included the middle-chain-elongated derivative I-8-p. I-8-p is characterized by an increased coupling efficiency at the expense of less signaling pathway-selectivity compared with I-6-p (Bock et al., 2012).

Fig. 4.
Fig. 4.

DMR response of CHO-hM2, CHO-hM2 after forskolin-pretreatment (+FSK), and MRC-5. (A, D, G) Indicated are representative real-time recordings of muscarinic agonist-induced DMR traces. The arrow marks the addition of the test compound after baseline read. (B, E, H) Bar graphs show mean maximal DMR responses of supramaximal agonist concentrations (OxoM: 100 µM; I-6-p: 100 µM; I-8-p: 10 µM; Pilo: 100 µM). Compared with OxoM, all ligands were tested for partial agonism; P < 0.05 was considered statistically significant according to one-way analysis of variance with Dunnett’s correction for comparison with OxoM (**P < 0.01). Indicated data are mean ± S.E.M. of at least n independent experiments. Experiments were performed in triplicate or quadruplicate. (C, F, I) Concentration–effect curves of the tested ligands resulting from DMR assays. The dashed lines mark the inflection points (pEC50) of the tested ligands in case of CHO-hM2. Indicated data are mean ± S.E.M. of at least three independent experiments. Experiments were performed in triplicate or quadruplicate. Potencies are listed in Supplemental Table 2 for CHO-hM2, CHO-hM2 + FSK, and MRC-5.

In CHO-hM2 cells, muscarinic agonists induced positive DMR signals with a characteristic peak (real-time recordings, Fig. 4A) representing M2 receptor activation (Bock et al., 2012). All tested ligands behaved as full agonists under basal conditions, as none of the compounds had a lower maximum effect than the full agonist OxoM (Fig. 4B). As reported previously, OxoM is as efficacious as the endogenous agonist acetylcholine (Schrage et al., 2013). In the presence of FSK, the absolute level of OxoM-induced DMR nearly doubled relative to control conditions. Additionally, DMR-signals fanned out and partial agonism emerged for I-6-p and Pilo (Fig. 4D), whereas efficacy of the middle chain-elongated dualsteric ligand I-8-p did not differ from the efficacy of OxoM (Fig. 4E).

In MRC-5 cells, the test compounds revealed a similar pattern of full/partial-DMR-agonism as found with FSK-pretreated CHO-hM2 cells (Fig. 4, G and H). However, a switch from full to partial agonism was observed for I-8-p (Fig. 4H). Regarding the relative potency of each agonist, inflection points (EC50) were shifted to higher concentrations in the following order: CHO-hM2 < CHO-hM2 + FSK < MRC-5 (compare Fig. 4, C, F, and G and Supplemental Table 2). A decrease of potency typically occurs when an agonist switches from full to partial agonism, because a rightward shift of the inflection point indicates a decline of spare receptors in the corresponding systems (Kenakin, 2007; Rajagopal et al., 2011).

Agonist-Encoded Coupling Efficiency Dictates Context-Sensitivity of Action.

Experimental findings suggested that the level of intracellular cAMP is critical for dualsteric ligand-induced M2 receptor activation. Coupling efficiency (τ) determines whether and to which extent a ligand transforms from a full to a partial agonist upon a change of cellular context. Coupling efficiency is a measure that includes the efficacy of an agonist and the sensitivity of the system to agonism (Kenakin, 2007). To quantify coupling efficiency of the ligands applied in this study, data from DMR experiments (see above) were analyzed according to the operational model of agonism (Black and Leff, 1983) as described elsewhere (Rajagopal et al., 2011; Schrage et al., 2013).

In Fig. 5, agonist coupling efficiency (as log τ) is plotted against the cellular context (upper panel) and the maximal response (Emax) of the agonists in the tested system (lower panel). The intracellular cAMP content and the receptor densities differ among the tested cellular systems (Supplemental Table 1): CHO-hM2 - (low cAMP and high receptor density), CHO-hM2 + FSK - (high cAMP and high receptor density), MRC-5 - (high cAMP and low receptor density). Ranking of the applied test compounds according to their coupling efficiencies (Fig. 5, upper panel) yielded a rather stable pattern irrespective of the cellular system: OxoM > I-8-p > I-6-p ∼ Pilo. Under FSK pretreatment and even more so in the MRC-5 context, coupling efficiency was reduced in case of each agonist compared with CHO-hM2 control conditions (upper panel; represented by the vertical dashed lines).

Fig. 5.
Fig. 5.

Context-sensitive Gi/o-signaling of muscarinic M2 receptor agonists. Upper panel: Coupling efficiency of agonist-bound receptors to induce dynamic mass redistribution in living cells is reflected by log τ-values for CHO-hM2 cells under basal conditions, for CHO-hM2 after FSK pretreatment to elevate intracellular cAMP, and for MRC-5 fibroblasts with high endogenous cAMP and a minor receptor reserve. Test compounds are designated by the indicated color code. Inclined stippled lines visualize context-dependent shifts of respective test compound coupling efficiency. Coupling efficiencies (log τ) were calculated according to eq. 1 with KA fixed to the agonist’s dissociation binding constant (Supplemental Table 3). Lower panel: Maximum effects (Emax) of the depicted agonists under the respective conditions (CHO-hM2, CHO-hM2 + FSK, MRC-5) expressed as a fraction of the respective system’s maximum response set to Emax = 1. Emax values of the tested compounds were taken from DMR recordings (Fig. 4B: CHO-hM2, 4E: CHO-hM2 + FSK, 4H: MRC-5). Coupling efficiencies correspond to log τ-values shown in the upper panel. Emax and log τ-values are mean values ± S.E.M. The curve indicates the theoretical dependence of the maximum effect Emax from the coupling efficiency “log τ” calculated on the basis of eq. 2. Of note, steepness and thus context-sensitivity of agonist action is highest at the inflection point of the Emax/log τ-relationship. Indicated are mean ± S.E.M. of at least three independent experimental days; further details are listed in Supplemental Table 3.

Most importantly, the maximum effect (Fig. 5, lower panel) was differently affected depending on the individual starting level of coupling efficiency. In particular, Emax was hardly changed in case of OxoM, diminished with I-8-p only in the MRC-5 context, and profoundly reduced with Pilo and I-6-p, both in the CHO-hM2 + FSK and the MRC-5 context. Taken together, the dualsteric building-block design opens an avenue for graded context-sensitive GPCR activation.

Data Availability.

All relevant data are available from the authors.

Discussion

Muscarinic acetylcholine receptors regulate the activity of numerous fundamental central and peripheral functions. Therefore, muscarinic agonists and antagonists possess several therapeutic indications and are currently approved for some clinical conditions (for reviews, see Wess et al., 2007; Kruse et al., 2014). However, the clinical usefulness of these agents is limited by side effects caused by the nonselective activation of all or multiple muscarinic acetylcholine receptors. For example, therapeutic systemic administration of conventional muscarinic agonists is mainly hampered by central tremorgenic activity (Gomeza et al., 1999) and peripheral cardiac depression (Barocelli et al., 2001). It is well known that conventional full agonists evoke profound cardiovascular depression (Barocelli et al., 2001). It was therefore of particular interest that a novel type of GPCR activator was recently introduced (Antony et al., 2009). Regarding the chemical design, this type of activator is a dualsteric compound consisting of an orthosteric and an allosteric building block, both linked via a hydrocarbon chain (chemical structure, Supplemental Fig. 1).

Quantification of M2 receptor-mediated signaling in three different cellular systems (i.e., CHO-hM2 cells, hippocampal brain slices, and eCM) revealed that the intrinsic efficacy of I-6-p was highly system and assay dependent, whereas the efficacy of OxoM remained largely stable over all systems. In comparison with direct G protein activation quantified in [35S]GTPγS-binding experiments, the efficacy of I-6-p to inhibit neurotransmitter release from cholinergic nerve endings was relatively weak (see Fig. 1D), whereas spontaneous beating of eCM was completely suppressed (see Fig. 1F). This powerful effect in eCM might result from a high receptor reserve and/or a strong signal amplification in cardiomyocytes.

The term receptor reserve (“spare receptors”) addresses the fraction of cellular receptors that is not needed for a maximum response of the system (Kenakin, 2013). Of note, we showed that the intrinsic efficacy of the dualsteric agonist I-6-p, in contrast to the orthosteric agonist OxoM, is reduced under elevated intracellular cAMP in primary cardiomyocytes (Fig. 2 and Supplemental Fig. 2). Furthermore, I-6-p induced less cardiac depression in vivo compared with OxoM and did not induce cardiac arrest (Fig. 3). This good cardiac tolerability of I-6-p makes a clinical application of dualsteric muscarinic agonists more likely than initially expected.

One potential indication for dualsteric ligands may be the treatment of pain disorders (Matera et al., 2014). Context-sensitive signaling offers a mechanistic explanation as to why the cardiovascular tolerability of I-6-p by far exceeded the tolerability of OxoM in anesthetized mice. As described by various researchers (Gehrmann et al., 2000; Rose et al., 2007; El Beheiry et al., 2011; Mabe and Hoover, 2011), application of the β-blocker metoprolol revealed a significant β-adrenergic tone acting on the mouse heart in our experiments. In consequence, β1-adrenergic signaling stimulates basal formation of intracellular cAMP in cardiomyocytes. In contrast, muscarinic antagonists have no significant effect on basal HR in mice (Wickman et al., 1998; Mabe and Hoover, 2011).

Ranking of the applied test compounds according to their coupling efficiencies shows a stable pattern irrespective of the cellular system: OxoM > I-8-p > I-6-p ∼ Pilo (Fig. 5 and Supplemental Table 3). Agonists with weak coupling efficiency have to activate more receptors for a given response than efficient activators. Thus, the system has a lower receptor reserve for such agonists. In particular, dualsteric (orthosteric/allosteric) agonists lose their receptor reserve under conditions of high intracellular cAMP (compare Fig. 4B, I-6-p, with Fig. 4E). This is not due to a reduction in absolute receptor number but due to an increased number of receptors which have to be activated to induce the maximum response of the system. If the intracellular cAMP level is increased, a stronger M2-signaling is needed to induce a similar effect than in systems with low cAMP levels. If high intracellular cAMP levels are combined with a low receptor density, as it is the case for MRC-5 cells, a switch from full to partial agonism is also detectable for I-8-p (compare Fig. 4E, I-8-p, with Fig. 4H). If receptor reserve is exhausted, partial agonism emerges.

In case of CHO-hM2, the sensitivity of the system to agonism was diminished by an increase of intracellular cAMP induced by FSK. As a consequence, compounds with lowest coupling efficiency in CHO-hM2 were the first that switched from full to partial agonism after FSK-pretreatment (Fig. 5 lower panel: half-filled circles of Pilo in gray and I-6-p in red). In case of MRC-5, besides high intracellular cAMP, additionally receptor density was reduced (Supplemental Table 1). As a consequence, not only Pilo and I-6-p but additionally I-8-p became a partial agonist (Fig. 4 lower panel, open circles of Pilo in gray, I-6-p in red, and I-8-p in green).

Taken together, we suggest the following mechanism of context-dependent coupling efficiency. Under conditions of low cAMP levels, a small fraction of activated M2 receptors will suffice to counterbalance cAMP-mediated actions: with high “M2 receptor reserve,” even M2 agonists with low signaling efficiency such as Pilo are able to generate a maximum muscarinic effect. With increasing cAMP levels to be counteracted, an increasing fraction of M2 receptors needs to be activated: reduced sensitivity of the system to agonism or, in other words, less “receptor reserve.” This mechanism can also be described as functional antagonism, if cAMP is increased due to adrenergic counterregulation (Pyne et al., 1992). A compound with less intrinsic efficacy for receptor activation (i.e., lower coupling efficiency) such as I-6-p and Pilo then fails to induce full agonism. Pilo behaved similar to I-6-p but is able to pass the blood–brain barrier and may exert central nervous side effects such as seizures (Bymaster et al., 2003).

Furthermore, the design of partial agonists in the past seemed to be fairly random. Now, the novel dualsteric concept allows the design of tailor-made agents for the exploitation of context-dependent signaling via linkage of orthosteric/allosteric building blocks and variation of the length of the hydrocarbon middle chain (Antony et al., 2009; Bock et al., 2012; Bock et al., 2014).

Irrespective of specific therapeutic perspectives, our study shows that pharmacologic modulators can be designed which are sensitive to the functional context of a target. Commonly, pharmacologically targeted receptors such as the muscarinic acetylcholine receptor species are classified into subtypes depending on differences in structure (for reviews, see Haga, 2013; Kruse et al., 2014). Exploitation of context-dependent signaling expands the repertoire for selective pharmacologic intervention in that one and the same receptor subtype may reveal different pharmacologic phenotypes, depending on the functional state of the cell population that harbors the receptor.

In conclusion, dualsteric (orthosteric/allosteric) targeting of muscarinic acetylcholine receptors generates new forms of pharmacologic modulation and selectivity. Dualsteric agents were first characterized as signaling pathway-selective (“biased”) activators of muscarinic receptors (Antony et al., 2009; Bock et al., 2012). Very recently, the dualsteric design concept was exploited to achieve the first designed dynamic partial agonists in the field of G protein-coupled receptors (Bock et al., 2014). Here we introduce context-sensitive signaling as an important mechanism of pharmacologic selectivity for clinical application, and we show that capacity to monitor cell function in real time both in vitro and in vivo was key to discovery of this.

Acknowledgments

The authors thank Corning Inc. for their support on the Epic system.

Authorship Contributions

Participated in research design: Seemann, Wenzel, Schrage, Sasse, Holzgrabe, Schlicker, Kostenis, Fleischmann, Mohr.

Conducted experiments: Seemann, Wenzel, Etscheid, Bödefeld, Bartol.

Contributed new agents and analytic tools: Warnken, Klöckner, Holzgrabe, DeAmici, Schlicker, Racké, Kostenis, Meyer, Fleischmann, Mohr.

Performed data analysis: Seemann, Wenzel, Schrage, Mohr

Wrote or contributed to the writing of the manuscript: Seemann, Wenzel, Schrage, Etscheid, Warnken, Holzgrabe, DeAmici, Schlicker, Racké, Kostenis, Meyer, Fleischmann, Mohr.

Footnotes

    • Received August 9, 2016.
    • Accepted November 10, 2016.

  • ↵1 Current affiliation: CNS Discovery Research, UCB Pharma, Chemin du Foriest R4, B-1420 Braine-l'Alleud, Belgium.

  • ↵2 Current affiliation: Clinical Study Core Unit, Study Center Bonn (SZB), Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Bonn, Germany.

  • This work was supported in part by the North-Rhine Westphalia International Graduate Research School Biotech-Pharma at the University of Bonn (to W.K.S.).

  • Part of this work was previously presented at the following workshop: Seemann W, Wenzel D, Sasse P, Warnken M, Kostenis E, Racké K, Fleischmann B, and Mohr K (2011) Dualsteric GPCR-targeting: whole cell response to M2 receptor activation is cell type-dependent. [Naunyn-Schmiedebergs Arch Pharmacol 381 (Suppl 1).] Deutsche Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie e.V. 77th Annual Meeting; 2011 Mar 30–April 1; Frankfurt a. M., Germany.

  • dx.doi.org/10.1124/jpet.116.237149.

  • ↵Embedded ImageThis article has supplemental material available at jpet.aspetjournals.org.

Abbreviations

bpm
beats per minute
CHO-hM2
Chinese hamster ovary cells stably transfected with the human M2 receptor
DMR
dynamic mass redistribution
Emax
maximal inducible effect
eCM
embryonic cardiomyocytes
FSK
forskolin
Gi/o
inhibitory G protein
Gs
stimulatory G protein
GPCR
G protein-coupled receptor
HBSS
Hank’s balanced salt solution
[3H]NMS
[3H]N-methylscopolamine bromide
HR
heart rate
IBMX
isobutylmethylxanthine
IC50
inflection point of sigmoidal binding curves
I-6-p
iper-6-phth
I-8-p
iper-8-phth
ISO
isoprenaline
KA
apparent binding constant
LVSP
left ventricular systolic pressure
MRC-5
human lung fibroblasts
OxoM
oxotremorine M
Pilo
pilocarpine
tau (τ)
operational efficacy

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Research ArticleCellular and Molecular

Context-Sensitive Agonism through a GPCR

Wiebke K. Seemann, Daniela Wenzel, Ramona Schrage, Justine Etscheid, Theresa Bödefeld, Anna Bartol, Mareille Warnken, Philipp Sasse, Jessica Klöckner, Ulrike Holzgrabe, Marco DeAmici, Eberhard Schlicker, Kurt Racké, Evi Kostenis, Rainer Meyer, Bernd K. Fleischmann and Klaus Mohr
Journal of Pharmacology and Experimental Therapeutics February 1, 2017, 360 (2) 289-299; DOI: https://doi.org/10.1124/jpet.116.237149
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