Allosteric modulation of the muscarinic M4 receptoras an approach to treating schizophreniaW. Y. Chan*, D. L. McKinzie†, S. Bose*, S. N. Mitchell*, J. M. Witkin†, R. C. Thompson‡, A. Christopoulos§, S. Lazareno¶,N. J. M. Birdsall�, F. P. Bymaster†, and C. C. Felder*†**

*Neuroscience Discovery Research, Lilly Research Centre, Surrey GU20 6PH, United Kingdom; †Neuroscience Division and ‡Discovery Chemistry and ResearchTechnologies, Lilly Corporate Center, Indianapolis 46285; §Department of Pharmacology, Drug Discovery Biology Laboratory, Monash University, Melbourne,Victoria 3010, Australia; ¶MRC Technology, Mill Hill, London NW7 1AD, United Kingdom; and �Division of Physical Biochemistry, Medical Research CouncilNational Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom

Edited by L. L. Iversen, University of Oxford, Oxford, United Kingdom, and approved April 17, 2008 (received for review January 23, 2008)

Current antipsychotics provide symptomatic relief for patientssuffering from schizophrenia and related psychoses; however,their effectiveness is variable and many patients discontinue treat-ment due to side effects. Although the etiology of schizophrenia isstill unclear, a leading hypothesis implicates an imbalanced dopa-minergic system. Muscarinic acetylcholine (ACh) receptors regulatedopamine levels in key areas of the brain involved in psychosis,with the M4 subtype emerging as a key regulator of dopaminergichyperactivity. Unfortunately, no selective small molecule toolsexist to provide pharmacological validation of this hypothesis.Here, we describe the discovery of a small molecule modulator,LY2033298, that is highly selective for human M4 receptors byvirtue of targeting an allosteric site on this receptor. Pharmaco-logical assays confirmed the selectivity of LY2033298 for the M4receptor and revealed the highest degree of positive allostericenhancement of ACh potency thus far identified. Radioligandbinding assays also show this compound to directly potentiateagonist binding while having minimal effects on antagonist bind-ing. Mutational analysis identified a key amino acid (D432) in thethird extracellular loop of the human M4 receptor to be critical forselectivity and agonist potentiation by LY2033298. Importantly,LY2033298 was active in animal models predictive of clinicalantipsychotic drug efficacy indicating its potential use as a first-in-class, selective, allosteric muscarinic antipsychotic agent.

cholinergic � GPCR � cooperativity

Schizophrenia is a complex disease presenting a broad spec-trum of endophenotypes that can be generalized to symp-toms in three major domains: positive (hallucinations, hearing ofvoices, delusions and disorganized thinking), negative (anhedo-nia, f lat affect), and cognitive (attention and working memorydeficits). The etiological basis for the disease is believed to derivepredominantly from dysregulation of dopamine and glutamateneurotransmission pathways in mesocortical and mesolimbicbrain areas (1–3). Atypical antipsychotics are the current front-line treatment for schizophrenia, ameliorating the positive symp-toms in approximately half of the treated patient population withlittle efficacy at the negative and cognitive symptoms. Mostatypical antipsychotics are broad spectrum G protein-coupledreceptor (GPCR) antagonists, and their therapeutic action ismediated primarily through inhibition of dopamine D2, D3 andD4, and serotonin 5HT2A, receptors (4). Their complex phar-macology, however, leads to significant undesirable side effects,including movement disorders and weight gain. Therefore, de-veloping antipsychotics through an alternative mechanism mayprovide better total symptom control and reduced side effects.

The family of five muscarinic acetylcholine (ACh) receptorsplays a prominent role in regulating neurotransmission in theCNS and may provide a mechanism for antipsychotic drugdiscovery. Studies using muscarinic receptor knock out micehave provided valuable insight into the potential role of thesereceptors, especially the muscarinic receptor M1 and M4 sub-

types, in the pathophysiology of schizophrenia (5, 6). Muscarinicreceptors are genetically linked to schizophrenia and cognitivedeficits (7, 8). Pharmacologically, relatively non-selective mus-carinic antagonists exacerbate, whereas agonists ameliorate,cognitive deficits and psychotic behaviors in animal models andin patients suffering from Alzheimer’s disease and schizophrenia(9–12). In particular, the M1/M4 preferring partial agonist,xanomeline, was found to be efficacious in animal modelspredictive of antipsychotic behaviors through modulation ofboth dopamine and glutamate transmission (13). In exploratoryclinical trials, xanomeline reduced psychotic behaviors andimproved cognitive measures in Alzheimer’s patients (9), andsignificantly improved positive, negative, and cognitive symp-toms in a small schizophrenia trial (12). Unfortunately, lack ofreceptor subtype selectivity led to undesirable side-effects (gas-trointestinal disturbances, blood pressure dysregulation) thatrendered xanomeline as an unsuitable candidate for furtherclinical development (14).

M4 receptors are most highly expressed in brain regions richin dopamine and dopamine receptors (15, 16). Experiments withmuscarinic knock out mice have implicated the M4 receptorsubtype in regulating dopaminergic neurons involved in move-ment control and cognition (17, 18); therefore, we reasoned thata highly selective M4 muscarinic receptor activator may representa useful candidate for the development of muscarinic-basedantipsychotic agents. Unfortunately, attempts at discoveringselective small molecule ligands for the five muscarinic receptorsubtypes have largely been unsuccessful when targeted at thereceptors’ orthosteric site, i.e., the binding site for the endoge-nous agonist ACh, which is highly conserved across all fivemuscarinic subtypes (19). This has been particularly true for theACh-mimetics, such as xanomeline, sabcomeline, and melame-line (20). However, it is also known that muscarinic receptorspossess allosteric binding sites that have the potential to showgreater sequence divergence across subtypes and, hence, mayprovide an alternative means of attaining receptor subtypeselectivity (21, 22). Allosteric modulators also offer the addi-tional potential of maintaining both spatial and temporal neu-rotransmission through modulation of physiologically relevantreceptor-mediated neural regulation, as opposed to direct (con-

Author contributions: W.Y.C., D.L.M., J.M.W., R.C.T., A.C., S.L., N.J.M.B., F.P.B., and C.C.F.designed research; W.Y.C., D.L.M., S.B., S.N.M., J.M.W., R.C.T., S.L., N.J.M.B., F.P.B., andC.C.F. performed research; R.C.T. contributed new reagents/analytic tools; W.Y.C., S.B.,S.N.M., A.C., S.L., N.J.M.B., and C.C.F. analyzed data; and W.Y.C., S.B., S.N.M., A.C., S.L.,N.J.M.B., and C.C.F. wrote the paper.

Conflict of interest statement: W.Y.C., D.L.M., S.B., S.N.M., J.M.W., R.C.T., F.P.B., and C.C.F.are or were employed by Eli Lilly & Co.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

**To whom correspondence should be addressed at: Eli Lilly & Co. Research Laboratories,Lilly Corporate Center, Indianapolis, IN 46285. E-mail: felder@lilly.com.

This article contains supporting information online at www.pnas.org/cgi/content/full/0800567105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

10978–10983 � PNAS � August 5, 2008 � vol. 105 � no. 31 www.pnas.org�cgi�doi�10.1073�pnas.0800567105

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tinuous) agonism or antagonism (23, 24). Here, we describe theexploitation of this concept to the discovery and molecularcharacterization of LY2033298 [3-amino-5-chloro-6-methoxy-4-methyl-thieno(2,3-b)pyridine-2-carboxylic acid cyclopropylam-ide (C13H14ClN3O2S; molecular weight � 311.8)] (Fig. 1A), ahighly selective positive allosteric potentiator of ACh actions atthe M4 muscarinic receptor that also shows significant efficacyin preclinical models predictive of antipsychotic drug behavior.

ResultsLY2033298 Is a Selective Positive Allosteric Modulator of ACh atHuman Muscarinic M4 Receptors. Initial screening quantified intra-cellular calcium mobilization as a readout of muscarinic receptor

activation, using cell lines stably expressing the hM1–M5 recep-tors coupled to intrinsic or engineered calcium signaling path-ways. No agonist activity at any muscarinic receptor subtype wasdetected when LY2033298 was applied alone. However, uponaddition of a submaximal concentration of ACh, an elevatedcalcium response was readily detected in the hM4 cell linesrelative to the effect of the same concentration of ACh in theabsence of LY2033298. Subsequent assays constructed completeconcentration-response curves to ACh in the presence of in-creasing concentrations of LY2033298. As shown in Fig. 1 A,these experiments revealed a prototypical characteristic of pos-itive allosteric interactions: a robust potentiation of agonistpotency that reached a limit at the highest concentrations ofallosteric modulator above which no further effect was obtained(24). Application of an allosteric ternary complex model to thedata yielded KB � 200 � 40 nM for LY2033298 at the allostericsite on the unoccupied hM4 receptor, and � � 35 � 4 for thedegree of allosteric enhancement when both orthosteric andallosteric sites are occupied; to our knowledge, this is the highestdegree of positive cooperativity reported for any allostericmodulator of a muscarinic receptor. In addition, these experi-ments also revealed the high selectivity of LY2033298 for thehM4 receptor, because there was no effect at hM1/3/5 receptors atany concentration of modulator and a very small allostericpotentiation at the closest homolog, hM2 (KB � 1.0 � 0.3 �M;� � 3.7 � 0.5). The positive allosteric effect of LY2033298 at thehM4 receptor was not unique to ACh, because it also selectivelypotentiated other full and partial orthosteric agonists, such ascarbachol, oxotremorine-M, and McN-A343 (data not shown).

To confirm that the allosteric effect detected in the calciumassay was neither unique to this signaling pathway or a conse-quence of engineering the coupling of the hM4 receptor to thispathway via the use of promiscuous G�15 proteins, additionalexperiments were performed measuring ACh-mediated[35S]guanosine-�-S–triphosphates (GTP�S) binding to native Gproteins in CHO cell membranes (Fig. 1B). As with the calciumassay, selectivity for the hM4 receptor was observed, with noquantifiable effect at the hM2 receptor. Interestingly, a smallincrease in basal [35S]GTP�S binding was also observed whenLY2033298 was applied alone to both hM4 and hM2 receptors,indicating a weak degree of allosteric agonism in addition to anyallosteric modulation. However, poor solubility of LY2033298 athigh concentrations limited the analysis and characterization ofthis allosteric agonist effect in this assay. Nonetheless, becausethe agonist effect of LY2033298 was small, we were still able toapply the simple allosteric ternary complex model to deriveapparent estimates of modulator affinity (KB � 870 � 310 nM)and cooperativity (� � 28 � 7) at the hM4 receptor.

LY2033298 Directly Enhances the Binding of Agonists but Not theAntagonist N-methyl-scopolamine (NMS). Radioligand bindingassays were performed by using the orthosteric agonist[3H]Oxotremorine-M (Oxo-M) and antagonist [3H]NMS toconfirm that the allosteric effects of LY2033298 are predom-inantly manifested directly at the level of orthosteric ligandaffinity. Fig. 2A shows that LY2033298 robustly potentiatedthe specific binding of [3H]Oxo-M in CHO hM4 cell mem-branes and in rat striatal membranes known to express a highproportion of M4 muscarinic receptor (F.P.B. and C.C.F.,unpublished data). Interestingly, although a significant poten-tiation of [3H]Oxo-M binding was retained in native rat tissue,the potency of LY2033298 at the rat M4 receptor was 5- to6-fold lower than that at the human M4 receptor, suggesting apossible species difference in the allosteric effect.

To assess the effect of LY2033298 on receptor affinity for theendogenous agonist at hM4 receptors, ACh inhibition of[3H]NMS binding was measured at equilibrium in the presenceof 0.2 mM GTP and with increasing concentrations of

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Fig. 1. LY2033298 enhances ACh responses selectively at hM4. The effect andselectivity of LY2033298 on ACh were tested by FLIPR (A) and [35S]GTP�S (B)binding, using recombinant cell lines (AV12 G�15 hM2, or hM4 and CHO hM1, hM3,or hM5) and CHO cell membranes of hM1–M5, respectively. Responses in thepresence of LY2033298 were normalized to the control maximal ACh response(100%)andbasal response (B;0%)foreachreceptor. Inbothassays,nosignificantallosteric effect was observed in hM1, hM3, or hM5 cell lines. A very modestallosteric effect on hM2 receptors was observed and a small agonist effect wasdetected at hM4. (A Inset) Illustrated is the paradigm of functional screening forthe concentration-dependent effect of LY2033298 on a submaximal dose of ACh(3 nM), which was obtained by plotting the data points along the dotted line.

Chan et al. PNAS � August 5, 2008 � vol. 105 � no. 31 � 10979

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LY2033298 (Fig. 2B). The inhibition curve in the absence ofLY2033298 had a logistic slope factor of 0.67 and, with increasingconcentrations of LY2033298, the curves were progressivelyshifted leftward without change in slope factor. The increase inACh potency was 40-fold at 3 �M LY2033298, illustrating thevery strong positive cooperativity between LY2033298 with AChfor binding to uncoupled hM4 receptors. In contrast, under theseincubation conditions, the binding of [3H]NMS was slightlyreduced at higher concentrations of LY2033298, compatiblewith a small negative cooperativity with [3H]NMS (� � 0.5).These assays demonstrated another characteristic of the alloste-ric interactions of LY2033298, probe-dependency (24). No ap-preciable radioligand displacement by LY2033298 was detectedin orthosteric binding assays for other GPCRs (e.g., dopamine,5HT, adrenergic receptor families), major ion channels (e.g.,nicotinic), signaling molecules, and enzymes (e.g., kinase family)(data not shown). Moreover, LY2033298 enhancement of[3H]Oxo-M binding was lost in M4 knockout and not in M2knockout or wild-type mouse membranes supporting the selec-tivity for M4 over the M2 receptor (data not shown).

Molecular Determinants of LY2033298 Selectivity and Allosterism atthe M4 Receptor. The orthosteric ligand binding pocket is highlyconserved between muscarinic receptor subtypes and acrossspecies (19). Among these receptors, hM2 and hM4 share theclosest sequence homology; however, we still observed signifi-cant differences in LY2033298 selectivity. Thus, we created aseries of hM4 receptor mutants [supporting information (SI)Table S1], in which divergent residues in the extracellular Nterminus and loop regions were replaced with those of hM2receptor at the corresponding sites, and measured ACh-stimulated functional responses. All mutants showed unalteredpharmacology to orthosteric agonists and antagonists comparedwith control wild-type hM4 receptors (Fig. S1). The mostdramatic loss of LY2033298 activity was noted upon progressivemutation of the third extracellular loop (o3) (Fig. 3A).LY2033298-mediated potentiation, both in terms of its potencyand magnitude of potentiation, was particularly reduced inmutant hM4-�c6.1, and a little further in hM4-�c7.1, indicatingresidue D432 is critical for this activity. Subsequent point muta-

tions confirmed that the size and charge of this residue are vitalto the potency of LY2033298 (Fig. S2).

Comparison of rat and human M4 receptor sequences at thisdomain also identified two nonconserved residues in the o3 loop,one of which corresponded to D432 in the hM4 receptor. Inagreement with the results of the [3H]Oxo-M binding assays, therM4 receptor demonstrated a decreased potency of LY2033298and a reduced potentiation of ACh-mediated calcium mobiliza-tion compared with the hM4 receptor, although the higherselectivity of LY2033298 for the M4 subtype relative to the M2subtype was retained (Fig. 3A). When the two divergent residueswithin the third extracellular loop of the hM4 receptor wereconverted to their rM4 counterparts (‘‘ratinized hM4’’), there wasa significant loss LY2033298 activity, albeit not to the degreeobserved at the wild-type rM4. However, when the reciprocalmutations were made in the rM4 receptor (‘‘humanized rM4’’),there was no gain of function (Fig. 3B). This discrepancy suggests

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Fig. 2. LY2033298 allosterically increases agonist binding to M4 receptors.(A) The specific binding of [3H]Oxo-M to CHO hM4 cell membranes and ratstriatal membranes was potentiated by LY2033298 in a dose-dependent man-ner. (B) The specific binding of [3H]NMS to hM4 receptors in the presence ofdifferent concentrations of unlabeled ACh and LY2033298 was measured inthe presence of 0.2 mM GTP. The incubation time was 2 h to allow equilibriumto be reached. The low slope factor of the ACh inhibition curve (0.67) wasunchanged in the presence of increasing concentrations of LY2033298. Thepresence of LY2033298 increased ACh potency up to 40-fold. At high concen-trations, LY2033298 appears to weakly and negatively modulate [3H]NMSbinding at hM4 receptors. The data are from one experiment repeated threetimes with quantitatively similar results.

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Fig. 3. Sequential mutations in the o3 loop of muscarinic receptors reveal acritical residue (D432) for LY2033298-mediated potentiation. Receptor mu-tants were transiently expressed in AV12 G�15 cells. The effect of LY2033298 onACh stimulated calcium mobilization was tested upon coapplication ofLY2033298 (1 nM to 1 �M) with the EC25 concentration of ACh for eachreceptor. The results were normalized to the potentiation mediated byLY2033298 on hM4 receptors, where 0% and 100% corresponded to zero andmaximum potentiation mediated by LY2033298 on hM4 receptors (see Fig. S4).(A) Mutants hM4-�6.1 and hM4-�7.1 were significantly less responsive toLY2033298 (*) when compared with hM4 receptors. The data points representsmean � S.E.M. of more than eight experiments. (B) The enhancement of AChfunction by LY2033298 for the humanized rM4 was not significantly differentfrom rM4 receptors, whereas ratinized hM4 was significantly different fromhM4 but not from rM4 receptors. The receptor selectivity of LY2033298-mediated potentiation between rM4 and rM2 receptors was sustained. Datashown are mean � SEM (n � 3).

10980 � www.pnas.org�cgi�doi�10.1073�pnas.0800567105 Chan et al.

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that the species difference observed likely involves a complexinterplay between the o3 loop and other domains/residues on therM4 receptor.

LY2033298 Displays in Vivo Efficacy in Preclinical Animal ModelsPredictive of Antispsychotic Drug Effects. When LY2033298 wasadministered alone to rats in conditioned avoidance responding(CAR), and prepulse inhibition (PPI) models and when per-forming microdialysis sampling of brain mono-amines, we didnot observe any effect- consistent with lower activity ofLY2033298 at the rodent vs. human M4 receptor. However, whencoadministered with a subeffective single dose of oxotremorine,LY2033298 was active in attenuation of CAR and reversal ofapomorphine-disrupted PPI in a dose-dependent manner (Fig. 4A and B), indicating that the compound is indeed effective in vivothrough a muscarinic mechanism. Similar results were observedin microdialysis experiments where LY2033298 positively mod-ulated the dopaminergic system in the prefrontal cortex in thepresence of an inactive dose of oxotremorine (Fig. S3), suggest-ing it had reached and affected the desired target pathway. Theeffectiveness of LY2033298 in rodent models predictive ofantipsychotic efficacy provides compelling proof-of-concept thatallosteric potentiation of the M4 muscarinic receptor is a viableapproach toward the development of muscarinic-based antipsy-chotic agents. Because of its significantly higher activity at thehuman receptor, this and related compounds may have efficacywhen given alone in human subjects.

DiscussionThis study has identified a functionally potent and selectiveallosteric potentiator of muscarinic receptors. The characteris-tics of this allosteric modulator LY2033298 include: (i) selectiv-ity for modulating ACh actions at the M4 receptor, attributableto epitopes on the receptor topographically distinct from thehighly conserved orthosteric site; (ii) saturability in the enhance-ment of ACh potency as a direct consequence of cooperativitybetween orthosteric and allosteric sites; (iii) probe dependencein the allosteric effect, because LY2033298 selectively potenti-ates the actions of orthosteric agonists at M4 receptors butinteracts very weakly with the orthosteric antagonist, [3H]NMS;and (iv) signal transduction pathway independence, because theeffects were observed in [35S]GTP�S binding and calcium mo-bilization assays.

Xanomeline is pharmacologically characterized as an M1/M4 �M2/M3 � M5 partial agonist with modest interaction at serotoninreceptors (6, 25). The clinical efficacy detailed in the Introductionraises the question as to which muscarinic receptor(s) to focus onfor the indication of schizophrenia. In the absence of selectivepharmacology, muscarinic receptor knockout mice have providedevidence encouraging a focus on the M4 receptor as a logical targetfor modulation of dopamine neurotransmission (17) in areasthought to be disrupted in schizophrenia (18). BuTAC and PTAC,potent molecules with M2/M4 agonist, M1/M3/M5 antagonist activ-ity, were active in models predictive of antipsychotic efficacy (26,27). Together with our data on LY2033298, these findings supporta role for M4 as potentially sufficient to treat symptoms of schizo-phrenia. Because M1 receptor involvement in cognitive processeshas been well documented (28, 29), a combination of M1 and M4agonistic activity may offer some additional efficacy in the clinic.

Previous investigations identified subtype-selective musca-rinic allosteric enhancers (23, 30–32). However, these moleculesdisplayed much lower affinity and cooperativity with ACh. Aparticularly striking finding from our functional assays was thehigh degree of positive cooperativity between the modulator andACh at the hM4 receptor (��30), which is the highest formuscarinic receptors reported to date and is sufficient to impartbehavioral responses in animal models predictive of antipsy-chotic drug effects at reasonable therapeutic doses. Radioligandbinding experiments using recombinant hM4 and native rM4membrane preparations confirmed that the allosteric effects ofLY2033298 are mediated predominantly by enhancement ofagonist binding while having close to neutral effects on antag-onist binding. [35S]GTP�S binding experiments indicate a lowintrinsic efficacy of LY2033298 at the hM2 and hM4 receptors inthe absence of added ACh consistent with an allosteric partialagonist of very low efficacy (33, 34).

To date, limited mutagenesis studies of muscarinic receptorshave focused on a common allosteric binding site on M1, M2, andM5 receptor subtypes (33, 35) and M3 (36, 37) and M4 receptors(38) used by prototypical negative allosteric modulators, such asgallamine, alcuronium, and C7/3-phth (21). Functional screeningof a wide range of receptor mutants systematically pointed to theacidic residue D432 of the hM4 receptor as being critical to thepotentiation of ACh by LY2033298 (Fig. 3A). Additional studieson the rM4 receptor, which shows a lower cooperativity withLY2033298, confirmed the importance of the o3 region of thereceptor in the actions of the modulator (Fig. 3B). However, ahumanized rM4 mutant did not show a gain-of-function inenhancing the actions of LY2033298, suggesting that a morecomplex network of interactions with other residues must also beinvolved. In general, the finding that D432 was critical to thecooperativity between LY2033298 and ACh at the hM4 receptorcontrasts with residues important for the action of less robustallosteric modulators, which, for the hM2 receptors, have beenlocalized to the o2 loop and the top of transmembrane domain7 (38, 39). However, an unequivocal demonstration thatLY2033298 interacts with a second allosteric site that is topo-graphically distinct from that used by prototypical muscarinicmodulators requires further pharmacological and structuralcharacterization.

A key finding of our study was the demonstration of in vivoefficacy of a muscarinic allosteric potentiator in rodent preclinicalmodels predictive of antipsychotic drug effects. Allosteric sites arenot expected to have evolved to accommodate a common endog-enous neurotransmitter or hormone; they are more likely to showhigher sequence divergence across receptor subtypes (which rep-resents a therapeutic targeting advantage) but may also showvariation between species, which represents a disadvantage in termsof target validation and/or preclinical efficacy and safety studies inanimal models. Transgenic approaches to knock-in humanizedreceptor proteins into mice may be helpful for progressing allosteric

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Fig. 4. LY2033298-mediated potentiation is effective in rat CAR and PPIpsychosis models. (A) LY2033298 reduced conditioned avoidance respond-ing (CAR) in trained male HSD rats in a dose-dependent manner in thepresence of an inactive dose of oxotremorine (*, P � 0.05 versus vehicle).(B) Apomorphine-induced suppression of the acoustic startle response wasreversed by LY2033298 in a dose-dependent manner in the presence of asubeffective dose of Oxo. Data are mean percentage prepulse inhibition(PPI) values (V, vehicle; Apo, apomorphine; Oxo, oxotremorine; LY,2033298) (#, P � 0.05 versus V/V/Apo; *, P � 0.05 versus V/V/V).

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drug leads showing high human receptor selectivity. Alternatively,allosteric compounds may be coadministered with inactive doses oforthosteric agonists.

Arguably, little innovation has been made in the treatment ofschizophrenia for the past several decades. Frontline treatments aregenerally compounds with complex broad GPCR antagonist phar-macology with highest affinity as antagonists for D2-type dopaminereceptors and 5HT2 serotonin receptors (4). GPCR agonist mech-anisms have largely failed or, at best, show temporary efficacybecause of issues of receptor desensitization or down regulation(40). Aripiprazole may be an exception because of its partial agonistactivity at D2 receptors, although the exact mechanism of action ofthis compound has been controversial (41). More recent prelimi-nary studies suggest antipsychotic efficacy through agonism of type2 and 3 metabotropic glutamate receptors (42). Muscarinic selec-tive agonism with xanomeline (although not completely muscarinicreceptor subtype selective) has shown interesting promise in man-aging Alzheimer’s-related psychosis and schizophrenia symptoms,including positive, negative, and cognitive domains (6, 9). AnM4-selective drug that provides modest or self-limiting agonismmay be preferable to a full orthosteric agonist, although it is notclear how much receptor agonism (or modulator affinity andcooperativity) will be required in patients to achieve maximaltherapeutic efficacy and tolerable side effects. Studies to dateindicate that M4 receptors are largely unchanged in schizophrenicpatients with the exception of a potential modest decrease inhippocampus, suggesting that M4 agonist-based therapies would

remain relevant during disease progression (43, 44). Our rodentstudies, although preliminary, provide support for a highly M4receptor-selective allosteric potentiator to control symptoms with-out inducing sedation or catalepsy observed with atypical antago-nist antipsychotics such as clozapine. The allosteric mechanisminherent in LY2033298 may appropriately modulate imbalanceddopamine and glutamate neurotransmission, thus representing apotential first-in-class muscarinic antipsychotic therapy that doesnot involve direct targeting of dopamine and serotonin receptors.

Materials and MethodsReagents. 3-Amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298; C13H14ClN3O2S; molecularweight � 311.8) (Fig. 1A) was synthesized by Eli Lilly & Co.. LY2033298 issoluble in buffer or in 100% DMSO up to 4 mM and is stable as a dry powderat room temperature or as a frozen DMSO stock solution. The human musca-rinic receptors were stably expressed in cell lines as described in SI Methods.

Methods. Signal transduction and radioligand binding assays and data anal-yses were performed as described in refs. 24, 45, and 46 with modifications asdetailed in SI Methods. Rat behavioral assays, conditioned avoidance respond-ing (CAR), and prepulse inhibition of the acoustic startle reflex (PPI) wereperformed as described in ref. 47 with modifications as detailed in SI Methods.Mutagenesis and receptor expression methods are described in SI Methods.

ACKNOWLEDGMENTS. We thank George Nomikos and Angela Popham fortheir key contributions to this manuscript. A.C. is a Senior Research Fellow ofthe National Health and Medical Research Council of Australia. This work wassupported by Eli Lilly & Co. and the Medical Research Council, United Kingdom(N.J.M.B. and S.L.).

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