Pharmacological Characterization of LY593093, an M1Muscarinic Acetylcholine Receptor-Selective Partial OrthostericAgonist□S

Marla L. Watt, Douglas A. Schober, Stephen Hitchcock,1 Bin Liu, Amy K. Chesterfield,David McKinzie, and Christian C. FelderLilly Research Laboratories, Eli Lilly & Co., Lilly Corporate Center, Indianapolis, Indiana

Received March 24, 2011; accepted May 9, 2011

ABSTRACTAlzheimer’s disease and schizophrenia are characterized byexpression of psychotic, affective, and cognitive symptoms.Currently, there is a lack of adequate treatment for the cognitivesymptoms associated with these diseases. Cholinergic signal-ing and, in particular, M1 muscarinic acetylcholine receptor(m1AChR) signaling have been implicated in the regulation ofmultiple cognitive domains. Thus, the M1AChR has been iden-tified as a therapeutic drug target for diseases, such as schizo-phrenia and Alzheimer’s disease, that exhibit marked cognitivedysfunction as part of their clinical manifestation. Unfortu-nately, the development of selective M1 agonist medicationshas not been successful, mostly because of the highly con-served orthosteric acetylcholine binding site among the fivemuscarinic receptor subtypes. More recent efforts have fo-cused on the development of allosteric M1AChR modulators

that target regions of the receptor distinct from the orthostericsite that are less conserved between family members. However,orthosteric and allosteric ligands may differentially modulate re-ceptor function and ultimately downstream signaling pathways.Thus, the need for highly selective M1AChR orthosteric agonistsstill exists, not only as a potential therapeutic but also as a phar-macological tool to better understand the physiologic conse-quences of M1AChR orthosteric activation. Here, we describe thenovel, potent and selective M1AChR orthosteric partial agonistLY593093 [N-[(1R,2R)-6-({(1E)-1-[(4-fluorobenzyl)(methyl)amino]ethylidene})amino)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]biphe-nyl-4-carboxamide]. This compound demonstrates modest to noactivity at the other muscarinic receptor subtypes, stimulates G�q-coupled signaling events as well as �-arrestin recruitment, anddisplays significant efficacy in in vivo models of cognition.

IntroductionSchizophrenia is characterized by positive (e.g., hallucina-

tions and delusions) and negative (e.g., apathy and socialwithdrawal) psychotic symptoms, as well as cognitive deficits(e.g., inattention and memory loss). Current antipsychotictreatments are most effective against the positive symptomsassociated with schizophrenia but are considerably less ef-fective in treating the negative symptoms and cognitive def-icits (Vohora, 2007). One rationale for the lack of observedcognitive improvement may be the anticholinergic activity

exerted by many of the current antipsychotics (Stahl, 2000).Acetylcholine signaling has been implicated in cognition, andmuscarinic acetylcholine receptor (mAChR) agonists have dem-onstrated antipsychotic efficacy in preclinical studies (McArthur etal., 2010). The neurotransmitter acetylcholine, along withthe two receptor families to which it binds, ionotropic nico-tinic receptors and metabotropic muscarinic receptors, com-prise the cholinergic system. The mAChRs are class A mem-bers of the G protein-coupled receptor superfamily andconsist of five subtypes designated M1-M5AChR (Wess, 1996;Kristiansen, 2004). These subtypes are separated into twodistinct classes based on the G proteins to which they pref-erentially couple. M1AChR, M3AChR, and M5AChR signalthrough G�q/11 and mobilize intracellular calcium, whereasM2AChR and M4AChR couple to G�i/o and inhibit adenylatecyclase and cAMP production (Caulfield, 1998).

1 Current affiliation: Envoy Therapeutics, Jupiter, Florida.Article, publication date, and citation information can be found at

http://jpet.aspetjournals.org.doi:10.1124/jpet.111.182063.□S The online version of this article (available at http://jpet.aspetjournals.org)

contains supplemental material.

ABBREVIATIONS: mAChR, muscarinic acetylcholine receptor; M1–5AChR, muscarinic; acetylcholine receptor subtypes 1–5; CHO, Chinesehamster ovary; GTP�[35S], guanosine 5�-O-(3-[35S]thiotriphosphate); ACh, acetylcholine; NMS, N-methylscopalamine; oxo-m, oxotremorine M;FLIPR, fluorometric imaging plate reader; LY593093, N-[(1R,2R)-6-({(1E)-1-[(4-fluorobenzyl)(methyl)amino]ethylidene})amino)-2-hydroxy-2,3-di-hydro-1H-inden-1-yl]biphenyl-4-carboxamide; PI, phosphoinositide; PAM, positive allosteric modulator; FCTx, frontal cortex; AC-42, 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]; 77-LH-28-1, 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone; McN-A-343, 4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride; TBPB, [1-(1�-2-methylbenzyl)-1,4�-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one].

0022-3565/11/3382-622–632$25.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 338, No. 2Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics 182063/3702576JPET 338:622–632, 2011 Printed in U.S.A.

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An imbalance in cholinergic synaptic transmission hasbeen associated with the cognitive decline apparent in bothAlzheimer’s disease and schizophrenia (Raedler et al., 2007;Langmead et al., 2008). Pharmacological activation of theM1AChR subtype may ameliorate the cognitive decline asso-ciated with these and other neurological illnesses (Langmeadet al., 2008). The M1AChR subtype is predominantly ex-pressed in the forebrain, including the hippocampus andcortex, and these brain regions have been implicated inlearning and memory deficits associated with the pathophys-iology of Alzheimer’s disease and schizophrenia (Levey, 1996;Dean et al., 2002; Porter et al., 2002).

The discovery and development of M1AChR-selective ago-nists has been challenging because of the high sequence simi-larity and identity shared among all five receptor subtypes attheir respective orthosteric acetylcholine binding sites (Hulmeet al., 1990; Heinrich et al., 2009). Although a number of arecho-line-based muscarinic agonists with M1 partial agonist activityentered the clinic, only xanomeline showed efficacy in the cog-nitive domain in both Alzheimer’s disease (Bodick et al., 1997)and schizophrenia (Bodick et al., 1997; Shekhar et al., 2008).However, the muscarinic subtype selectivity and drug-like qual-ities of xanomeline and related compounds were less than ideal,leading to discontinuation of further development.

More recently, allosteric mAChR agonists and modulatorshave been investigated as a means to gain greater selectivityfor individual subtypes (Chan et al., 2008; Conn et al.,2009b). Allosteric site sequences are less conserved amongthe mAChR subtypes, allowing for greater potential com-pound specificity. Moreover, allosteric modulation allows re-tention of spatial and temporal signaling patterns critical fornormal neurophysiology (Conn et al., 2009a; Valant et al.,2009). Allosteric binding sites are distinct from orthostericsites and either regulate receptor-mediated signaling directlyor through cooperativity with simultaneous orthosteric li-gand binding and signaling.

We sought new chemotypes with improved prospects for se-lectivity and conducted a high throughput functional screen forM1AChR agonists against a sample collection composed of Lillyarchival compounds and libraries prepared via parallel synthe-sis. Here we describe the pharmacological characterization ofthe novel, potent and highly selective M1AChR partial agonistLY593093 [N-[(1R,2R)-6-({(1E)-1-[(4-fluorobenzyl)(methyl)amino]ethylidene})amino)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]biphenyl-4-carboxamide] from a 1,6-diamino-indane-2-olchemical platform. LY593093 competes with ACh for [3H]N-methylscopalamine ([3H]NMS) binding and also selectivelystimulates M1AChR-mediated G protein activation and cal-cium mobilization. Furthermore, LY593093 stimulates �-arres-tin recruitment, a signaling pathway postulated to be pertinentfor behavioral and cognitive efficacy, as well as inositol phos-phate turnover in vivo (Ma et al., 2009). Finally, we demon-strate that the compound facilitates spatial learning in ratsafter oral administration, suggesting therapeutic potential indisorders involving cognitive dysfunction.

Materials and MethodsMaterials. All reagents were purchased from Sigma-Aldrich (St.

Louis, MO) unless otherwise indicated. All compounds were synthe-sized at Eli Lilly & Co. unless otherwise noted. Acetylcholine chloridewas purchased from MP Biomedicals (Solon, OH). Oxotremorine M

was purchased from Tocris Bioscience (Ellisville, MO). PathHunterdetection kit and CHO-K1 cells stably-expressing both the �-arrestinfusion protein and M1AChR-ProLink fusion protein were licensedfrom DiscoveRx (Fremont, CA). M1-M5AChR-expressing CHO cellmembrane preparations, GTP�[35S], [3H]myoinositol, and MeltiLexA scintillation sheets were purchased from PerkinElmer Life andAnalytical Sciences (Waltham, MA). Anti-G�i-3 and anti-G�q anti-bodies were generated by Eli Lilly & Co. Scintillation proximityassay beads were purchased from GE Healthcare (Chalfont St. Giles,Buckinghamshire, UK). Fluo-4 dye, probenecid, Opti-MEM I, andtissue culture reagents were purchased from Invitrogen (Carlsbad,CA) unless otherwise indicated. Ready Solv HP scintillation fluidwas purchased from Beckman Coulter, Inc. (Brea, CA). Wild-type,M1AChR knockout, and M3AChR knockout mice were purchasedfrom Taconic Farms Inc. (Hudson, NY).

Inhibition of [3H]NMS Binding at Equilibrium. [3H]NMSbinding assays were performed as described previously (Chan et al.,2008) with the following modifications. In brief, frozen membranepreparations were thawed and resuspended in assay buffer (20 mMHEPES, 100 mM NaCl, 10 mM MgCl2, pH 7.4). Twenty-five micro-grams per well of protein was incubated with [3H]NMS and unla-beled test compounds for 2 h at 22°C in a total volume of 200 �l inpolypropylene 96-deep well plates. Membranes were collected byrapid filtration using a Tomtec cell harvester (Tomtec, Inc., Hamden,CT) through GF/A filters that had been presoaked in 0.3% polyeth-yleneimine. The filters were washed with 5 ml of ice-cold 50 mM Trisbuffer, pH 7.4, and air-dried overnight. The dried filters were treatedwith MeltiLex A melt-on scintillator sheets, and the radioactivityretained on the filters was counted using a Wallac 1205 Betaplatescintillation counter (PerkinElmer Life and Analytical Sciences). Ki

values were determined from the Cheng and Prusoff (1973) relation-ship Ki � IC50/1 � [ligand]/Kd, where IC50 is determined from afour-parameter fit of displacement curves, [ligand] � 1 nM [3H]NMS,and Kd is the equilibrium dissociation constant of [3H]NMS for eachreceptor determined by saturation binding experiments carried outby the membrane supplier. Nonspecific binding was determined us-ing 1 or 10 �M atropine, as indicated in the figure legend.

Cell-Based and Native Tissue GTP�[35S] Binding Assays.The GTP�[35S] binding assay measures the level of G protein acti-vation after agonist treatment by determining the amount of nonhy-drolyzable GTP�[35S] bound to G� subunit subtypes of interest (seeHarrison and Traynor, 2003; for a thorough review). GTP�[35S] bind-ing in cells was determined using the antibody capture technique ina 96-well plate format as described previously by DeLapp et al.(1999). In brief, 100 �l (20–40 fmol/well) of membrane preparationfrom CHO cells that overexpress human M1, M2, M3, M4, or M5muscarinic receptors were incubated for 30 min with 50 �l of testcompound. The receptor densities (Bmax) for the ectopically ex-pressed M1-M5AChRs were 9, 3, 0.7, 0.6, 0.9, and 4.8 pmol/mgmembrane protein, respectively. Representative receptor densitiesfor the M1 and M3AChR knockout animals were reported previously(Oki et al., 2005). In the case of the M2 and M4 receptor membranes,1 �M GDP (final concentration) was added to the membranes. (NoGDP was added to M1–, M3–, or M5– membranes.) After the incuba-tion period, 50 �l/well (500 pM) diluted GTP�[35S] was added to eachwell, and the mixture was allowed to incubate for 30 min. Thelabeled membranes were then solubilized with 0.27% Nonidet P-40for 30 min followed by the addition of 20 �l (final dilution of 1:400) ofthe appropriate anti-G�q antibody. For the M1–, M3–, or M5AChRmembranes, anti-G�q/11 antibody was used, whereas anti G�i-3 an-tibody was used for the M2– and M4AChR membranes. After the60-min incubation, 50 �l/well (1.25 mg/well) scintillation proximityassay beads were added, and the plates were incubated for an addi-tional 3 h. The plates were centrifuged for 10 min at 1000 rpm usingan Accuspin 1R centrifuge (Thermo Fisher Scientific, Waltham MA)and counted for 1 min/well using a Wallac MicroBeta TriLux scin-tillation counter (PerkinElmer Life and Analytical Sciences–Wallac,Gaithersburg, MD). All incubations took place at room temperature

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in GTP-binding assay buffer (20 mM HEPES, 100 mM NaCl, 5 mMMgCl2, pH 7.4). GTP�[35S] binding in native tissue was performedessentially as described previously (Overk et al., 2010).

Calcium Mobilization Assay. Calcium mobilization was mea-sured using assay guidance according to the National Institutes ofHealth’s Chemical Genomics Assay Guidance Manual for FLIPRAssays to Measure GPCR and Ion Channel Targets (http://www.ncgc.nih.gov/guidance/section9.html). CHO cells stably overexpressing ei-ther M1, M3, or M5AChR were plated at 50,000 cell/well in poly-D-lysine-coated 96-well black, clear-bottom plates and grown overnightat 37°C with 5% CO2 and 95% humidity in growth media [Dulbecco’smodified Eagle’s medium/F-12 (3:1), 10% FBS, 20 mM HEPES, 2 mML-glutamine (GlutaMAX), 40 �g/ml L-proline, 250 �g/ml G418(Genetecin), 0.5% penicillin-streptomycin]. Twenty-four hours later,media were removed and cells were preloaded with 50 �l of Fluo-4Dye (5 �g/�l) for 45 min before replacing with 100 �l of fresh assaybuffer (1� Hanks’ balanced salt solution, 20 mM HEPES, 250 �Mprobenecid. Cells were allowed to equilibrate at room temperature inbuffer for 6 min before the addition of compounds using the FLIPRinstrument.

�-Arrestin Recruitment Assay. �-Arrestin 3 recruitment wasmeasured using the PathHunter �-arrestin complementation assaysystem, including a �-arrestin-induced CHO-K1 cell line stably ex-pressing M1AChR and two fragments of �-galactosidase, one fused toM1AChR and the other fused to �-arrestin 3 (DiscoveRx, Fremont,CA). In brief, 40,000 cells/well were treated in Opti-MEM I withligand for 90 min at 37°C with 5% CO2. �-Arrestin 3 recruitment, asdetermined by �-galactosidase complementation, was measured af-ter the addition of PathHunter detection reagent for 60 min atroom-temperature followed by luminescence signal detection using aGENiosPro (Tecan, Mannedorf, Switzerland).

Animal Experiments. Rats used were maintained in facilitiesfully accredited by the Association for the Assessment and Accredi-tation of Laboratory Animal Care (AAALAC). All research protocolswere approved by the institutional animal care and use committeeand are in accordance with the guidelines of the Institutional Careand Use Committee of the National Institute on Drug Abuse (Na-tional Research Council, 1996).

In Vivo Phosphoinositide Hydrolysis. LY593093 was assessedfor its ability to induce phosphoinositide (PI) hydrolysis in rats usingmethods described previously (Bymaster et al., 1998). In brief, sixcolony-acclimated male Sprague-Dawley rats (95–110 g) were anes-thetized and prepared for stereotaxic administration of 2 �Ci of[3H]myoinositol (18.6 Ci/mmol) in 8 �l of saline injected into theventricle (1.5 mm lateral, 1.4 mm posterior from bregma, and 4 mmdeep from the surface of the skull). Twenty-four hours later, ratsreceived saline or lithium chloride (10 mmol/kg s.c.). Vehicle [water �10% caprylocaproyl macrogol-8 glyceride(Labrasol)] or LY593093 (3or 10 mg/kg p.o.) was administered 1 h after lithium administration.Three hours later, rats were sacrificed and the hippocampus wasdissected and placed on dry ice. After weighing, hippocampal tissueswere rapidly homogenized by sonication in 1.25 ml of 10 mM lithiumchloride. After centrifugation at 10,000g for 10 min, the [3H]inositolmonophosphates in 1 ml of supernatant were determined by chro-matography. Total radioactivity in the tissue was determined in an80-�l aliquot of the homogenate. Aqueous aliquots or column eluateswere added to scintillation vials containing 15 ml of Ready Solv HPscintillation fluid, and radioactivity was determined by liquid scin-tillation spectrometry with approximately 40% counting efficiency.Effects of in vivo phosphoinositide hydrolysis were calculated usinga within-animal design by determining percentage conversion of 3Hradioactivity to inositol monophosphates and treatment differencesanalyzed via analysis of variance.

Radial Arm Maze Acquisition. Six male Sprague-Dawley rats,approximately 200 to 225 g, were tested for eight-arm radial mazelearning using food reward. Rats were single-housed and acclimatedfor at least 4 days to a 12:12 light/dark colony with onset of light at6:00 AM. During this time, rats were reduced to and maintained at

85% of their arrival body weight. Rats received a single maze sessioneach day for a total of 4 consecutive days. A session began with anindividual rat being placed into the hub of the maze (CoulbournInstruments, Allentown, PA), and then all guillotine doors wereraised, allowing free access to all areas of the maze. A food hopperwas located at the end of each of the eight runway arms, and a singlefood pellet (45 mg; Bio-Serv, Frenchtown, NJ) was placed in eachfood hopper. Each daily session terminated when either all eight foodhoppers had been visited or when the rat timed out (15 min on day1; 5 min on days 2–4). The number of arm entries was recorded.Errors were counted as repeat arm entries or failures to visit an armin the session period. An animal was excluded from the study if itfailed to visit at least one arm on day 1, 2 arms on day 2, and at least4 arms on days 3 and 4. Each rat was pseudo-randomly assigned toeither a vehicle or drug group and received the same treatmentthroughout the experimental period. Vehicle consisted of 10% cap-rylocaproyl macrogol-8 glyceride within sterile water. Injectionswere administered orally 3 h before each daily session.

ResultsLY593093 Is Selective for the M1AChR

The selectivity of LY593093 (Fig. 1A) for the variousmAChR subtypes was assessed in vitro by three methods:functional stimulation of both G protein activation and cal-cium mobilization as well as Ki determination following dis-placement of NMS binding.

Ca2� Mobilization

Among the three G�q-coupled subtypes (M1AChR, M3AChR,and M5AChR), LY593093 preferentially stimulated M1AChR-mediated calcium mobilization (85.3 2.86% control) (Fig. 1C).LY593093 displayed 100- and 2000-fold better potency atM1AChR (EC50 � 49.9 6.32 nM) versus either M3AChR(EC50 � 6053 2490 nM) or M5AChR (EC50 � 100,000 nM),respectively, distinguishing itself from the pre-existing mol-ecules in terms of M1AChR functional selectivity (Figs. 1Cand 2; Table 1). Whereas both alvameline and talsaclidinedisplayed relatively no activity in terms of M5AChR-medi-ated calcium mobilization, none of the control compoundsshowed selectivity for M1AChR over both M3- and M5AChRwith the combined affinity and percentage of maximal efficacy(Emax) demonstrated by LY593093 (Fig. 2; Table 1). EC50 (log10,nM) values for the various compounds at the M1-/M3-/M5AChRwere observed as follows: LY593093 � 7.3/5.2/� 4.0; ace-tylcholine � 7.6/8.8/7.4; alvameline � 6.4/5.8/� 4.0;sabcomeline � 8.3/8.4/7.4; talsaclidine � 6.6/6.0/�4.0; xanomeline � 7.2/6.6/5.9. The percentage of Emax

values at the M1-/M3-/M5AChR for those compounds listedabove with the highest affinity at M1AChR versus the M3- orM5AChR (LY593093, alvameline, and xanomeline) were ob-served as follows (%Emax): LY593093 � 85.3 2.86/43.9 0.98/not applicable; alvameline � 46.9 7.72/44.0 8.99/notapplicable; and xanomeline � 115 4.32/92.4 9.20/64.2 8.77. It is noteworthy that xanomeline behaved as a partialagonist at M5AChR (Fig. 2), indicating that it may also actas a partial antagonist at this receptor subtype. As was ex-pected, the M1AChR allosteric agonist TBPB, was a high-affin-ity (EC50 � 2.8 1.1 nM) full agonist (118 6.6% Emax) atM1AChR in terms of calcium mobilization (Jones et al., 2006,2008) (Fig. 2). It is surprising that the M1AChR positive allo-steric modulator (PAM) benzyl quinolone carboxylic acid(BQCA) also displayed weak partial agonist activity (EC50 �

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1.8 1.7 �M; 73 7.2% Emax) with respect to M1AChR-mediated calcium mobilization (Fig. 2).

GTP�[35S] Binding

The concentration response curves for LY593093-stimu-lated GTP�[35S] binding in cells heterologously expressingthe various receptor subtypes demonstrate that LY593093 isa functionally selective partial agonist for the M1AChR sub-type, with modest (M2AChR, M4AChR) or no (M3AChR,M5AChR) agonist activity at the other muscarinic subtypes(Fig. 1B). In terms of G protein activation, at M1AChR,LY593093 (EC50 � 219 31.9 nM) demonstrated �95% ofthe control Emax observed with the full orthosteric agonistoxotremorine M (oxo-m; 30 �M), whereas LY593093 dis-played less than 25% Emax at both M2AChR and M4AChR,(EC50 � 1.07 0.17 �M and �10 �M, respectively) (Fig. 3;Table 2). These functional results contrast those of theM1AChR-preferring control agonists, which failed to exhibitstrong functional selectivity for M1AChR over both M2AChRand M4AChR (Fig. 3; Table 2). Xanomeline displayed rela-tively no functional selectivity for the M1AChR subtype(EC50 � 43.6 7.33 nM) over either M2- or M4AChR(EC50 � 78.4 10.4 and 63.1 16.9 nM, respectively) butdisplayed the greatest degree of G protein activation atM2AChR (82.6 3.12% Emax) compared with M1AChR orM4AChR in these studies (63.7 3.48 and 46.1 4.02%control, respectively) (Table 2). Alvameline, sabcomeline, andtalsaclidine also demonstrated activity at M2AChR (EC50 �296 5.69, 56.3 6.58, and 848 69.0 nM, respectively)with sabcomeline and talsaclidine exhibiting high partialagonist activity at M2AChR (89.9 2.85 and 94.4 2.47%Emax, respectively) (Table 2). The augmented M3AChR activ-ity observed with the calcium mobilization assay (Figs. 1Cand 2; Table 2), relative to the GTP�[35S] binding assay (Figs.1B and 4), probably demonstrates the amplified downstreamcalcium mobilization response measured with the FLIPRassay, whereas the GTP�[35S] binding assay measures initialreceptor activation.

Native GTP�[35S] binding studies performed using mem-brane preparations of hippocampi (Hippo) and frontal cortex(FCTx) isolated from the brains of wild-type, M1AChR knockout,and M3AChR knockout mice further define the functionally selec-tive nature of LY593093 for M1AChR (Fig. 4). Frontal cortexmembrane preparations from M3AChR knockout and wild-typeanimals treated with oxo-m displayed similar EC50 and Emax val-ues with respect to G�q activation (EC50FCTx-oxo(WT) � 99.7 23.8nM, % EmaxFCTx-oxo(WT) � 102.4 3.2; EC50FCTx-oxo(M3KO) �

TABLE 1Summary of effects of LY593093 and reference muscarinic agonist compounds on stimulation of calcium mobilization in whole cells (CHO) stablyexpressing human M1, M3, or M5 muscarinic receptorsData were analyzed using nonlinear regression for a sigmoidal concentration-response curve (Prism version 4.0) and are expressed as mean S.E.M. from n � 3 separateexperiments. Potency data are expressed in log10 molar (M) units, and efficacy data are expressed as a percentage of response at 10 �M oxotremorine M.

CompoundM1AChR M3AChR M5AChR

EC50 Emax EC50 Emax EC50 Emax

log10 M % log10 M % log10 M %

LY593093 7.30 8.20 85.3 2.86 5.21 5.60 43.9 0.98 �4.00 *Acetylcholine 7.62 8.64 130 3.90 8.77 9.82 124 10.0 7.39 7.76 124 18.8Alvameline 6.36 7.28 46.9 7.72 5.82 6.43 44.0 8.99 �4.00 *Sabcomeline 8.27 9.30 87.1 4.41 8.39 9.30 99.3 3.96 7.44 8.19 27.6 3.03Talsaclidine 6.57 7.42 104 4.37 5.96 6.64 97.5 8.17 �4.00 *Xanomeline 7.22 7.76 115 4.32 6.58 7.42 92.4 9.20 5.86 6.26 64.2 8.77

* Indicates 20% efficacy at the highest doses tested (100 nM).

Fig. 1. LY593093 is functionally selective for M1AChRs. A, structure ofLY593093; B, LY593093-induced GTP�[35S] binding activity at the M1-M5AChRs [receptor densities (Bmax) for the M1-M5AChRs were 9,3, 0.7,0.6, 0.9, and 4.8 pmol/mg membrane protein, respectively]. Data areexpressed as a percentage of response in presence of 30 �M oxotremorineM. C, effect of LY593093 on stimulation of calcium mobilization in wholecells (CHO) stably expressing human M1, M3, or M5 muscarinic recep-tors. Data are expressed as a percentage of response in presence of 10 �Moxotremorine M. All data were analyzed using nonlinear regression for asigmoidal concentration response curve (Prism version 4.0; GraphPadSoftware, Inc., La Jolla, CA) and are displayed as the mean S.E.M.from n � 3 separate experiments.

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69.9 22.1 nM, % EmaxFCTx-oxo(M3KO) � 99.2 5.2). G�q

activation in M3AChR knockout and wild-type hippocampalmembrane preparations treated with oxo-m was relativelysimilar, both in terms of percentage of Emax and potency(EC50Hippo-oxo(WT) � 70.5 17.4 nM, % EmaxHippo-oxo(WT) �102.8 3.3; EC50Hippo-oxo(M3KO) � 66.8 21.3 nM, %EmaxHippo-oxo(M3KO) � 101.7 6.5) (Fig. 4, A and C). Oxo-msignaling in M1AChR knockout membranes (both hippocam-pal and frontal cortex) was dramatically decreased (�80%

reduction of wild-type efficacy in both tissues) (Fig. 4, A andC). LY593093-mediated G�q signaling was nearly identical inM3AChR and wild-type membrane preparations of both hip-pocampal and frontal cortex (EC50FCTx-LY593093(WT) � 3.6 3.3, %EmaxFCTx-LY593093(WT) � 92.9 4.5; EC50FCTx-LY593093(M3KO) �2.7 1.6 nM, % EmaxFCTx-LY593093(M3KO) � 90.9 6.6;EC50Hippo-LY593093(WT) � 3.2 1.6 nM, % EmaxHippo-LY593093(WT) �93.5 4.5; EC50Hippo-LY593093(M3KO) � 1.6 0.2 nM, %EmaxHippo-LY593093(M3KO) � 95.0 5.9) (Fig. 4, B and D).

Fig. 2. Effect of LY593093 and referencemuscarinic agonists on stimulation of cal-cium mobilization in whole cells (CHO)stably expressing human M1-, M3-, orM5AChRs. Data were analyzed usingnonlinear regression for a sigmoidal con-centration response curve (Prism version4.0) and are expressed as a percentage ofresponse in presence of either maximalacetylcholine (50 �M ACh, for BQCA andTBPB) or maximal oxotremorine M (10�M oxotremorine M, for remaining com-pounds). Data are displayed as themean S.E.M. from n � 3 separateexperiments.

Fig. 3. Effects of LY593093 and referencemuscarinic agonists on stimulation ofGTP�[35S] binding by G�i-3 in membranesprepared from CHO cells stably express-ing human M1- (Bmax � 9 pmol/mg pro-tein), M2- (Bmax � 3 pmol/mg protein), orM4AChRs (Bmax � 0.6 pmol/mg protein).Data were analyzed using nonlinear re-gression for a sigmoidal concentration re-sponse curve (Prism version 4.0) and areexpressed as the mean S.E.M. from n �3 separate experiments. Data are dis-played as a percentage of response inpresence of 30 �M oxotremorine M.

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LY593093-mediated G�q signaling in M1AChR knockout tissueswas dramatically diminished in both frontal cortex and hippocam-pus, relative to wild type (Fig. 4, B and D).

[3H]NMS Displacement

LY593093 dose-dependently competed with the nonselec-tive mAChR antagonist [3H]NMS for binding at the M1AChRand M2AChR orthosteric sites (Ki � 622 23.2 and 890 28.6 nM, respectively) (Fig. 5A). However, LY593093displayed no appreciable affinity for receptor subtypesM3AChR-M5AChR, distinguishing itself from the otherM1AChR-preferring agonists tested (Table 3). Whereas sab-comeline, talsaclidine, and xanomeline all displayed rela-tively similar binding affinities for the different mAChR sub-types, only LY593093 exhibited no appreciable affinity forreceptor subtypes 3 to 5.

Curve-shift analysis was performed to determine whetherLY593093 had any modulatory role in ACh functional activ-ity. Curve-shift analyses indicated that LY593093 does notpotentiate ACh-mediated displacement of [3H]NMS as doesthe prototypical M1AChR-selective PAM BQCA (Fig. 5, Band C) but rather competed with acetylcholine for binding tothe M1AChR (Fig. 5B). It is noteworthy that BQCA did not

modulate the competitive nature of LY593093 binding (Fig.5D), indicating that the interaction between LY593093 andM1AChR is different from the interaction of the receptor withthe endogenous ligand ACh.

In addition to determining the affinity of LY593093 for thevarious mAChR subtypes, the affinity of LY593093 against apanel of 64 various human proteins, including neurotrans-mitter receptors and transporters, ion channels, second mes-senger receptors, enzymes, and peptides, was determined(CEREP, Paris, France). At 10 �M, LY593093 was relatively inef-ficient at displacing the binding of radioligand selective for any ofthe non-mAChR human proteins in the panel. LY593093 (10 �M)inhibited the binding of radioligand by greater than 50% at onlythree of the �60 proteins tested: dopamine transporter (58.8%inhibition); 5-hydroxytryptamine2A receptor (53.99% inhibition);and glutamate receptor, chloride-dependent site (54.06% inhibi-tion) (Supplemental Table 1).

LY593093 Stimulates �-Arrestin 3 Recruitment atM1AChR

Paralleling the effect on GTP�[35S] binding and calciummobilization, LY593093 displayed both partial agonist andantagonist activity with respect to M1AChR-mediated

Fig. 4. Effect of LY593093 (B and D) andoxotremorine M (A and C) on stimulationof GTP�[35S] binding by G�q in mem-branes prepared from the hippocampiand frontal cortex of wild-type, M1AChRknockout, and M3AChR knockout mice.Data were analyzed using nonlinear re-gression for a sigmoidal concentration re-sponse curve with the minimal value setto zero (Prism version 4.0) and aregraphed as the mean S.E.M. from n � 2separate experiments. Data are displayedas a percentage of the signal observed inwild-type membrane preparations fromeach tissue type using oxotremorine M andLY593093, respectively.

TABLE 2Summary of effects of LY593093 and reference muscarinic agonist compounds on stimulation of GTP��35S� binding activity in membranesprepared from CHO cells stably expressing human M1, M2, or M4 muscarinic receptorsData were analyzed using nonlinear regression for a sigmoidal concentration-response curve (Prism version 4.0) and are expressed as mean S.E.M. from n � 3 separateexperiments. Potency data are expressed in log10 molar (M) units, and efficacy data are expressed as a percentage of response at 10 �M oxotremorine M.

CompoundM1AChR M2AChR M4AChR

EC50 Emax EC50 Emax EC50 Emax

log10 M % log10 M % log10 M %

LY593093 6.66 7.50 94.3 2.29 5.97 6.76 23.2 1.39 �5.00 *Acetylcholine 7.59 8.61 103 1.86 8.16 9.31 101.7 2.20 7.28 7.87 87.4 3.88Alvameline �4.52 * 6.53 8.24 49.4 2.33 �4.52 *Sabcomeline 7.25 8.03 38.9 2.18 7.25 8.18 89.9 2.85 7.17 7.95 20.0 1.41Talsaclidine 6.09 7.04 69.3 2.42 6.07 7.16 94.4 2.47 6.10 7.03 21.6 0.78Xanomeline 7.36 8.13 63.7 3.48 7.11 7.98 82.6 3.12 7.20 7.77 46.1 4.02

* 20% efficacy at the highest doses tested (10 �m for LY593093 and 30 �M for alvameline).

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�-arrestin 3 recruitment, as did the M1/M4AChR-prefer-ring orthosteric agonist xanomeline (Fig. 6, A and B, anddata not shown). LY593093 and xanomeline partially stim-ulated �-arrestin 3 recruitment at M1AChR (EC50 � 1.95�M and 19.91 nM, respectively). The M1AChR-selectivePAM BQCA potentiated the effect of ACh on M1AChR-mediated �-arrestin 3 recruitment in a dose-dependentfashion, similar to its effect on ACh-stimulated calciummobilization (Fig. 6A) and confirming the observationmade previously by Ma et al. (2009). The EC20 concentra-tion of ACh used when testing BQCA in potentiator modewas determined based on previous experiments and not

immediately before the potentiator assay was run. Assay-to-assay variability probably explains why, at low concen-trations of BQCA, 20% Emax was observed in the pres-ence of what we previously observed in this assay to be anEC20 concentration of ACh.

Unlike xanomeline and BQCA, not every control com-pound tested uniformly activated all M1AChR-coupled sig-naling pathways. For example, TBPB did not stimulate�-arrestin 3 recruitment at M1AChR, as had been demon-strated previously (Fig. 6C) (Ma et al., 2009). It is surpris-ing that the full agonists oxo-m and talsaclidine also ex-hibited partial agonist activity at M1ACh with respect to

Fig. 5. Displacement of NMS binding to membranes prepared from CHO cells stably expressing human M1AChR (Bmax � 1.8 pmol/mg protein). A, LY593093,unlike the PAM BQCA, dose-dependently competes with NMS for M1AChR binding. B, LY593093 dose-dependently competes with acetylcholine for an orthostericbinding site on M1AChR. C, BQCA dose-dependently potentiates acetylcholine binding at M1AChR. D, BQCA has no effect on LY593093 binding at M1AChR,indicating that LY593093 binding cannot be allosterically modulated by compounds acting at the BQCA-interacting allosteric site. Nonspecific binding wasdetermined in the presence of 10 �M atropine. Data were analyzed using nonlinear regression for a sigmoidal concentration-response curve (Prism version 4.0) andare expressed as the mean S.E.M. from n � 2 experiments (A) and n � 3 separate experiments (B–D), each performed in duplicate.

TABLE 3Summary of effects of LY593093 and reference muscarinic agonist compounds on displacement of N-methylscopalamine binding to membranesprepared from CHO cells stably expressing human M1–M5 muscarinic receptorsNonspecific binding was measured in the presence of 1 �M atropine. Ki values were determined from the Cheng-Prusoff relationship Ki � IC50/1 � �ligand�/Kd, where IC50is determined from a four-parameter fit of displacement curves, �ligand� � 1 nM �3H�NMS, and Kd is the equilibrium dissociation constant of �3H�NMS for each receptordetermined by saturation binding experiments carried out by the membrane supplier. Data are expressed as the mean S.E.M. from n � 3 separate experiments in log10molar (M) units.

CompoundKi

M1AChR M2AChR M3AChR M4AChR M5AChR

log10 M

LY593093 6.21 7.63 6.05 7.54 �5.00 �5.00 �5.00Alvameline 5.72 6.67 6.49 7.48 5.96 6.88 5.33 6.25 5.53 6.48Sabcomeline 7.69 8.39 8.11 8.75 7.91 8.60 7.62 8.36 7.72 8.29Talsaclidine 6.86 7.69 7.76 8.50 7.08 8.14 6.95 8.02 6.46 7.51Xanomeline 6.81 7.92 7.16 8.11 7.36 8.23 7.24 8.33 6.88 8.10

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�-arrestin 3 recruitment (Fig. 6C). This observation wasindependent of the supplier or lot number of oxo-m tested(data not shown; multiple lot numbers not tested fortalsaclidine).

LY593093 Is Active at M1AChR In Vivo

LY593093 Effects on In Vivo Hippocampal Phospho-inositide Hydrolysis. Conversion of 3H radioactivity to[3H]inositol monophosphates was increased in a dose-relatedmanner with the 10 mg/kg dose of LY593093 producing a 165%increase compared with the lithium-alone group (Fig. 7; P 0.034 versus lithium alone control). These data suggest thatLY593093 activates muscarinic M1 receptors and stimulatesthe formation of inositol triphosphate in a brain region rich inM1AChRs.

LY593093 Effects on Spatial Learning. In this 4-daytraining paradigm, untreated rats typically do not show sig-nificant improvement in maze performance (i.e., fewer er-rors), whereas nootropic agents accelerate learning acquisi-tion and statistical separation in number of errors committedcan be observed by day 4 of training. Planned independentgroup t test, using total errors on day 4, was conducted ontest groups for two separate studies. In experiment 1 (Fig. 8,left), 3 and 10 mg/kg LY593093 significantly reduced errorsrelative to vehicle controls [t(13) � 2.11, P 0.03 and t(14) �2.23, P 0.02, respectively]. In experiment 2 (Fig. 8, right),the top dose of 3 mg/kg LY593093 significantly reduced error[t(13) � 2.73, P 0.01]. These data show consistent efficacyof LY593093 within a 3 to 10 mg/kg dose range under thepresent experimental conditions.

DiscussionThe deregulation of the cholinergic system has been impli-

cated in the development of cognitive symptoms associatedwith mental illnesses, such as Alzheimer’s disease andschizophrenia (Rainer and Mucke, 1998; Raedler et al., 2007;Langmead et al., 2008). The cholinergic M1AChR receptorsubtype is widely expressed in the cortical and limbic brainregions, and extensive evidence indicates a mediating role incognitive processes (Buckley et al., 1988; Anagnostaras et al.,2003; Conn et al., 2009b). Thus, the M1AChR receptor has

Fig. 7. Stimulation of in vivo phosphoinositide hydrolysis by oralLY593093 administration. LY593093 (3 or 10 mg/kg) was given to rats 1 hafter lithium (10 mmol/kg), and hippocampus was collected 2 h later.Data represent percentage conversion of [3H] radioactivity to [3H]inositolmonophosphates.�, P 0.034 versus lithium alone group.

Fig. 6. Ligand-induced signaling bias for M1AChR-coupled �-arrestinrecruitment. A, LY593093 and xanomeline behave as partial agonists for�-arrestin recruitment at M1AChR, and BQCA dose-dependently aug-ments ACh-stimulated �-arrestin recruitment when incubated with anempirically determined EC20 concentration of ACh. B, LY593093 displaysboth partial agonist and partial antagonist (when incubated with anemipirically determined EC90 concentration of ACh). C, TBPB, oxotremo-rine M, and talsaclidine behave as partial agonists in terms of M1AChR-mediated �-arrestin recruitment. Data were analyzed using nonlinearregression for a sigmoidal concentration-response curve (Prism version4.0) and are expressed as a percentage of response at 1.0 mM ACh. Dataare displayed as the mean S.E.M. of n � 3 separate experiments[except for BQCA (agonist mode), for which n � 2], each performed ineither duplicate or quadruplicate.

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emerged as a potential pharmacological target for diseasescharacterized by cognitive impairment. Unfortunately, thehigh degree of conservation between muscarinic receptorsubtypes at their orthosteric ligand binding sites has hin-dered the development of M1AChR-selective drugs. TheM1/M2/M4AChR-preferring muscarinic agonist xanomelineshowed symptom reduction efficacy in patients with bothAlzheimer’s disease and schizophrenia; however, parasympa-thetic side effects, particularly in elderly patients, have lim-ited its therapeutic potential (Bodick et al., 1997; Shekhar etal., 2008; Heinrich et al., 2009). Results of previous studiesperformed with M1-M5AChR knockout mice indicate thatthe M2AChR and M3AChR receptor subtypes are responsiblefor mediating the physiological processes involved in many ofthe side effects observed during testing of the pre-existing ofM1AChR-preferring agonists, including xanomeline (Wess etal., 2007). Thus, significant improvement in M1 selectivityover M2- and M3AChR receptor subtypes is probably essen-tial in the successful development of a tolerable M1AChRorthosteric agonist (Heinrich et al., 2009). Although a role forthe M3AChR in cognition has recently emerged (Poulin et al.,2010), its role in peripheral parasympathetic regulation ofsmooth muscle and exocrine glands precludes it as an attrac-tive drug target.

We have demonstrated that LY593093 is a potent, func-tionally selective partial orthosteric agonist at the M1AChRreceptor subtype. Comparing the ability of LY593093 to stim-ulate calcium mobilization at the endogenously G�q-coupledsubtypes, LY593093 displayed selectivity for M1AChR, ex-hibiting extremely modest or no activity at M3AChR andM5AChR, respectively (Fig. 1C). By use of the GTP�[35S]binding assay to assess mAChR-coupled G-protein activa-tion, LY593093 displayed potent partial activation ofM1AChR [while demonstrating only modest (M2AChR,M4AChR) or no (M3AChR, M5AChR) activity at the othersubtypes] (Fig. 1B). These results were further supported bya decrease in LY593093-stimulated GTP�[35S] G�q activa-tion in M1AChR knockout, but not M3AChR or wild-type,hippocampal and frontal cortex membrane preparations (Fig.4). Similar results were shown previously for the nonselec-tive full agonist oxo-m (Felder et al., 2001; Porter et al.,2002). The rationale for the initial modest, yet, seeminglydose-dependent increase in G�q activation in LY593093-treated M1AChR knockout tissues followed by a decrease tonear zero stimulation at higher concentrations is unknown.Although the results clearly show that genetic ablation of

M1AChR drastically affects LY593093-mediated G�q activa-tion, other nonobvious signaling pathways may also be mod-ulated by LY593093 treatment in the absence of theM1AChR that affect G protein activation and the results ofthis native tissue-based assay, but with modest effect.

Varying assay methods to evaluate LY593093 activity atthe various mAChR subtypes may also explain the discrep-ancy that exists between our results here and those pub-lished previously by Heinrich et al. (2009). In an initialpharmacological characterization of LY593093 functional se-lectivity, Heinrich et al. (2009) observed LY593093 affinity atM2AChR (Ki � 0.79 �M) to be similar to that which we reporthere (0.62 �M). However, in terms of functional selectivity,they reported (Heinrich et al., 2009) modest LY593093 activ-ity (�35-fold less potent compared with M1AChR) at the M2-and M4AChRs, which conflicts with our observations re-ported here. Once again, these differences may be explainedby the assay method chosen to assess LY593093 activity. Fortheir experiments, Heinrich et al. (2009) used assays mea-suring amplified downstream responses (cAMP production)to assess mAChR activity instead of an initial event such asG protein activation measured here using the GTP�[35S]binding assay. Assaying calcium mobilization, Heinrich et al.(2009) also reported modest activation of M3AChR byLY593093 (EC50 � 1.42 �M; 102% Emax), which is similar toour results (6.05 2.5 �M; Table 1), despite the fact that inour hands, the maximal agonist effect of LY593093 (43.9%Emax; Table 1) was 2-fold less (102% Emax) than that observedby Heinrich et al. (2009). Although these data highlight theimportance of investigating receptor activation by more thanone detection method, these discrepancies do not negate thehighly selective and potent activity of LY593093 for theM1AChR subtype observed here and previously (Heinrich etal. (2009).

A CEREP screen of LY593093 against various human pro-teins, including neurotransmitter receptors and transport-ers, ion channels, and enzymes indicated that LY593093 haslittle-to-no affinity for off-target proteins (Supplemental Ta-ble 1). The selective nature of LY593093 for M1AChR overthe other mAChR subtypes suggests improved tolerabilitywithout the undesirable off-target effects that have beenobserved with many of the pre-existing muscarinic com-pounds (Bymaster et al., 2003). LY593093 was competitivewith acetylcholine and displaced binding of the antagonist[3H]NMS at M1AChR with no effect on [3H]NMS binding atthe M3-M5AChRs (Fig. 5, A and B; Table 3). Furthermore,

Fig. 8. Total errors on day 4 radial armmaze performance in two independentstudies. Errors are plotted on the ordi-nate and dose of LY593093 (LY) on theabscissa. LY was administered orally, 3 hbefore each training session. LY wastested across a range of doses to establishthe effective dose range and establish re-peatability.�, P 0.05 versus vehicle con-trol group for each experiment.

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the observation that LY593093 binding was unaffectedby the M1AChR-selective PAM BQCA further supports thelikelihood that LY593093 binds at additional sites distinctfrom ACh binding determinants (Fig. 5D). LY593093 alsodisplaced [3H]NMS binding at M2AChR (Table 3). However,in terms of functional activity, LY593093 displayed between5- and 120-fold better potency and demonstrated at least2-fold higher Emax values at M1AChR versus any of the othersubtypes, including M2AChR (Tables 1 and 2).

Ma et al. (2009) recently suggested that ligand-stimulated�-arrestin recruitment at M1AChR may be an importantindicator of pharmacological efficacy in improved cognition.The requirement for this pathway in memory and learningwas further supported in studies with wild-type and mutantM3AChRs in a fear-conditioning behavioral paradigm (Pou-lin et al., 2010). Here, we show that LY593093 acts as apartial agonist with respect to M1AChR-mediated �-arrestin3 recruitment, indicating that LY593093 may induce thesignaling events proposed to be pertinent for cognitive im-provement. To that end, our in vivo studies demonstratepharmacological activity of LY593093 in stimulating PI turn-over after oral dosing in brain regions important for learningand memory—an effect shown to be M1AChR-dependent (By-master et al., 2003). PI turnover is used as a measure ofG�q-mediated stimulation of phospholipase C signaling. Thedoses of LY593093 that produced PI hydrolysis effects wereconsistent with the dose range that improved acquisition of aspatial learning task. These data support the premise that�-arrestin recruitment at M1AChR may indeed be essentialfor cognitive restoration and continue to highlight a role forM1AChR-mediated signaling in cognitive processing.

Allosteric agonists and PAMs have shown promise as ameans of selectively modulating M1AChR signaling (Spald-ing et al., 2002; Ma et al., 2009; Shirey et al., 2009). However,controversy exists concerning the possibility that differencesin G protein coupling occur when the M1AChR receptor isstimulated by allosteric versus orthosteric agonists (Thomaset al., 2009). For example, the M1AChR-selective allostericagonist 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] (AC-42) was shown to cause slightly altered G protein coupling toM1AChRs compared with orthosteric agonists and exhibits adecreased likelihood to induce receptor desensitization andinternalization (Thomas et al., 2009). Furthermore, theM1AChR-selective PAM BQCA was shown to potentiateACh-mediated �-arrestin recruitment, whereas the selectiveM1AChR allosteric agonist TBPB did not, despite similarstimulation of other M1AChR-mediated signaling pathways(Fig. 6, A and C) (Ma et al., 2009). We also show that, in ourhands, the prototypical full orthosteric agonist oxo-m be-haves as only a partial agonist in terms of stimulating �-ar-restin recruitment at M1AChR (Fig. 6C). These phenomenamay be explained by a “bitopic” mode of ligand binding,whereby allosteric compounds differentially interact withboth the allosteric and orthosteric sites, possibly inferringunique receptor conformations and, subsequently, differen-tial signaling events (Avlani et al., 2010). Such bitopic inter-actions have been previously proposed regarding 77-LH-28-1[1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quino-linone] at M1AChR and McN-A-343 [4-(N-(3-chlorophenyl)carbamoyloxy)-2-butynyltrimethylammonium chloride] atM2AChR (Valant et al., 2008; Avlani et al., 2010). Suchligand-specific signaling bias at M1AChR may help to illu-

minate particular amino acid residues responsible for thesuccessful coupling of M1AChR activation and its variousassociated downstream signaling events, as has been ex-plored previously for M2AChR (Gregory et al., 2010). Futurestudies should focus on determining the specific amino acidsresponsible for M1AChR ligand-specific signaling bias.

In summary, in this study we have presented the pharma-cological characterization of the partial M1AChR orthostericagonist LY539039. In comparison with the other orthostericagonists tested here, LY539039 displays selectivity forM1AChR versus the remaining four mAChR subtypes (Figs.1–3; Tables 1 and 2). Furthermore, LY593093 stimulates�-arrestin recruitment at M1AChR and displays in vivo ac-tivity in pharmacological and cognition animal models (Figs.6–8). We propose that LY539039 may be worthy of subse-quent clinical investigation as a therapeutic agent in ill-nesses characterized, at least in part, by cognitive decline,including Alzheimer’s disease and schizophrenia. Further-more, the highly selective nature of this ligand lends itself asa pharmacological tool to better evaluate the role of M1AChRsignaling in cognition and other physiological processes.

Acknowledgments

We thank Frank Bymaster, Mary Wolff, Robert Crile, and PetraCarter for technical contributions.

Authorship Contributions

Participated in research design: Watt, Schober, Hitchcock, Ches-terfield, McKinzie, and Felder.

Conducted experiments: Watt, Schober, Chesterfield, and McKinzie.Contributed new reagents or analytic tools: Hitchcock, Liu, and

Felder.Performed data analysis: Watt, Schober, Chesterfield, McKinzie,

and Felder.Wrote or contributed to the writing of the manuscript: Watt,

McKinzie, and Felder.Other: Liu designed LY593093.

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Address correspondence to: Christian C. Felder, Lilly Corporate Center,Indianapolis, IN 46285. E-mail: felder@lilly.com

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