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Bitopic Binding Mode of an M1 Muscarinic Acetylcholine Receptor Agonist Associated with Adverse Clinical Trial Outcomes

The realization of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer’s disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one), described previously as a potent M1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that “pure” positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses.


Introduction
The M 1 muscarinic acetylcholine receptor (mAChR) has emerged as an attractive molecular target to overcome cognitive decline associated with cholinergic degeneration in Alzheimer disease (AD) (Anagnostaras et al., 2003). Activation of M 1 mAChRs, which are abundantly expressed in the amygdala, cerebral cortex, striatum, and hippocampus (Buckley et al., 1988;Levey et al., 1995), has been reported to rescue learning and memory deficits associated with neurodegeneration in a number of mouse models (Lange et al., 2015;Puri et al., 2015;Vardigan et al., 2015;Bradley et al., 2017). Translating these promising findings to successful clinical candidates has, however, been challenging due to adverse effects associated with a lack of selectivity of orthosteric M 1 mAChR agonists. This is exemplified by the M 1/ M 4 -preferring mAChR agonist xanomeline, which significantly improved cognitive function in patients with AD (Bodick et al., 1997) but ultimately failed due to adverse effects attributed to activation of peripheral cholinergic signaling likely through M 2 and M 3 mAChRs (Langmead et al., 2008).
There is, therefore, an urgent need to develop novel approaches to build selectivity into M 1 mAChR ligands. Two related approaches to this problem have been taken: 1) the development of positive allosteric modulators (PAMs) that bind to a site that is topographically distinct from that of the endogenous ligand acetylcholine (ACh) (May et al., 2007) and 2) development of a newer generation of more selective agonists (whose mechanism of action is not always well defined) (Langmead et al., 2008). Allosteric modulators enhance ACh binding and/or signaling, the magnitude of which can vary with different degrees of positive cooperativity (Langmead and Christopoulos, 2006;May et al., 2007;Conn et al., 2009). They can also possess direct allosteric agonist activity (intrinsic activity). M 1 mAChR allosteric modulators with high functional selectivity over other muscarinic receptor subtypes (i.e., M 2 -M 5 mAChRs) have been reported to reverse phenotypes associated with neurodegenerative disease (Ma et al., 2009;Puri et al., 2015;Vardigan et al., 2015;Bradley et al., 2017) while showing no adverse side effects in animal models (Bradley et al., 2017).
Less well described in the literature is the development of a new generation of M 1 mAChR agonists, which have variously been described as "ectopic" (Spalding et al., 2002), "allosteric" (Langmead and Christopoulos, 2006;Jones et al., 2008;Budzik et al., 2010), "atypical" (Lebon et al., 2009), and "bitopic" (Keov et al., 2011). With but a few exceptions , this broad nomenclature reflects a paucity in understanding of mechanism(s) of action; it is not clear whether some of these ligands exert true allosteric agonism (i.e., bind solely to an allosteric binding site to activate the receptor), are subtype-selective orthosteric agonists, or represent a hybrid of the two (with a pharmacophore that engages both orthosteric and allosteric binding pockets). GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one) has been described as an allosteric M 1 mAChR agonist; it was identified from a series of benzimidazolones and reversed scopolamine-induced amnesia in rodents (Budzik et al., 2010) and had positive effects on cognitive function in humans. Unfortunately, it also induced gastrointestinal adverse effects, consistent with activation of peripheral mAChRs (Nathan et al., 2013).
Here we conduct a comprehensive pharmacological analysis of GSK1034702 and show that this molecule is not a purely allosteric ligand as previously suggested. Radioligand binding, using [ 3 H]-N-methylscopolamine (NMS) and [ 3 H]-GSK1034702, and functional inositol phosphate (IP) accumulation studies at the muscarinic M 1 receptor reveal that GSK1034702 interacts with the orthosteric ACh binding site and likely, concomitantly, with an allosteric binding site. Importantly, this bitopic mode of action is able to mediate beneficial effects on learning and memory but the lower degree of selectivity of GSK1034702 compared with ligands that engage solely with an allosteric binding site, together with (A-C) Effects of xanomeline (1, 3, 10, and 30 mg/kg) (A), GSK1034702 (0.3, 1, 3, 10, and 30 mg/kg) (B), and TBPB (1, 3, 10, and 30 mg/kg) (C) on scopolamine (1.5 mg/kg)-induced impairments in contextual fear conditioning (inset chemical structures of compounds). Data are expressed as the means 6 S.E.M. of eight or more mice per group. Data were analyzed using a one-way analysis of variance with the Tukey multiple comparison test. *P , 0.05; ** P , 0.01; ***P , 0.001 vs. vehicle alone; # P , 0.05 vs. 1.5 mg/kg scopolamine. (D-F) Unbound concentrations (Log M) of xanomeline (D), GSK1034702 (E), and TBPB (F) measured in plasma or brain samples 30 minutes after intraperitoneal injection with increasing concentrations of respective compound. Data are expressed as the means 6 S.E.M. of three mice per concentration. the intrinsic agonist activity reported here, may account for the adverse effects observed with this molecule in the clinic.
Mouse Maintenance and Diet. All experiments were performed under a project license from the British Home Office (United Kingdom) under the Animals (Scientific Procedures) Act of 1986. C57Bl/6J mice used in this study were purchased from Charles River (Margate, UK). Mice were fed ad libitum with a standard mouse chow and were maintained within the animal facility at least 1 week prior to experiments.
Fear Conditioning. C57Bl/6J male mice (aged 8-12 weeks) were acclimatized to the behavioral testing suite at least 2 hours prior to the test. Mice were injected (intraperitoneally) with vehicle (5% glucose) or scopolamine (1.5 mg/kg) alone or in combination with xanomeline, GSK1034702, or TBPB 30 minutes prior to training. Mice were placed in the conditioning chamber (ANY-maze Fear Conditioning System; Stoelting, Dublin, Ireland); after a 2-minute adaptation period, they received three tone/foot shock pairings where the foot shock (unconditioned stimulus; 2 seconds; 0.4 mA) always co-terminated with a tone (conditioned stimulus; 2.8 kH; 85 dB; 30 seconds). The conditioned stimulus-unconditioned stimulus pairings were separated by 1-minute intervals. After the mice completed training, they remained in the conditioning chamber for 1 minute and were then returned to their home cages. The next day, the mice were placed back in the conditioning chamber, and time spent immobile was recorded for 3 minutes to assess context-dependent learning. Data were analyzed using ANY-maze software (Stoelting).
Mouse Pharmacokinetics. Pharmacokinetic analyses were conducted as previously described (Witkin et al., 2017). Compounds were administered via intraperitoneal injection (in 5% glucose) 30 minutes prior to blood collection. Mice were anesthetized with 3% isoflurane (2 l/min), and blood was collected by cardiac puncture of the left ventricle. Blood was immediately transferred to EDTA tubes and centrifuged at 1000g for 10 minutes at 4°C; the supernatant was collected and frozen. Brains from each mouse were also dissected and snap-frozen on dry ice.
Brain samples were homogenized in three volumes of methanol/water [1:4 (v/v)] by weight. A 25-ml aliquot of each study sample, calibration standard, and control sample was added to a 96-well plate and mixed with 180 ml acetonitrile/methanol [1:1 (v/v)] containing internal standard. The samples were subsequently centrifuged, and the resulting supernatants were diluted 12.5-fold with methanol/water [1:1 (v/v)] prior to analyzing 10-ml aliquots by liquid chromatography-tandem mass spectrometry as previously described (Bradley et al., 2017).
Equilibrium [ 3 H]-NMS Binding. Chinese hamster ovary (CHO) Flp-In cells expressing the wild-type M 1 mAChR (B max 5 870 fmol/mg) or the M 1 designer receptor exclusively activated by designer drug (DREADD) (B max 5 2400 fmol/mg) were plated at 7500 cells/well in clear 96-well plates and grown to confluence. Prior to the experiment, cells were washed with 100 ml phosphate-buffered saline. Increasing concentrations of test compounds and an approximate equilibrium dissociation constant (K D ) concentration of [ 3 H]-NMS (K D (nM) for [ 3 H]-NMS binding to CHO Flp-In cells expressing wild-type M 1 mAChR or M 1 DREADD were 0.37 6 0.10 and 18.70 6 3.49 (n 5 3) were incubated with cells overnight at room temperature in a final volume of 100 ml binding buffer of the following composition: 110 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl 2 , 1 mM MgSO 4 , 25 mM glucose, 20 mM HEPES, and 58 mM sucrose, pH 7.4. Binding was terminated by rapid aspiration followed by two washes with 200 ml ice-cold 0.9% NaCl. Bound radioactivity was determined by liquid scintillation (Ultima Gold; PerkinElmer, Boston, MA) counting. Nonspecific binding was determined in the presence of 10 mM atropine.
For competition binding experiments at M 1 , M 2 , M 3 , M 4 , and M 5 mAChRs, CHO membranes were purchased from PerkinElmer. All experiments were performed in assay buffer of the following composition: 20 mM HEPES, 100 mM NaCl, and 10 mM MgCl 2 , pH 7.5, and used 10 mg protein/well in a total assay volume of 1 ml using deep well blocks. CHO cell membranes overexpressing human mAChR M 1 -M 5 subtypes were incubated with a concentration of [ 3 H]-NMS that was close to the calculated K D for each receptor (M 1 : 200 pM, K D 5 196 pM; M 2 : 700 pM, K D 5 769 pM; M 3 : 700 pM, K D 5 642 pM; M 4 : 200 pM, K D 143 pM; M 5 : 400 pM, K D 5 410 pM), in the presence or absence of 11 different concentrations of compound. Nonspecific binding was determined in the presence of 10 mM atropine. All assay incubations were initiated by the addition of membrane suspensions and deep well blocks were shaken for 5 minutes to ensure complete mixing. Incubation was then carried out for 2 hours at 21°C. Binding reactions were terminated by rapid filtration through GF/A filters (PerkinElmer) presoaked with 0.5% (w/v) polyethylenimine for 1 hour. Filters were then washed three times with 1 ml ice-cold assay buffer. Dried filters were counted with Meltilex A scintillant using a Trilux 1450 scintillation counter (PerkinElmer). The specific bound counts (in disintegrations per minute) were expressed as a percentage of the maximal binding observed in the absence of test compound (total) and nonspecific binding determined in the presence of 10 mM atropine.
Kinetic [    6.5 6 0.2 3 TBPB 6.8 6 0.1 3 Novel Bitopic Mode of an M 1 Agonist by the addition of atropine (10 mM) alone or atropine (10 mM) plus 100 mM GSK1034702 added in a reverse time course protocol. Reactions were terminated by rapid filtration onto GF/B filter paper (Whatman, Maidstone, UK) and three washes with 3 ml ice-cold 0.9% NaCl using a Brandel harvester (M-24TI; Brandel, Fort Lauderdale, FL). Membrane bound radioactivity was determined by liquid scintillation (Ultima Gold; PerkinElmer) counting. IP-One Accumulation Assay. Stimulation of IP accumulation was determined using the Cisbio IP-One Gq assay kit per the manufacturer's instructions. For agonist concentration-response curves, agonists (2Â concentrated) were added to 384-well white ProxiPlates (PerkinElmer) in 7 ml stimulation buffer. CHO Flp-In cells stably expressing the human M 1 mAChR were grown to confluence in T75 cell culture flasks at 37°C. Cells were washed with warm phosphate-buffered saline and detached using phosphate-buffered saline with 0.1 M EDTA. Detached cells were centrifuged at 1000g and the cells were resuspended in stimulation buffer. Seven microliters of this cell suspension (1.43 Â 10 6 cells/ml) was added to each well, and cells were stimulated for 45 minutes at 37°C.
For functional interaction studies, CHO Flp-In cells stably expressing the human M 1 mAChR were seeded at 5000 cells/well in 384-well white ProxiPlates. Experiments were conducted 48 hours later. Cells were washed once with 50 ml phosphate-buffered saline and then incubated in F12 media containing phenoxybenzamine (where applicable) at 37°C for 30 minutes. Cells were washed with 50 ml phosphatebuffered saline and incubated in stimulation buffer containing agonists in a final volume of 14 ml for 45 minutes at 37°C.
All IP-One stimulations were terminated by the addition of 3 ml/well IP 1 -d2 solution, followed by 3 ml/well anti-IP 1 -cryptate solution and incubation for 1 hour at room temperature with shaking. Fluorescence emission at two different wavelengths (665 and 620 nm) was measured with a PHERAstar plate reader (BMG Labtech, Ortenberg, Germany).
ERK1/2 Phosphorylation. Stimulation of phospho-ERK1/2 (Thr 202/Tyr 204) was determined using the Cisbio Phospho-ERK Cellular Assay Kit. CHO Flp-In cells stably expressing the human M 1 receptor were seeded onto transparent 96-well plates at 20,000 cells/well and grown to confluence. Cells were serum starved overnight prior to the experiment. Prior to the stimulations, cells were washed with 100 ml phosphate-buffered saline and then incubated in serum free F12 medium at 37°C. Cells were stimulated with ligands for 5 minutes at 37°C in a final volume of 200 ml. The stimulations were terminated by rapid aspiration and addition of 50 ml lysis buffer supplemented with blocking reagent per the manufacturer's instructions, followed by gentle agitation at room temperature for 30 minutes. Subsequently, 16 ml of this lysate was transferred to a 384-well white ProxiPlate (PerkinElmer) and incubated with 4 ml premixed antibody solution for 2 hours at room temperature. Fluorescence emission at two different wavelengths (665 and 620 nm) was measured with a PHERAstar plate reader (BMG Labtech).
Native  (DeLapp et al., 1999). Native rat membranes were prepared as follows. All procedures were performed at 4°C. Ten to fifteen milliliters of sucrose buffer (10 mM HEPES, 1 mM EGTA, 1 mM dithiothreitol, 10% sucrose, and one tablet/50 ml Complete Protease Inhibitor Cocktail, pH 7.4) was added to each tissue sample and homogenized for 10 strokes using an electric IKA RW20 homogenizer (800 rpm) (IKA, Staufen, Germany) with glass homogenizer. The homogenate was centrifuged at 1000g for 10 minutes at 4°C and the supernatant collected. The pellet was rehomogenized and centrifuged again as above and the supernatant pooled and centrifuged at 11,000g for 20 minutes at 4°C. The resulting pellet was suspended in 40 ml final storage buffer (10 mM HEPES, 1 mM EGTA, 1 mM MgCl 2 , and 1 mM dithiothreitol, pH 7.4) and centrifuged at 27,000g for 20 minutes at 4°C. The supernatant was removed and the final pellet was suspended in 2 ml final storage buffer. The protein concentration was measured using the Bradford method (Coomassie Plus, Bio-Rad protein assay kit; Bio-Rad, Hercules, CA) with bovine gamma globulin standards. Samples were aliquoted and stored at 280°C. Membrane aliquots (15 mg/well) were then incubated with test compound and GTPɣ[ 35 S] (500 pM/well) for 30 minutes. Labeled membranes were then solubilized with 0.27% Nonidet P-40 plus Gqa antibody (E17; Santa Cruz Biotechnology, Dallas, TX) at a final dilution of 1:200 and 1.25 mg/well anti-rabbit scintillation proximity beads. Plates were left to incubate for 3 hours and then centrifuged for 10 minutes at 2000 rpm. Plates were counted for 1 minute/well using a Wallac MicroBeta Trilux scintillation counter (Perki-nElmer). All incubations took place at room temperature in GTPbinding assay buffer of the following composition: 20 mM HEPES, 100 mM NaCl, and 5 mM MgCl 2 , pH 7.5. Data were converted to the percentage of response compared with oxotremorine-M (100 mM) or the percentage over basal and EC 50 values were generated (fourparameter logistic curve) using GraphPad Prism 6 software (Graph-Pad Software Inc., La Jolla, CA).

648
[ 3 H]-GSK1034702 Binding. [ 3 H]-GSK1034702 (specific activity 3.65 TBq/mmol) was synthesized by direct titration on Pd Black (performed by Quotient Bioresearch, Manchester, UK). All experiments were performed in assay buffer of the following composition: 20 mM HEPES, 100 mM NaCl, and 10 mM MgCl 2 , pH 7.5, and used 50 mg protein/well in a total assay volume of 250 ml.
CHO cell membranes overexpressing human M 1 mAChR (PerkinElmer) were incubated with [ 3 H]-GSK1034702 (20 nM) in the presence of 11 concentrations of test compound. All assay incubations were initiated by the addition of membrane suspensions. Incubation was then carried out for 4 hours at 21°C. Binding reactions were terminated by rapid filtration through GF/A filters (PerkinElmer) presoaked with 0.5% (w/v) polyethylenimine for 1 hour. Filters were then washed six times with 1 ml ice-cold assay buffer. Dried filters were counted using Meltilex A scintillant using a Trilux 1450 scintillation counter (PerkinElmer). The specific bound counts (in disintegrations per minute) were expressed as a percentage of the maximal binding observed in the absence of test compound (total) and nonspecific binding determined in the presence of 100 mM nonradiolabeled GSK1034702.
Isolated Rat Atria and Ileum Contraction Experiments. Adult Wistar rats were humanely euthanized and the left atria and the ileum were placed in an organ bath containing oxygenated Tyrode solution. Measurement of negative inotropic responses in atria or contraction of ileum was performed as described by Lambrecht et al. (1989). For atrial responses, ligands were administered for 5 minutes at 32°C in McEwen's buffer (pH 7.4) in a bath volume of 10 ml. Agonist responses were measured as negative inotropy relative to 1 mM methacholine, and antagonism was measured as inhibition of methacholine (1 mM)-induced negative ionotropic response. For rat ileum experiments, ligands were administered for 5 minutes at 32°C in Krebs' buffer (pH 7.4) in a bath volume of 10 ml. Contraction of ileum as a percentage of methacholine-induced contraction was measured, and antagonistic effects were measured by inhibition of methacholineinduced responses.
Data Analysis. Inhibition binding data were curve-fit using Graph-Pad Prism 6 to derive the potency (IC 50 ) of the test compound. The equilibrium dissociation constant (K I ) of the test compound was then calculated with the Cheng-Prusoff equation: K I 5 IC 50 /[1 1 ([L]/K D )] using the K D value derived separately from saturation binding studies.
Functional concentration-response curves were fitted according to a four-parameter logistic equation (to determine minimum and maximum asymptotes, LogEC 50 , and slope; GraphPad Prism 6). For ACh curves in the presence of multiple concentrations of GSK1034702 or TBPB (after phenoxybenzamine treatment), the following form of the Gaddum and Schild equations was applied globally to the datasets: [ 3 H]-NMS binding interaction studies with benzyl quinolone carboxylic acid (BQCA) were fitted to an allosteric ternary complex model (Leach et al., 2010): where B max represents the total number of receptors; [A], [B], and [I] are concentrations of radioligand, allosteric modulator, and orthosteric ligand, respectively; and K A , K B , and K I represent equilibrium dissociation constants of radioligand, allosteric modulator, and orthosteric ligand, respectively. a9 and a are the binding cooperativities between the allosteric modulator and radioligand and the allosteric modulator and the orthosteric ligand, respectively. An a value of .1 denotes positive cooperativity, a value of ,1 denotes negative cooperativity, and a value of 1 denotes neutral cooperativity of binding.
To assess agonist bias, the same concentration-response curves were analyzed according to a modified form of the operational model of agonism, recast to directly yield a transduction ratio (Log where basal represents the response in the absence of agonist, E m represents the maximal response of the assay system, K A represents the equilibrium dissociation constant of the agonist, [A] represents the concentration of agonist, t is an index of the coupling efficiency (or efficacy) of the agonist, and n is the slope of the transducer function linking agonist occupancy to response. For the analysis, all families of agonist curves at each pathway were globally fitted to the model with the parameters, basal, E m , and n shared between all agonists. For full agonists, the LogK A was constrained to a value of zero, whereas for  Novel Bitopic Mode of an M 1 Agonist partial agonists this was directly estimated by the curve fitting procedure; the Log(t/K A ) parameter was estimated as a unique measure of activity for each agonist. Agonist bias factors (10^DDLog[t/K A ]) were calculated as described in van der Westhuizen et al. (2014)).

Results
GSK1034702, TBPB, and Xanomeline Reverse Scopolamine-Induced Deficits in Fear Conditioning. It is well established that muscarinic receptor agonists and PAMs can reverse deficits in learning and memory induced by the administration of a broad-spectrum muscarinic antagonist such as scopolamine (Young et al., 1995;Ma et al., 2009). Here, doses of scopolamine above 1 mg/kg administered to mice 30 minutes prior to fear conditioning training were sufficient to induce a significant reduction in contextual fear conditioning learning and memory (Supplemental Fig. 1). The effects of muscarinic receptor agonists on this deficit were tested by the coadministration of scopolamine (1.5 mg/kg) with escalating intraperitoneal doses of xanomeline (Fig. 1A), GSK1034702 (Fig. 1B), or TBPB (Fig. 1C). All three agents significantly improved learning and memory compared with vehicle controls (5% glucose solution in double distilled water) (P , 0.05 vs. administration of 1.5 mg/kg scopolamine alone; one-way analysis of variance with the Tukey multiple comparisons test). Free brain concentrations of xanomeline and GSK1034702 determined 30 minutes after administration were seen to increase linearly with escalating doses (Fig. 1, D and E). In contrast, the effects of these compounds on learning and memory were bell shaped, with lower doses improving learning and memory and high doses showing reduced effect (Fig. 1, A and B). This bell-shaped response is characteristic of procognitive agents. Interestingly, brain exposure of TBPB could not be increased beyond that observed at 10 mg/kg, remaining relatively low even after intraperitoneal injection of higher doses (Fig. 1F). This resulted in TBPB effects on learning and memory being similar at both low and high-administered doses with no evidence of a bell-shaped dose response (Fig. 1C).
GSK1034702 and TBPB Interact Competitively with [ 3 H]-NMS at M 1 mAChRs. GSK1034702 and TBPB have previously been described as allosteric agonists of the M 1 mAChR (Jones et al., 2008;Budzik et al., 2010;Nathan et al., 2013). To test this assertion, [ 3 H]-NMS binding studies were conducted on monolayers of CHO Flp-In cells expressing the human M 1 mAChR to determine the nature of their interaction at the receptor. Both GSK1034702 and TBPB fully inhibited binding of [ 3 H]-NMS (0.5 nM) to M 1 mAChRs, with estimated pK i values of 6.5 6 0.2 and 6.8 6 0.1, respectively, and in a similar manner to the orthosteric agonist, xanomeline ( Fig. 2; Table 1). These data suggest that GSK1034702, in contrast to the allosteric mechanism of action previously reported, binds instead in a competitive manner consistent with interaction at the orthosteric site of the M 1 mAChR.
GSK1034702 and TBPB Do Not Alter [ 3 H]-NMS Dissociation Kinetics. As allosteric ligands with high negative cooperativity can still fully inhibit orthosteric ligand binding, kinetic binding experiments were performed to probe any allosteric interactions of GSK1034702 or TBPB with the M 1 mAChR. Membranes of CHO Flp-In cells expressing the human M 1 mAChR were pre-equilibrated with [ 3 H]-NMS, and bound radioligand dissociated from the receptor with atropine (10 mM) with a rate constant of k off 5 0.188 6 0.009 minute 21 . The presence of either GSK1034702 (10 mM) or TBPB (10 mM) had no effect on the [ 3 H]-NMS dissociation rate (Fig. 3). These data further argue against an allosteric mode of action of GSK1034702 at M 1 mAChRs as previously reported (Nathan et al., 2013).
To verify the mechanism of action, we performed receptor alkylation experiments with the orthosteric site covalent binder, phenoxybenzamine, to deplete the level of available  and functional muscarinic receptors. Phenoxybenzamine, at a concentration of 3 mM (for 30 minutes), reduced the functional human M 1 mAChR population in CHO Flp-In cells by approximately 80%, to an expression level where GSK1034702 had no agonist effect in an IP 1 accumulation assay but where ACh still yielded a response (Supplemental Fig. 2, A and B). Under these conditions, establishing whether GSK1034702 and TBPB acted as competitive antagonists with respect to ACh would verify the interaction of these compounds with the orthosteric site.
In phenoxybenzamine-treated cells, GSK1034702 caused a concentration-dependent, parallel rightward shift in AChstimulated IP 1 accumulation (Fig. 5A) consistent with a competitive antagonist. This effect was similar to that of TBPB (Fig. 5B), which was reported previously to act as a competitive antagonist in a similar preparation (Keov et al., 2013). Analysis of these data using a modified form of the Gaddum and Schild equations yielded Schild slopes approximating to unity and pA 2 values of 6.2 6 0.2 and 7.0 6 0.1 for GSK1034702 and TBPB antagonism of ACh-stimulated responses, respectively (Table 3).
The Prototypical PAM, BQCA, Potentiates ACh, But Not GSK1034702 Affinity. Having established that GSK1034702 interacts competitively with ACh at the orthosteric site, radioligand binding experiments were designed to establish whether the mode of GSK1034702 binding at the orthosteric pocket was equivalent to ACh. In these studies, the potentiation of orthosteric agonist binding by a PAM was used to probe the nature of GSK1034702 and TBPB binding. BQCA, a PAM selective for the M 1 mAChR, has previously been shown to potentiate ACh affinity by approximately 100-fold (Ma et al., 2009;Butcher et al., 2016). Consistent with these previous studies, we show here that BQCA potentiates the AChmediated displacement of [ 3 H]-NMS, thereby demonstrating positive cooperativity for ACh binding of approximately 35-fold, consistent with previous reports of modulation according to a two-state model (Canals et al., 2012;Fig. 6A). Such actions would predict a similar (if less substantial) effect on GSK1034702 or TBPB. However, the displacement of [ 3 H]-NMS by GSK1034702 (Fig. 6B) or TBPB (Fig. 6C) was not modulated by BQCA. These data demonstrate the probe dependency of BQCA and indicate that either: 1) there is neutral cooperativity between BQCA and GSK1034702/TBPB, or 2) that the binding site of GSK1034702 or TBPB simultaneously overlaps with those of both ACh and BQCA.
[ 3 H]-GSK1034702 Binding Studies Further Confirm Novel Orthosteric Binding Pose. To further characterize the binding site of GSK1034702 at the M 1 mAChR, we generated a radiolabeled version of the GSK1034702 compound ([ 3 H]-GSK1034702) and conducted binding interaction experiments in membranes expressing the human M 1 mAChR. [ 3 H]-GSK1034702 bound in a monophasic manner with moderate affinity (K D 5 550 nM; B max 5 2.6 pmol/mg protein) (Supplemental Fig. 3, A-C). Membranes were incubated with 20 nM [ 3 H]-GSK1034702 in the absence and presence of increasing concentrations of unlabeled GSK1034702, TBPB, ACh, and xanomeline (Fig. 7). GSK1034702 and TBPB fully displaced specific [ 3 H]-GSK1034702 binding to the M 1 mAChR in a monophasic manner, whereas ACh and xanomeline only partially displaced [ 3 H]-GSK1034702 binding. These data indicate either an allosteric interaction between GSK1034702 and ACh/xanomeline or a bitopic mechanism consistent with GSK1034702 spanning a binding pocket at the M 1 mAChR partially shared with that of ACh.
DREADD Pharmacology Confirms an Atypical Mechanism of Action of GSK1034702. By introducing point mutations (Y106C and A196G) into the orthosteric binding pocket of the M 1 mAChR, an M 1 DREADD mutant is created (Abdul-Ridha et al., 2013) that displays reduced responsiveness to ACh but instead is activated by clozapine-N-oxide, a ligand that shows little activity at the wild-type M 1 mAChR. We investigated the ability of GSK1034702 to interact with the M 1 DREADD by conducting [ 3 H]-NMS binding, functional IP 1 accumulation, and ERK1/2 phosphorylation studies in CHO Flp-In cells expressing the humanized M 1 DREADD (Figs. 8 and 9; Supplemental Tables 1 and 2). The affinity of GSK1034702 for the M 1 DREADD was not significantly different from the affinity for binding at the  wild-type M 1 mAChR (pK i 5 6.5 6 0.2 and 6.0 6 0.2 for the wild type and DREADD, respectively; Fig. 8). In addition, as reported previously (Armbruster et al., 2007;Abdul-Ridha et al., 2013), M 1 mAChR orthosteric agonists, ACh and xanomeline, showed a significant reduction in potency at the M 1 DREADD (Fig. 9). In contrast, GSK1034702 activated the M 1 DREADD with comparable potency and efficacy compared with its activity at the wild-type receptor. Similar results were obtained with TBPB, confirming previous observations for this compound (Abdul-Ridha et al., 2014) (Fig. 9, D and E). Furthermore, we assessed the ability of ACh and GSK1034702 to stimulate phosphorylation of the M 1 mAChR at serine 228 using a phosphorylation-specific antibody (Butcher et al., 2016). Both ACh and GSK1034702 stimulated a concentration-dependent increase in pSer228 immunoreactivity at the wild-type receptor (Fig. 10). The potency of GSK1034702 to stimulate phosphorylation at serine 228 was unchanged at the M 1 DREADD, whereas ACh failed to stimulate a response. These data support the notion that GSK1034702 has a distinct binding mode at the orthosteric site from that of ACh.
GSK1034702 and TBPB Are Differentially Biased Agonists at M 1 mAChR. Convergent evidence from radioligand binding and functional studies (vide supra and Supplemental Fig. 4; Supplemental Table 3) suggests that GSK1034702 and TBPB bind to the orthosteric site of the M 1 mAChR, but with an orientation or pose that distinguishes them from ACh and xanomeline. To further interrogate their pharmacology, we assessed their ability to engender biased signaling by application of an operational model of agonism to the concentration-response curves of either xanomeline, GSK1034702, or TBPB in both IP 1 and ERK1/2 phosphorylation assays (Evans et al., 2011;Keov et al., 2011;Kenakin et al., 2012) (Table 4). These analyses generated transduction coefficient values for each of these agonists at the two different pathways and allowed us to calculate the bias factor between IP 1 and ERK1/2 phosphorylation, revealing significant differences between ACh and xanomeline/TBPB (P , 0.001; one-way analysis of variance), with the latter displaying bias toward IP 1 responses (bias factor IP 1 2 ERK1/2 phosphorylation 5 1.1 and 1.4 for xanomeline and TBPB, respectively). However, no significant differences were revealed for GSK1034702 and ACh, suggesting that GSK1034702 and TBPB, despite apparently similar binding modes, engender differential signaling from the receptor.   GSK1034702 Shows a Lack of Selectivity for M 1 mAChRs. We evaluated the ability of GSK1034702 to bind to other mAChR subtypes by conducting equilibrium-binding studies on membranes expressing the M 1 , M 2 , M 3 , M 4 , or M 5 mAChR, and we found that GSK1034702 could inhibit [ 3 H]-NMS binding at all muscarinic receptor subtypes, albeit with much lower affinity for the M 3 mAChR ( Fig. 11A; Table 5). We further assessed the functional activity of GSK1034702 at the M 2 , M 3 , M 4 , and M 5 mAChRs in the ERK1/2 phosphorylation assay. GSK1034702 exhibited partial agonist activity at M 2 , M 4 , and M 5 mAChRs but was devoid of activity at the M 3 mAChR in this assay (Fig. 11B). Finally, we investigated the ability of GSK1034702 to stimulate negative inotropic responses in isolated rat atria (Fig. 12, A and B) or contraction of rat ileum (Fig. 12, C and D), indicative of activity at M 2 and M 3 mAChRs, respectively. GSK1034702 elicited a robust response in the rat atria, reaching a maximal response equivalent to that of methacholine, with micromolar potency (Fig. 12A). Furthermore, GSK1034702 could inhibit methacholine-induced responses with an IC 50 of 8 mM (Fig.  12B). In the rat ileum, GSK1034702 stimulated approximately 50% of the maximal methacholine-induced contraction, with an EC 50 of 7 mM (Fig. 12C), and inhibited methacholine-induced contraction with an IC 50 of 46 mM (Fig. 12D).

Discussion
The development of the selective M 1 mAChR allosteric agonist GSK1034702 provided the opportunity to test the hypothesis that allosteric M 1 mAChR drugs might provide a clinical advantage over orthosteric M 1 mAChR agents due to increased selectivity while yielding fewer side effects. In the nicotine abstinence model of cognitive dysfunction, GSK1034702 significantly improved immediate memory recall but also induced adverse responses consistent with activation of other muscarinic receptor subtypes (Nathan et al., 2013). At first glance, these data might suggest that allosteric M 1 mAChR drugs offer little or no safety benefit compared with previous investigational agents targeting mAChRs. However, here we provide direct pharmacological evidence that GSK1034702 is not a pure allosteric agonist as previously reported, but rather interacts with the orthosteric binding site and broadly mimics the pharmacology of the known bitopic ligand, TBPB. Based on radioligand binding and functional studies, coupled with the structural similarity between GSK1034702 and TBPB, we conclude that GSK1034702 likely interacts concomitantly with both allosteric and orthosteric sites on the M 1 mAChR in a bitopic manner.
The conclusion that GSK1034702 interacts with the orthosteric site is primarily based on the full inhibition of [ 3 H]-NMS binding and a lack of cooperative effects on [ 3 H]-NMS, features consistent with an orthosteric rather than prototypical allosteric mechanism. In functional assays after receptor alkylation (to diminish its agonist response), GSK1034702 causes a nonsaturable, concentration-dependent parallel rightward shift in the ACh-mediated IP response, further confirming an orthosteric mode of action.
That GSK1034702 might also interact with a site distinct from the orthosteric site was indicated most clearly in functional assays at the M 1 mAChR DREADD and by characterizing the binding of [ 3 H]-GSK1034702 to the wild-type receptor. The M 1 mAChR DREADD contains mutations at key residues within the orthosteric binding pocket, which yields a receptor that is poorly responsive to the cognate ligand, ACh, but instead is activated by an otherwise inert chemical ligand, clozapine-noxide (Armbruster et al., 2007;Roth, 2016). As predicted from previous reports (Abdul-Ridha et al., 2014), the potencies of orthosteric ligands ACh and xanomeline in IP signaling, ERK1/2 phosphorylation, and M 1 receptor phosphorylation were significantly reduced at the M 1 DREADD. However, the potency and efficacy of GSK1034702 and TBPB was unaffected by the DREADD mutations, suggesting that GSK1034702, like TBPB, is able to activate the M 1 mAChR with a binding mode that is subtly distinct from that of prototypical orthosteric ligands. In support of this conclusion, GSK1034702 and TBPB fully inhibit the binding of [ 3 H]-GSK1034702 to the M 1 mAChR, whereas the prototypical orthosteric agonists, ACh and xanomeline, only partially inhibit its binding. This indicates that GSK1034702 can still bind to the M 1 mAChR when ACh or xanomeline occupy the orthosteric site, suggesting that it can interact with the receptor via an allosteric binding site. The display of apparently both orthosteric (competitive) and allosteric behaviors depending on test system is typical of bitopic ligands that are able to "flip-flop" between binding poses (Valant et al., 2012).  Greater clarity around the receptor mechanism of action of GSK1034702, revealed here, has implications for drug design aimed at the treatment of AD. We have previously demonstrated that the learning and memory deficit observed in murine prion disease is due to a loss of cholinergic signaling in the hippocampus and as such, this model replicates one of the key pathologic hallmarks associated with human AD (Bradley et al., 2017). In the prion model, we found that both M 1 mAChR orthosteric agonists and allosteric modulators completely rescue the learning and memory deficit observed in prion disease. However, we also found that the orthosteric ligand, xanomeline, gave adverse responses consistent with the activation of other muscarinic receptor subtypes, whereas the PAM, BQCA, gave no detectable adverse responses at doses that rescued learning and memory (Bradley et al., 2017). Although these results together with other studies on the cognitive responses of M 1 mAChR allosteric modulators (Ma et al., 2009;Lange et al., 2015;Puri et al., 2015;Vardigan et al., 2015) support the potential clinical benefit of this class of ligand, it is also clear that allosteric modulators that show direct agonism in addition to cooperativity in rodent models result in adverse effects (Alt et al., 2016;Davoren et al., 2016Davoren et al., , 2017. Hence, we conclude here that to avoid adverse effects, clinical candidates targeting the M 1 mAChR in AD would require the following properties: 1) high levels of receptor subtype selectivity as would be seen with an allosteric modulator and 2) low levels of intrinsic agonist activity.