Allosteric Modulation of the Muscarinic M4 Receptor As an Approach to Treating Schizophrenia

Allosteric modulation of the muscarinic M4 receptor as an approach to treating schizophrenia

W. Y. Chan*, D. L. McKinzie†, S. Bose*, S. N. Mitchell*, J. M. Witkin†, R. C. Thompson‡, A. Christopoulos§, S. Lazareno¶, N. J. M. Birdsallʈ, F. P. Bymaster†, and C. C. Felder*†**

*Neuroscience Discovery Research, Lilly Research Centre, Surrey GU20 6PH, United Kingdom; †Neuroscience Division and ‡Discovery Chemistry and Research Technologies, 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 ʈDivision of Physical Biochemistry, Medical Research Council National 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 patients types, in the pathophysiology of schizophrenia (5, 6). Muscarinic suffering from schizophrenia and related psychoses; however, receptors are genetically linked to schizophrenia and cognitive their effectiveness is variable and many patients discontinue treat- deficits (7, 8). Pharmacologically, relatively non-selective mus- ment due to side effects. Although the etiology of schizophrenia is carinic antagonists exacerbate, whereas agonists ameliorate, still unclear, a leading hypothesis implicates an imbalanced dopa- cognitive deficits and psychotic behaviors in animal models and minergic system. Muscarinic acetylcholine (ACh) receptors regulate in patients suffering from Alzheimer’s disease and schizophrenia dopamine levels in key areas of the brain involved in psychosis, (9–12). In particular, the M1/M4 preferring partial agonist, with the M4 subtype emerging as a key regulator of dopaminergic xanomeline, was found to be efficacious in animal models hyperactivity. Unfortunately, no selective small molecule tools predictive of antipsychotic behaviors through modulation of exist to provide pharmacological validation of this hypothesis. both dopamine and glutamate transmission (13). In exploratory Here, we describe the discovery of a small molecule modulator, clinical trials, xanomeline reduced psychotic behaviors and improved cognitive measures in Alzheimer’s patients (9), and LY2033298, that is highly selective for human M4 receptors by virtue of targeting an allosteric site on this receptor. Pharmaco- significantly improved positive, negative, and cognitive symptoms in a small schizophrenia trial (12). Unfortunately, lack of logical assays confirmed the selectivity of LY2033298 for the M 4 receptor subtype selectivity led to undesirable side-effects (gas- receptor and revealed the highest degree of positive allosteric trointestinal disturbances, blood pressure dysregulation) that enhancement of ACh potency thus far identified. Radioligand rendered xanomeline as an unsuitable candidate for further binding assays also show this compound to directly potentiate clinical development (14). agonist binding while having minimal effects on antagonist bind- M receptors are most highly expressed in brain regions rich 432 4 ing. Mutational analysis identified a key amino acid (D )inthe in dopamine and dopamine receptors (15, 16). Experiments with third extracellular loop of the human M receptor to be critical for 4 muscarinic knock out mice have implicated the M4 receptor selectivity and agonist potentiation by LY2033298. Importantly, subtype in regulating dopaminergic neurons involved in move- LY2033298 was active in animal models predictive of clinical ment control and cognition (17, 18); therefore, we reasoned that antipsychotic drug efficacy indicating its potential use as a first- a highly selective M4 muscarinic receptor activator may represent in-class, selective, allosteric muscarinic antipsychotic agent. a useful candidate for the development of muscarinic-based antipsychotic agents. Unfortunately, attempts at discovering cholinergic ͉ GPCR ͉ cooperativity selective small molecule ligands for the five muscarinic receptor subtypes have largely been unsuccessful when targeted at the chizophrenia is a complex disease presenting a broad spec- receptors’ orthosteric site, i.e., the binding site for the endoge- Strum of endophenotypes that can be generalized to symp- nous agonist ACh, which is highly conserved across all five toms in three major domains: positive (hallucinations, hearing of muscarinic subtypes (19). This has been particularly true for the voices, delusions and disorganized thinking), negative (anhedo- ACh-mimetics, such as xanomeline, sabcomeline, and melame- line (20). However, it is also known that muscarinic receptors nia, flat affect), and cognitive (attention and working memory possess allosteric binding sites that have the potential to show deficits). The etiological basis for the disease is believed to derive greater sequence divergence across subtypes and, hence, may predominantly from dysregulation of dopamine and glutamate provide an alternative means of attaining receptor subtype neurotransmission pathways in mesocortical and mesolimbic selectivity (21, 22). Allosteric modulators also offer the addi- brain areas (1–3). Atypical antipsychotics are the current front- tional potential of maintaining both spatial and temporal neu- line treatment for schizophrenia, ameliorating the positive symp- rotransmission through modulation of physiologically relevant toms in approximately half of the treated patient population with receptor-mediated neural regulation, as opposed to direct (con- little efficacy at the negative and cognitive symptoms. Most atypical antipsychotics are broad spectrum G protein-coupled receptor (GPCR) antagonists, and their therapeutic action is 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., and mediated primarily through inhibition of dopamine D2,D3 and C.C.F. performed research; R.C.T. contributed new reagents/analytic tools; W.Y.C., S.B., D4, and serotonin 5HT2A, receptors (4). Their complex phar- 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., macology, however, leads to significant undesirable side effects, N.J.M.B., and C.C.F. wrote the paper. including movement disorders and weight gain. Therefore, de- 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. veloping antipsychotics through an alternative mechanism may are or were employed by Eli Lilly & Co. provide better total symptom control and reduced side effects. This article is a PNAS Direct Submission. The family of five muscarinic acetylcholine (ACh) receptors Freely available online through the PNAS open access option. plays a prominent role in regulating neurotransmission in the **To whom correspondence should be addressed at: Eli Lilly & Co. Research Laboratories, CNS and may provide a mechanism for antipsychotic drug Lilly Corporate Center, Indianapolis, IN 46285. E-mail: [email protected]. discovery. Studies using muscarinic receptor knock out mice This article contains supporting information online at www.pnas.org/cgi/content/full/ have provided valuable insight into the potential role of these 0800567105/DCSupplemental. receptors, especially the muscarinic receptor M1 and M4 sub- © 2008 by The National Academy of Sciences of the USA

10978–10983 ͉ PNAS ͉ August 5, 2008 ͉ vol. 105 ͉ no. 31 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800567105 Downloaded by guest on September 25, 2021 activation, using cell lines stably expressing the hM1–M5 recep- A tors coupled to intrinsic or engineered calcium signaling pathways. No agonist activity at any muscarinic receptor subtype was detected when LY2033298 was applied alone. However, upon addition of a submaximal concentration of ACh, an elevated calcium response was readily detected in the hM4 cell lines relative to the effect of the same concentration of ACh in the absence of LY2033298. Subsequent assays constructed complete concentration-response curves to ACh in the presence of increasing concentrations of LY2033298. As shown in Fig. 1A, these experiments revealed a prototypical characteristic of positive allosteric interactions: a robust potentiation of agonist potency that reached a limit at the highest concentrations of allosteric modulator above which no further effect was obtained (24). Application of an allosteric ternary complex model to the data yielded KB ϭ 200 Ϯ 40 nM for LY2033298 at the allosteric site on the unoccupied hM4 receptor, and ␣ ϭ 35 Ϯ 4 for the degree of allosteric enhancement when both orthosteric and allosteric sites are occupied; to our knowledge, this is the highest degree of positive cooperativity reported for any allosteric modulator of a muscarinic receptor. In addition, these experiments also revealed the high selectivity of LY2033298 for the hM4 receptor, because there was no effect at hM1/3/5 receptors at B any concentration of modulator and a very small allosteric potentiation at the closest homolog, hM2 (KB ϭ 1.0 Ϯ 0.3 ␮M; ␣ ϭ 3.7 Ϯ 0.5). The positive allosteric effect of LY2033298 at the hM4 receptor was not unique to ACh, because it also selectively

potentiated other full and partial orthosteric agonists, such as NEUROSCIENCE carbachol, oxotremorine-M, and McN-A343 (data not shown). To confirm that the allosteric effect detected in the calcium assay was neither unique to this signaling pathway or a consequence of engineering the coupling of the hM4 receptor to this pathway via the use of promiscuous G␣15 proteins, additional experiments were performed measuring ACh-mediated [35S]guanosine-␥-S–triphosphates (GTP␥S) binding to native G proteins in CHO cell membranes (Fig. 1B). As with the calcium assay, selectivity for the hM4 receptor was observed, with no quantifiable effect at the hM2 receptor. Interestingly, a small increase in basal [35S]GTP␥S binding was also observed when LY2033298 was applied alone to both hM4 and hM2 receptors, indicating a weak degree of allosteric agonism in addition to any allosteric modulation. However, poor solubility of LY2033298 at high concentrations limited the analysis and characterization of this allosteric agonist effect in this assay. Nonetheless, because Fig. 1. LY2033298 enhances ACh responses selectively at hM4. The effect and the agonist effect of LY2033298 was small, we were still able to selectivity of LY2033298 on ACh were tested by FLIPR (A) and [35S]GTP␥S(B) apply the simple allosteric ternary complex model to derive binding, using recombinant cell lines (AV12 G␣15 hM2,orhM4 and CHO hM1,hM3, apparent estimates of modulator affinity (KB ϭ 870 Ϯ 310 nM) or hM5) and CHO cell membranes of hM1–M5, respectively. Responses in the ␣ ϭ Ϯ presence of LY2033298 were normalized to the control maximal ACh response and cooperativity ( 28 7) at the hM4 receptor. (100%) and basal response (B; 0%) for each receptor. In both assays, no signiﬁcant allosteric effect was observed in hM1,hM3,orhM5 cell lines. A very modest LY2033298 Directly Enhances the Binding of Agonists but Not the allosteric effect on hM2 receptors was observed and a small agonist effect was Antagonist N-methyl-scopolamine (NMS). Radioligand binding detected at hM4.(A Inset) Illustrated is the paradigm of functional screening for assays were performed by using the orthosteric agonist the concentration-dependent effect of LY2033298 on a submaximal dose of ACh [3H]Oxotremorine-M (Oxo-M) and antagonist [3H]NMS to (3 nM), which was obtained by plotting the data points along the dotted line. confirm that the allosteric effects of LY2033298 are predominantly manifested directly at the level of orthosteric ligand affinity. Fig. 2A shows that LY2033298 robustly potentiated tinuous) agonism or antagonism (23, 24). Here, we describe the the specific binding of [3H]Oxo-M in CHO hM cell mem- exploitation of this concept to the discovery and molecular 4 branes and in rat striatal membranes known to express a high characterization of LY2033298 [3-amino-5-chloro-6-methoxy-4- methyl-thieno(2,3-b)pyridine-2-carboxylic acid cyclopropylam- proportion of M4 muscarinic receptor (F.P.B. and C.C.F., ϭ unpublished data). Interestingly, although a significant poten- ide (C13H14ClN3O2S; molecular weight 311.8)] (Fig. 1A), a 3 highly selective positive allosteric potentiator of ACh actions at tiation of [ H]Oxo-M binding was retained in native rat tissue, the potency of LY2033298 at the rat M receptor was 5- to the M4 muscarinic receptor that also shows significant efficacy 4 in preclinical models predictive of antipsychotic drug behavior. 6-fold lower than that at the human M4 receptor, suggesting a possible species difference in the allosteric effect. Results To assess the effect of LY2033298 on receptor affinity for the LY2033298 Is a Selective Positive Allosteric Modulator of ACh at endogenous agonist at hM4 receptors, ACh inhibition of 3 Human Muscarinic M4 Receptors. Initial screening quantified intra- [ H]NMS binding was measured at equilibrium in the presence cellular calcium mobilization as a readout of muscarinic receptor of 0.2 mM GTP and with increasing concentrations of

Chan et al. PNAS ͉ August 5, 2008 ͉ vol. 105 ͉ no. 31 ͉ 10979 Downloaded by guest on September 25, 2021 A B A hM4(o3) NTFCQSCIPDTVWS hM4-Δ5.1 NTFCQSCIPDTVWT hM4-Δ6.1 NTFCQSCIPNTVWT hM4-Δ7.1 NTFCASCIPNTVWT 110 hM2(o3) NTFCAPCIPNTVWT 100 ) ( ) hM4 90 80 ( ) hM4-Δ5.1 70 60 50 40 ( ) ∗ Δ 30 hM4- 6.1 ∗ 20 ( ) hM4-Δ7.1 10 ∗ ( ) hM2 LY2033298 mediated potentiation mediated LY2033298 (%) Fig. 2. LY2033298 allosterically increases agonist binding to M4 receptors. mediatedLY2033298 potentiation(% 0 (A) The specific binding of [3H]Oxo-M to CHO hM4 cell membranes and rat striatal membranes was potentiated by LY2033298 in a dose-dependent man- -9 -8 -7 -6 3 ner. (B) The specific binding of [ H]NMS to hM4 receptors in the presence of log [LY2033298], M different concentrations of unlabeled ACh and LY2033298 was measured in the presence of 0.2 mM GTP. The incubation time was2htoallow equilibrium B hM4(o3) NTFCQSCIPDTVWS to be reached. The low slope factor of the ACh inhibition curve (0.67) was 110 rM4(o3) NTFCQSCIPERVWS unchanged in the presence of increasing concentrations of LY2033298. The ) 100 ( ) hM4 presence of LY2033298 increased ACh potency up to 40-fold. At high concen- 90 3 trations, LY2033298 appears to weakly and negatively modulate [ H]NMS 80 binding at hM4 receptors. The data are from one experiment repeated three 70 ( )Ratinized hM4 ( ) ratinized hM4 times with quantitatively similar results. 60 (o3) DT(o3)DT to ER to ER 50 rM4 40 ( ) humanized rM4 edi ated potent i at i on (% ( ) LY2033298 (Fig. 2B). The inhibition curve in the absence of 30 ( )Humanized(o3)ER to DT rM4 LY2033298 had a logistic slope factor of 0.67 and, with increasing 20 (o3) ER to DT concentrations of LY2033298, the curves were progressively 10 ( ) rM2 shifted leftward without change in slope factor. The increase in 0 ACh potency was 40-fold at 3 ␮M LY2033298, illustrating the m LY2033298 -10 very strong positive cooperativity between LY2033298 with ACh potentiation mediated LY2033298 (%) -9 -8 -7 -6 Log[LY2033298], M for binding to uncoupled hM4 receptors. In contrast, under these 3 incubation conditions, the binding of [ H]NMS was slightly Fig. 3. Sequential mutations in the o3 loop of muscarinic receptors reveal a reduced at higher concentrations of LY2033298, compatible critical residue (D432) for LY2033298-mediated potentiation. Receptor mu- 3 ␣ Х with a small negative cooperativity with [ H]NMS ( 0.5). tants were transiently expressed in AV12 G␣15 cells. The effect of LY2033298 on These assays demonstrated another characteristic of the alloste- ACh stimulated calcium mobilization was tested upon coapplication of ␮ ric interactions of LY2033298, probe-dependency (24). No ap- LY2033298 (1 nM to 1 M) with the EC25 concentration of ACh for each preciable radioligand displacement by LY2033298 was detected receptor. The results were normalized to the potentiation mediated by in orthosteric binding assays for other GPCRs (e.g., dopamine, LY2033298 on hM4 receptors, where 0% and 100% corresponded to zero and maximum potentiation mediated by LY2033298 on hM4 receptors (see Fig. S4). 5HT, adrenergic receptor families), major ion channels (e.g., (A) Mutants hM4-⌬6.1 and hM4-⌬7.1 were significantly less responsive to nicotinic), signaling molecules, and enzymes (e.g., kinase family) LY2033298 (*) when compared with hM4 receptors. The data points represents (data not shown). Moreover, LY2033298 enhancement of mean Ϯ S.E.M. of more than eight experiments. (B) The enhancement of ACh 3 [ H]Oxo-M binding was lost in M4 knockout and not in M2 function by LY2033298 for the humanized rM4 was not significantly different knockout or wild-type mouse membranes supporting the selec- from rM4 receptors, whereas ratinized hM4 was significantly different from hM4 but not from rM4 receptors. The receptor selectivity of LY2033298- tivity for M4 over the M2 receptor (data not shown). mediated potentiation between rM4 and rM2 receptors was sustained. Data shown are mean Ϯ SEM (n Ն 3). Molecular Determinants of LY2033298 Selectivity and Allosterism at the M4 Receptor. The orthosteric ligand binding pocket is highly conserved between muscarinic receptor subtypes and across tions confirmed that the size and charge of this residue are vital species (19). Among these receptors, hM2 and hM4 share the to the potency of LY2033298 (Fig. S2). closest sequence homology; however, we still observed signifi- Comparison of rat and human M4 receptor sequences at this cant differences in LY2033298 selectivity. Thus, we created a domain also identified two nonconserved residues in the o3 loop, 432 series of hM4 receptor mutants [supporting information (SI) one of which corresponded to D in the hM4 receptor. In Table S1], in which divergent residues in the extracellular N agreement with the results of the [3H]Oxo-M binding assays, the terminus and loop regions were replaced with those of hM2 rM4 receptor demonstrated a decreased potency of LY2033298 receptor at the corresponding sites, and measured ACh- and a reduced potentiation of ACh-mediated calcium mobiliza- stimulated functional responses. All mutants showed unaltered tion compared with the hM4 receptor, although the higher pharmacology to orthosteric agonists and antagonists compared selectivity of LY2033298 for the M4 subtype relative to the M2 with control wild-type hM4 receptors (Fig. S1). The most subtype was retained (Fig. 3A). When the two divergent residues dramatic loss of LY2033298 activity was noted upon progressive within the third extracellular loop of the hM4 receptor were mutation of the third extracellular loop (o3) (Fig. 3A). converted to their rM4 counterparts (‘‘ratinized hM4’’), there was LY2033298-mediated potentiation, both in terms of its potency a significant loss LY2033298 activity, albeit not to the degree and magnitude of potentiation, was particularly reduced in observed at the wild-type rM4. However, when the reciprocal mutant hM4-⌬c6.1, and a little further in hM4-⌬c7.1, indicating mutations were made in the rM4 receptor (‘‘humanized rM4’’), residue D432 is critical for this activity. Subsequent point muta- there was no gain of function (Fig. 3B). This discrepancy suggests

10980 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800567105 Chan et al. Downloaded by guest on September 25, 2021 Xanomeline is pharmacologically characterized as an M1/M4 Ͼ 100 80 A B M2/M3 Ͼ M5 partial agonist with modest interaction at serotonin receptors (6, 25). The clinical efficacy detailed in the Introduction 75 60 * raises the question as to which muscarinic receptor(s) to focus on 50 40 for the indication of schizophrenia. In the absence of selective pharmacology, muscarinic receptor knockout mice have provided 25 * 20 evidence encouraging a focus on the M4 receptor as a logical target

Avoidance Response (%) Response Avoidance for modulation of dopamine neurotransmission (17) in areas 0 Percent Prepulse Inhibition 0

o o o o thought to be disrupted in schizophrenia (18). BuTAC and PTAC, /V p p p p V A A Vehicle / /A /A / / potent molecules with M2/M4 agonist, M1/M3/M5 antagonist activ- V V o o o + 3 mg/kg / x x x + 10 mg/kg+ 30 mg/kg V O O O Oxo+ 0 mg/kg / / / ity, were active in models predictive of antipsychotic efficacy (26, V 0 0 1 3 Y Y 27). Together with our data on LY2033298, these findings support L L a role for M4 as potentially sufficient to treat symptoms of schizo- Fig. 4. LY2033298-mediated potentiation is effective in rat CAR and PPI phrenia. Because M1 receptor involvement in cognitive processes psychosis models. (A) LY2033298 reduced conditioned avoidance respond- has been well documented (28, 29), a combination of M1 and M4 ing (CAR) in trained male HSD rats in a dose-dependent manner in the agonistic activity may offer some additional efficacy in the clinic. presence of an inactive dose of oxotremorine (*, P Ͻ 0.05 versus vehicle). Previous investigations identified subtype-selective musca- (B) Apomorphine-induced suppression of the acoustic startle response was rinic allosteric enhancers (23, 30–32). However, these molecules reversed by LY2033298 in a dose-dependent manner in the presence of a displayed much lower affinity and cooperativity with ACh. A subeffective dose of Oxo. Data are mean percentage prepulse inhibition particularly striking finding from our functional assays was the (PPI) values (V, vehicle; Apo, apomorphine; Oxo, oxotremorine; LY, high degree of positive cooperativity between the modulator and 2033298) (#, P Ͻ 0.05 versus V/V/Apo; *, P Ͻ 0.05 versus V/V/V). ACh at the hM4 receptor (␣Ϸ30), which is the highest for muscarinic receptors reported to date and is sufficient to impart that the species difference observed likely involves a complex behavioral responses in animal models predictive of antipsy- interplay between the o3 loop and other domains/residues on the chotic drug effects at reasonable therapeutic doses. Radioligand binding experiments using recombinant hM and native rM rM4 receptor. 4 4 membrane preparations confirmed that the allosteric effects of LY2033298 Displays in Vivo Efficacy in Preclinical Animal Models LY2033298 are mediated predominantly by enhancement of NEUROSCIENCE Predictive of Antispsychotic Drug Effects. When LY2033298 was agonist binding while having close to neutral effects on antag- 35 ␥ administered alone to rats in conditioned avoidance responding onist binding. [ S]GTP S binding experiments indicate a low intrinsic efficacy of LY2033298 at the hM and hM receptors in (CAR), and prepulse inhibition (PPI) models and when per- 2 4 the absence of added ACh consistent with an allosteric partial forming microdialysis sampling of brain mono-amines, we did agonist of very low efficacy (33, 34). not observe any effect- consistent with lower activity of To date, limited mutagenesis studies of muscarinic receptors LY2033298 at the rodent vs. human M4 receptor. However, when have focused on a common allosteric binding site on M1,M2, and coadministered with a subeffective single dose of oxotremorine, M5 receptor subtypes (33, 35) and M3 (36, 37) and M4 receptors LY2033298 was active in attenuation of CAR and reversal of (38) used by prototypical negative allosteric modulators, such as apomorphine-disrupted PPI in a dose-dependent manner (Fig. 4 gallamine, alcuronium, and C7/3-phth (21). Functional screening A and B), indicating that the compound is indeed effective in vivo of a wide range of receptor mutants systematically pointed to the 432 through a muscarinic mechanism. Similar results were observed acidic residue D of the hM4 receptor as being critical to the in microdialysis experiments where LY2033298 positively mod- potentiation of ACh by LY2033298 (Fig. 3A). Additional studies ulated the dopaminergic system in the prefrontal cortex in the on the rM4 receptor, which shows a lower cooperativity with presence of an inactive dose of oxotremorine (Fig. S3), suggest- LY2033298, confirmed the importance of the o3 region of the ing it had reached and affected the desired target pathway. The receptor in the actions of the modulator (Fig. 3B). However, a effectiveness of LY2033298 in rodent models predictive of humanized rM4 mutant did not show a gain-of-function in antipsychotic efficacy provides compelling proof-of-concept that enhancing the actions of LY2033298, suggesting that a more allosteric potentiation of the M4 muscarinic receptor is a viable complex network of interactions with other residues must also be approach toward the development of muscarinic-based antipsy- involved. In general, the finding that D432 was critical to the chotic agents. Because of its significantly higher activity at the cooperativity between LY2033298 and ACh at the hM4 receptor human receptor, this and related compounds may have efficacy contrasts with residues important for the action of less robust when given alone in human subjects. allosteric modulators, which, for the hM2 receptors, have been localized to the o2 loop and the top of transmembrane domain Discussion 7 (38, 39). However, an unequivocal demonstration that This study has identified a functionally potent and selective LY2033298 interacts with a second allosteric site that is topographically distinct from that used by prototypical muscarinic allosteric potentiator of muscarinic receptors. The characteris- modulators requires further pharmacological and structural tics of this allosteric modulator LY2033298 include: (i) selectiv- characterization. ity for modulating ACh actions at the M4 receptor, attributable A key finding of our study was the demonstration of in vivo to epitopes on the receptor topographically distinct from the efficacy of a muscarinic allosteric potentiator in rodent preclinical highly conserved orthosteric site; (ii) saturability in the enhance- models predictive of antipsychotic drug effects. Allosteric sites are ment of ACh potency as a direct consequence of cooperativity not expected to have evolved to accommodate a common endog- between orthosteric and allosteric sites; (iii) probe dependence enous neurotransmitter or hormone; they are more likely to show in the allosteric effect, because LY2033298 selectively potenti- higher sequence divergence across receptor subtypes (which rep- ates the actions of orthosteric agonists at M4 receptors but resents a therapeutic targeting advantage) but may also show interacts very weakly with the orthosteric antagonist, [3H]NMS; variation between species, which represents a disadvantage in terms and (iv) signal transduction pathway independence, because the of target validation and/or preclinical efficacy and safety studies in effects were observed in [35S]GTP␥S binding and calcium mo- animal models. Transgenic approaches to knock-in humanized bilization assays. receptor proteins into mice may be helpful for progressing allosteric

Chan et al. PNAS ͉ August 5, 2008 ͉ vol. 105 ͉ no. 31 ͉ 10981 Downloaded by guest on September 25, 2021 drug leads showing high human receptor selectivity. Alternatively, remain relevant during disease progression (43, 44). Our rodent allosteric compounds may be coadministered with inactive doses of studies, although preliminary, provide support for a highly M4 orthosteric agonists. receptor-selective allosteric potentiator to control symptoms with- Arguably, little innovation has been made in the treatment of out inducing sedation or catalepsy observed with atypical antago- schizophrenia for the past several decades. Frontline treatments are nist antipsychotics such as clozapine. The allosteric mechanism generally compounds with complex broad GPCR antagonist phar- inherent in LY2033298 may appropriately modulate imbalanced macology with highest affinity as antagonists for D2-type dopamine dopamine and glutamate neurotransmission, thus representing a potential first-in-class muscarinic antipsychotic therapy that does receptors and 5HT2 serotonin receptors (4). GPCR agonist mechanisms have largely failed or, at best, show temporary efficacy not involve direct targeting of dopamine and serotonin receptors. because of issues of receptor desensitization or down regulation Materials and Methods (40). Aripiprazole may be an exception because of its partial agonist Reagents. 3-Amino-5-chloro-6-methoxy-4-methyl-thieno[2,3-b]pyridine-2- activity at D2 receptors, although the exact mechanism of action of carboxylic acid cyclopropylamide (LY2033298; C13H14ClN3O2S; molecular this compound has been controversial (41). More recent prelimi- weight ϭ 311.8) (Fig. 1A) was synthesized by Eli Lilly & Co.. LY2033298 is nary studies suggest antipsychotic efficacy through agonism of type soluble in buffer or in 100% DMSO up to 4 mM and is stable as a dry powder 2 and 3 metabotropic glutamate receptors (42). Muscarinic selec- at room temperature or as a frozen DMSO stock solution. The human musca- tive agonism with xanomeline (although not completely muscarinic rinic receptors were stably expressed in cell lines as described in SI Methods. receptor subtype selective) has shown interesting promise in man- aging Alzheimer’s-related psychosis and schizophrenia symptoms, Methods. Signal transduction and radioligand binding assays and data anal- including positive, negative, and cognitive domains (6, 9). An yses were performed as described in refs. 24, 45, and 46 with modifications as detailed in SI Methods. Rat behavioral assays, conditioned avoidance respond- M4-selective drug that provides modest or self-limiting agonism ing (CAR), and prepulse inhibition of the acoustic startle reflex (PPI) were may be preferable to a full orthosteric agonist, although it is not performed as described in ref. 47 with modifications as detailed in SI Methods. clear how much receptor agonism (or modulator affinity and Mutagenesis and receptor expression methods are described in SI Methods. cooperativity) will be required in patients to achieve maximal therapeutic efficacy and tolerable side effects. Studies to date ACKNOWLEDGMENTS. We thank George Nomikos and Angela Popham for indicate that M receptors are largely unchanged in schizophrenic their key contributions to this manuscript. A.C. is a Senior Research Fellow of 4 the National Health and Medical Research Council of Australia. This work was patients with the exception of a potential modest decrease in supported by Eli Lilly & Co. and the Medical Research Council, United Kingdom hippocampus, suggesting that M4 agonist-based therapies would (N.J.M.B. and S.L.).

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