Translational Pharmacology of the Metabotropic Glutamate 2 Receptor-Preferring Agonist LY2812223 in the Animal and Human Brain.

LY2812223 [(1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid] was identified via structure-activity studies arising from the potent metabotropic glutamate mGlu2/3 receptor agonist LY354740 [(+)-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylic acid] as an mGlu2-preferring agonist. This pharmacology was determined using stably transfected cells containing either the human mGlu2 or mGlu3 receptor. We extended the pharmacological evaluation of LY2812223 to native brain tissues derived from relevant species used for preclinical drug development as well as human postmortem brain tissue. This analysis was conducted to ensure pharmacological translation from animals to human subjects in subsequent clinical studies. A guanosine 5'-O-(3-[35S]thio)triphosphate (GTPγS) functional binding assay, a method for measuring Gi-coupled signaling that is inherent to the group 2 mGlu receptors, was used to evaluate LY2812223 pharmacology of native mGlu receptors in mouse, rat, nonhuman primate, and human cortical brain tissue samples. In native tissue membranes, LY2812223 unexpectedly acted as a partial agonist across all species tested. Activity of LY2812223 was lost in cortical membranes collected from mGlu2 knockout mice, but not those from mGlu3 knockout mice, providing additional support for mGlu2-preferring activity. Other signal transduction assays were used for comparison with the GTP binding assay (cAMP, calcium mobilization, and dynamic mass redistribution). In ectopic cell line-based assays, LY2812223 displayed near maximal agonist responses at the mGlu2 receptor across all assay formats, while it showed no functional agonist activity at the mGlu3 receptor except in the cAMP assay. In native brain slices or membranes that express both mGlu2 and mGlu3 receptors, LY2812223 displayed unexpected partial agonist activity, which may suggest a functional interplay between these receptor subtypes in the brain.

Characterization of PCS1055, a novel muscarinic M4 receptor antagonist.

Identification of synthetic ligands selective for muscarinic receptor subtypes has been challenging due to the high sequence identity and structural homology among the five muscarinic acetylcholine receptors. Here, we report the pharmacological characterization of PCS1055, a novel muscarinic M4 receptor antagonist. PCS1055 inhibited radioligand [(3)H]-NMS binding to the M4 receptor with a Ki=6.5nM. Though the potency of PCS1055 is lower than that of pan-muscarinic antagonist atropine, it has better subtype selectivity over previously reported M4-selective reagents such as the muscarinic-peptide toxins (Karlsson et al., 1994; Santiago and Potter, 2001a) at the M1 subtype, and benzoxazine ligand PD102807 at the M3-subtype (Bohme et al., 2002). A detailed head-to-head comparison study using [(3)H]-NMS competitive binding assays characterizes the selectivity profiles of PCS1055 to that of other potent muscarinic-antagonist compounds PD102807, tropicamide, AF-DX-384, pirenzapine, and atropine. In addition to binding studies, the subtype specificity of PCS1055 is also demonstrated by functional receptor activation as readout by GTP-γ-[(35)S] binding. These GTP-γ-[(35)S] binding studies showed that PCS1055 exhibited 255-, 69.1-, 342- and >1000-fold greater inhibition of Oxo-M activity at the M4 versus the M1-, M2(-), M3-or M5 receptor subtypes, respectively. Schild analyses indicates that PCS1055 acts as a competitive antagonist to muscarinic M4 receptor, and confirms the affinity of the ligand to be low nanomolar, Kb=5.72nM. Therefore, PCS1055 represents a new M4-preferring antagonist that may be useful in elucidating the roles of M4 receptor signaling.

Characterization of the novel positive allosteric modulator, LY2119620, at the muscarinic M(2) and M(4) receptors.

The M(4) receptor is a compelling therapeutic target, as this receptor modulates neural circuits dysregulated in schizophrenia, and there is clinical evidence that muscarinic agonists possess both antipsychotic and procognitive efficacy. Recent efforts have shifted toward allosteric ligands to maximize receptor selectivity and manipulate endogenous cholinergic and dopaminergic signaling. In this study, we present the pharmacological characterization of LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethoxy] thieno[2,3-b]pyridine-2-carboxamide), a M(2)/M(4) receptor-selective positive allosteric modulator (PAM), chemically evolved from hits identified through a M4 allosteric functional screen. Although unsuitable as a therapeutic due to M(2) receptor cross-reactivity and, thus, potential cardiovascular liability, LY2119620 surpassed previous congeners in potency and PAM activity and broadens research capabilities through its development into a radiotracer. Characterization of LY2119620 revealed evidence of probe dependence in both binding and functional assays. Guanosine 5'-[γ-(35)S]-triphosphate assays displayed differential potentiation depending on the orthosteric-allosteric pairing, with the largest cooperativity observed for oxotremorine M (Oxo-M) LY2119620. Further [(3)H]Oxo-M saturation binding, including studies with guanosine-5'-[(ß,γ)-imido]triphosphate, suggests that both the orthosteric and allosteric ligands can alter the population of receptors in the active G protein-coupled state. Additionally, this work expands the characterization of the orthosteric agonist, iperoxo, at the M(4) receptor, and demonstrates that an allosteric ligand can positively modulate the binding and functional efficacy of this high efficacy ligand. Ultimately, it was the M(2) receptor pharmacology and PAM activity with iperoxo that made LY2119620 the most suitable allosteric partner for the M(2) active-state structure recently solved (Kruse et al., 2013), a structure that provides crucial insights into the mechanisms of orthosteric activation and allosteric modulation of muscarinic receptors.

The muscarinic acetylcholine receptor agonist BuTAC mediates antipsychotic-like effects via the M4 subtype.

The generation of muscarinic acetylcholine receptor (mAChR) subtype-selective compounds has been challenging, requiring use of nonpharmacological approaches, such as genetically engineered animals, to deepen our understanding of the potential that members of the muscarinic receptor subtype family hold as therapeutic drug targets. The muscarinic receptor agonist 'BuTAC' was previously shown to exhibit efficacy in animal models of psychosis, although the particular receptor subtype(s) responsible for such activity was unclear. Here, we evaluate the in vitro functional agonist and antagonist activity of BuTAC using an assay that provides a direct measure of G protein activation. In addition, we employ the conditioned avoidance response paradigm, an in vivo model predictive of antipsychotic activity, and mouse genetic deletion models to investigate which presynaptic mAChR subtype mediates the antipsychotic-like effects of BuTAC. Our results show that, in vitro, BuTAC acts as a full agonist at the M2AChR and a partial agonist at the M1 and M4 receptors, with full antagonist activity at M3- and M5AChRs. In the mouse conditioned avoidance response (CAR) assay, BuTAC exhibits an atypical antipsychotic-like profile by selectively decreasing avoidance responses at doses that do not induce escape failures. CAR results using M2(-/-), M4(-/-), and M2/M4 (M2/M4(-/-)) mice found that the effects of BuTAC were near completely lost in M2/M4(-/-) double-knockout mice and potency of BuTAC was right-shifted in M4(-/-) as compared with wild-type and M2(-/-) mice. The M2/M4(-/-) mice showed no altered sensitivity to the antipsychotic effects of either haloperidol or clozapine, suggesting that these compounds mediate their actions in CAR via a non-mAChR-mediated mechanism. These data support a role for the M4AChR subtype in mediating the antipsychotic-like activity of BuTAC and implicate M4AChR agonism as a potential novel therapeutic mechanism for ameliorating symptoms associated with schizophrenia.

N-(4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a novel metabotropic glutamate 2 potentiator with potential anxiolytic/antidepressant properties: in vivo profiling suggests a link between behavioral and central nervous system neurochemical changes.

The normalization of excessive glutamatergic neurotransmission through the activation of metabotropic glutamate 2 (mGlu2) receptors may have therapeutic potential in a variety of psychiatric disorders, including anxiety/depression and schizophrenia. Here, we characterize the pharmacological properties of N-(4-((2-(trifluoromethyl)-3-hydroxy-4-(isobutyryl)phenoxy)methyl)benzyl)-1-methyl-1H-imidazole-4-carboxamide (THIIC), a structurally novel, potent, and selective allosteric potentiator of human and rat mGlu2 receptors (EC(50) = 23 and 13 nM, respectively). THIIC produced anxiolytic-like efficacy in the rat stress-induced hyperthermia assay and the mouse stress-induced elevation of cerebellar cGMP and marble-burying assays. THIIC also produced robust activity in three assays that detect antidepressant-like activity, including the mouse forced-swim test, the rat differential reinforcement of low rate 72-s assay, and the rat dominant-submissive test, with a maximal response similar to that of imipramine. Effects of THIIC in the forced-swim test and marble burying were deleted in mGlu2 receptor null mice. Analysis of sleep electroencephalogram (EEG) showed that THIIC had a sleep-promoting profile with increased non-rapid eye movement (REM) and decreased REM sleep. THIIC also decreased the dark phase increase in extracellular histamine in the medial prefrontal cortex and decreased levels of the histamine metabolite tele-methylhistamine (t-MeHA) in rat cerebrospinal fluid. Collectively, these results indicate that the novel mGlu2-positive allosteric modulator THIIC has robust activity in models used to predict anxiolytic/antidepressant efficacy, substantiating, at least with this molecule, differentiation in the biological impact of mGlu2 potentiation versus mGlu2/3 orthosteric agonism. In addition, we provide evidence that sleep EEG and CSF t-MeHA might function as viable biomarker approaches to facilitate the translational development of THIIC and other mGlu2 potentiators.