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To characterize the effects of blocking calcitonin gene-related peptide (CGRP) activity in a mouse model of gastrointestinal transport.Migraine management using CGRP modulating therapies can cause constipation of varying frequency and severity. This variation might be due to the different mechanisms through which therapies block CGRP activity (e.g., blocking CGRP, or the CGRP receptor) with antibodies or receptor antagonists. The charcoal meal gastrointestinal transit assay was used to characterize constipation produced by these modes of therapy in transgenic mice expressing the human receptor activity-modifying protein 1 (hRAMP1) subunit of the CGRP receptor complex.Male and female hRAMP1 mice were dosed with compound or vehicle and challenged with a charcoal meal suspension via oral gavage. The mice were then humanely euthanized and the proportion of the length of the large intestine that the charcoal meal had traveled indicated gastrointestinal transit.Antibody to the CGRP receptor produced % distance traveled (mean ± standard deviation) of 31.8 ± 8.2 (4 mg/kg; p = 0.001) and 33.2 ± 6.0 (30 mg/kg; p 0.001) compared to 49.7 ± 8.3 (control) in female mice (n = 6-8), and 35.6 ± 13.5 (30 mg/kg, p = 0.019) compared to 50.2 ± 14.0 (control) in male mice (n = 10). Telcagepant (5 mg/kg, n = 8) resulted in % travel of 30.6 ± 14.7 versus 41.2 ± 8.3 (vehicle; p = 0.013) in male mice. Atogepant (3 mg/kg, n = 9) resulted in % travel of 30.6 ± 12.0, versus 41.2 ± 3.7 (control; p = 0.030) in female mice. The CGRP antibody galcanezumab (n = 7-10; p = 0.958 and p = 0.929) did not have a statistically significant effect.These results are consistent with reported clinical data. Selectively blocking the CGRP receptor may have a greater impact on gastrointestinal transit than attenuating the activity of the ligand CGRP. This differential effect may be related to physiologically opposing mechanisms between the CGRP and AMY1 receptors, as the CGRP ligand antibody could inhibit the effects of CGRP at both the CGRP and AMY1 receptors.

Clinical development of catechol-based orthosteric agonists of the dopamine D1 receptor has thus far been unsuccessful due to multiple challenges. To address these issues, we identified LY3154207 (...

SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization when administered early during COVID-19 disease. However, the emergence of variants of concern has negatively impacted the therapeutic use of some authorized mAbs. Using a high throughput B-cell screening pipeline, we isolated a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody called LY-CoV1404 (also known as bebtelovimab). LY-CoV1404 potently neutralizes authentic SARS-CoV-2 virus, including the prototype, B.1.1.7, B.1.351 and B.1.617.2). In pseudovirus neutralization studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant and retains binding to spike proteins with a variety of underlying RBD mutations including K417N, L452R, E484K, and N501Y. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved with the exception of N439 and N501. Notably, the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of reactivity to amino acid substitutions present among current VOC together with broad and potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants causing COVID-19.In BriefLY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated.HighlightsLY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and known variants of concern including the B.1.1.529 (Omicron), the BA.2 Omicron subvariant, and B.1.617.2 (Delta) variantsNo loss of potency against currently circulating variantsBinding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID databaseBreadth of neutralizing activity and potency supports clinical development

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour-1 kg-1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

LY-CoV555, a SARS-CoV-2 spike protein–specific antibody, neutralizes SARS-CoV-2 and protects the airways of nonhuman primates against infection., A monoclonal for SARS-CoV-2 Among the most promising therapeutic options for individuals with coronavirus disease 2019 (COVID-19) are monoclonal antibodies (mAbs). In this study, Jones et al. identified, characterized, and tested one such mAb, LY-CoV555, in vitro and in vivo. They found that LY-CoV555 bound to the severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2) spike protein and prevented its interaction with angiotensin-converting enzyme 2. Prophylactic treatment with LY-CoV555 protected the upper and lower respiratory tracts of nonhuman primates from becoming infected with SARS-CoV-2. Together, these data support the clinical use of LY-CoV555 for treating patients with COVID-19., Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour−1 kg−1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

Multiple therapeutic opportunities have been suggested for compounds capable of selective activation of metabotropic glutamate 3 (mGlu3) receptors, but small molecule tools are lacking. As part of our ongoing efforts to identify potent, selective, and systemically bioavailable agonists for mGlu2 and mGlu3 receptor subtypes, a series of C4β-N-linked variants of (1S,2S,5R,6S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 1 (LY354740) were prepared and evaluated for both mGlu2 and mGlu3 receptor binding affinity and functional cellular responses. From this investigation we identified (1S,2S,4S,5R,6S)-2-amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 8p (LY2794193), a molecule that demonstrates remarkable mGlu3 receptor selectivity. Crystallization of 8p with the amino terminal domain of hmGlu3 revealed critical binding interactions for this ligand with residues adjacent to the glutamate binding site, while pharmacokinetic assessment of 8p combined with its effect in an mGlu2 ...

The novel mGlu2/3 receptor antagonist, LY3020371, has been shown to produce antidepressant-like effects comparable to that of the clinically-effective antidepressant ketamine. In the present study, we investigated whether LY3020371 would be predicted to be free of the side-effects and safety pharmacology issues associated with ketamine. In contrast to ketamine, LY3020371 produced small increases in locomotion and did not impair motor performance on an inverted screen. Ketamine, but not LY3020371, increased dopamine efflux in the nucleus accumbens of rats. Ketamine also produced cognitively-impairing effects in rats in a T-maze and in a psychomotor vigilance task and altered theta synchrony between the hippocampus and mPFC, whereas LY3020371 had either no significant impact or lesser effects in these assays. In mice, ketamine, but not LY3020371, negatively affected spontaneous alternation in a Y-maze. Rats were trained to discriminate LY3020371 from vehicle where 30mg/kg produced 100% drug-appropriate responding and the ED50 for LY3020371 was 9.4mg/kg, i.p. In rats discriminating LY3020371, neither d-amphetamine nor phencyclidine fully substituted for LY3020371 (35-45%) and the mGlu2/3 receptor agonist LY354740 partially attenuated the discriminative stimulus effects of LY3020371. These are the first data to demonstrate the discriminative stimulus effects of an mGlu2/3 receptor antagonist. Some alterations were suggested to occur in the density of mGlu2/3 receptor binding sites in the drug discrimination rats relative to their age-matched non-drug-exposed controls. In preclinical toxicology studies of 14day dosing of doses up to 1000mg/kg, i.v. in rats and up to 500m/kg, i.v. in Cynomologous monkeys, LY3020371 produced uM plasma exposures without producing critical toxicological findings. It is concluded that LY3020371 does not recapitulate the motor, cognitive, subjective, neurochemical, electrophysiological, or toxicological findings reported with ketamine. Thus, LY3020371 possesses both the efficacy signatures of a rapidly-acting antidepressant and a safety profile enabling proof of concept studies in patients.

Metabotropic glutamate 2/3 (mGlu2/3) receptors are of considerable interest owing to their role in modulating glutamate transmission via presynaptic, postsynaptic and glial mechanisms. As part of our ongoing efforts to identify novel ligands for these receptors, we have discovered (1S,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid; (LY3020371), a potent and selective orthosteric mGlu2/3 receptor antagonist. In this account, we characterize the effects of LY3020371 in membranes and cells expressing human recombinant mGlu receptor subtypes as well as in native rodent and human brain tissue preparations, providing important translational information for this molecule. In membranes from cells expressing recombinant human mGlu2 and mGlu3 receptor subtypes, LY3020371.HCl competitively displaced binding of the mGlu2/3 agonist ligand [3H]-459477 with high affinity (hmGlu2 Ki = 5.26 nM; hmGlu3 Ki = 2.50 nM). In cells expressing hmGlu2 receptors, LY3020371.HCl potently blocked mGlu2/3 agonist (DCG-IV)-inhibited, forskolin-stimulated cAMP formation (IC50 = 16.2 nM), an effect that was similarly observed in hmGlu3-expressing cells (IC50 = 6.21 nM). Evaluation of LY3020371 in cells expressing the other human mGlu receptor subtypes revealed high mGlu2/3 receptor selectivity. In rat native tissue assays, LY3020371 demonstrated effective displacement of [3H]-459477 from frontal cortical membranes (Ki = 33 nM), and functional antagonist activity in cortical synaptosomes measuring both the reversal of agonist-suppressed second messenger production (IC50 = 29 nM) and agonist-inhibited, K+-evoked glutamate release (IC50 = 86 nM). Antagonism was fully recapitulated in both primary cultured cortical neurons where LY3020371 blocked agonist-suppressed spontaneous Ca2+ oscillations (IC50 = 34 nM) and in an intact hippocampal slice preparation (IC50 = 46 nM). Functional antagonist activity was similarly demonstrated in synaptosomes prepared from epileptic human cortical or hippocampal tissues, suggesting a translation of the mGlu2/3 antagonist pharmacology from rat to human. Intravenous dosing of LY3020371 in rats led to cerebrospinal fluid drug levels that are expected to effectively block mGlu2/3 receptors in vivo. Taken together, these results establish LY3020371 as an important new pharmacological tool for studying mGlu2/3 receptors in vitro and in vivo. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

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.

Clinical development of catechol-based orthosteric agonists of the dopamine D1 receptor has thus far been unsuccessful due to multiple challenges. To address these issues, we identified LY3154207 (

As part of our ongoing efforts to identify novel ligands for the metabotropic glutamate 2 and 3 (mGlu2/3) receptors, we have incorporated substitution at the C3 and C4 positions of the (1S,2R,5R,6R)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid scaffold to generate mGlu2/3 antagonists. Exploration of this structure–activity relationship (SAR) led to the identification of (1S,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid hydrochloride (LY3020371·HCl, 19f), a potent, selective, and maximally efficacious mGlu2/3 antagonist. Further characterization of compound 19f binding to the human metabotropic 2 glutamate (hmGlu2) site was established by cocrystallization of this molecule with the amino terminal domain (ATD) of the hmGlu2 receptor protein. The resulting cocrystal structure revealed the specific ligand–protein interactions, which likely explain the high affinity of 19f for this site and support its functional mGlu2 antagonist pharm...

Negative modulators of metabotropic glutamate 2 & 3 receptors demonstrate antidepressant-like activity in animal models and hold promise as novel therapeutic agents for the treatment of major depressive disorder. Herein we describe our efforts to prepare and optimize a series of conformationally constrained 3,4-disubstituted bicyclo[3.1.0]hexane glutamic acid analogs as orthosteric (glutamate site) mGlu2/3 receptor antagonists. This work led to the discovery of a highly potent and efficacious tool compound 18 (hmGlu2 IC50 46±14.2nM, hmGlu3 IC50=46.1±36.2nM). Compound 18 showed activity in the mouse forced swim test with a minimal effective dose (MED) of 1mg/kg ip. While in rat EEG studies it exhibited wake promoting effects at 3 and 10mg/kg ip without any significant effects on locomotor activity. Compound 18 thus represents a novel tool molecule for studying the impact of blocking mGlu2/3 receptors both in vitro and in vivo.

Pharmacological manipulation of specific neural circuits to optimize therapeutic index is an unrealized goal in neurology and psychiatry. AMPA receptors are important for excitatory synaptic transmission, and their antagonists are antiepileptic. Although efficacious, AMPA-receptor antagonists, including perampanel (Fycompa), the only approved antagonist for epilepsy, induce dizziness and motor impairment. We hypothesized that blockade of forebrain AMPA receptors without blocking cerebellar AMPA receptors would be antiepileptic and devoid of motor impairment. Taking advantage of an AMPA receptor auxiliary protein, TARP γ-8, which is selectively expressed in the forebrain and modulates the pharmacological properties of AMPA receptors, we discovered that LY3130481 selectively antagonized recombinant and native AMPA receptors containing γ-8, but not γ-2 (cerebellum) or other TARP members. Two amino acid residues unique to γ-8 determined this selectivity. We also observed antagonism of AMPA receptors expressed in hippocampal, but not cerebellar, tissue from an patient with epilepsy. Corresponding to this selective activity, LY3130481 prevented multiple seizure types in rats and mice and without motor side effects. These findings demonstrate the first rationally discovered molecule targeting specific neural circuitries for therapeutic advantage.

The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one], a positive allosteric modulator (PAM) of the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111 [N-(6-tert-butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide], a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a β2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state β2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously.

Multiple therapeutic opportunities have been suggested for compounds capable of selective activation of metabotropic glutamate 3 (mGlu

Identification of orthosteric mGlu(2/3) receptor agonists capable of discriminating between individual mGlu2 and mGlu3 subtypes has been highly challenging owing to the glutamate-site sequence homology between these proteins. Herein we detail the preparation and characterization of a series of molecules related to (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate 1 (LY354740) bearing C4-thiotriazole substituents. On the basis of second messenger responses in cells expressing other recombinant human mGlu2/3 subtypes, a number of high potency and efficacy mGlu2 receptor agonists exhibiting low potency mGlu3 partial agonist/antagonist activity were identified. From this, (1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 14a (LY2812223) was further characterized. Cocrystallization of 14a with the amino terminal domains of hmGlu2 and hmGlu3 combined with site-directed mutation studies has clarified the underlying molecular basis of this unique pharmacology. Evaluation of 14a in a rat model responsive to mGlu2 receptor activation coupled with a measure of central drug disposition provides evidence that this molecule engages and activates central mGlu2 receptors in vivo.

As part of our ongoing research to identify novel agents acting at metabotropic glutamate 2 (mGlu2) and 3 (mGlu3) receptors, we have previously reported the identification of the C4α-methyl analog of mGlu2/3 receptor agonist 1 (LY354740). This molecule, 1S,2S,4R,5R,6S-2-amino-4-methylbicyclo[3.1.0]hexane-2,6-dicarboxylate 2 (LY541850), exhibited an unexpected mGlu2 agonist/mGlu3 antagonist pharmacological profile, whereas the C4β-methyl diastereomer (3) possessed dual mGlu2/3 receptor agonist activity. We have now further explored this structure–activity relationship through the preparation of cyclic and acyclic C4-disubstituted analogs of 1, leading to the identification of C4-spirocyclopropane 5 (LY2934747), a novel, potent, and systemically bioavailable mGlu2/3 receptor agonist which exhibits both antipsychotic and analgesic properties in vivo. In addition, through the combined use of protein–ligand X-ray crystallography employing recombinant human mGlu2/3 receptor amino terminal domains, molecular mod...

Scopolamine produces rapid and significant symptom improvement in patients with depression, and most notably in patients who do not respond to current antidepressant treatments. Scopolamine is a nonselective muscarinic acetylcholine receptor antagonist, and it is not known which one or more of the five receptor subtypes in the muscarinic family are mediating these therapeutic effects. We used the mouse forced-swim test, an antidepressant detecting assay, in wild-type and transgenic mice in which each muscarinic receptor subtype had been genetically deleted to define the relevant receptor subtypes. Only the M1 and M2 knockout (KO) mice had a blunted response to scopolamine in the forced-swim assay. In contrast, the effects of the tricyclic antidepressant imipramine were not significantly altered by gene deletion of any of the five muscarinic receptors. The muscarinic antagonists biperiden, pirenzepine, and VU0255035 (N-[3-oxo-3-[4-(4-pyridinyl)-1-piper azinyl]propyl]-2,1,3-benzothiadiazole-4-sulfonamide) with selectivity for M1 over M2 receptors also demonstrated activity in the forced-swim test, which was attenuated in M1 but not M2 receptor KO mice. An antagonist with selectivity of M2 over M1 receptors (SCH226206 [(2-amino-3-methyl-phenyl)-[4-[4-[[4-(3 chlorophenyl)sulfonylphenyl]methyl]-1-piperidyl]-1-piperidyl]methanone]) was also active in the forced-swim assay, and the effects were deleted in M2 (-/-) mice. Brain exposure and locomotor activity in the KO mice demonstrated that these behavioral effects of scopolamine are pharmacodynamic in nature. These data establish muscarinic M1 and M2 receptors as sufficient to generate behavioral effects consistent with an antidepressant phenotype and therefore as potential targets in the antidepressant effects of scopolamine.

As part of our ongoing interest in identifying novel agonists acting at metabotropic glutamate (mGlu) 2/3 receptors, we have explored the effect of structural modifications of 1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740), a potent and pharmacologically balanced mGlu2/3 receptor agonist. Incorporation of relatively small substituents (e.g., F, O) at the C4 position of this molecule resulted in additional highly potent mGlu2/3 agonists that demonstrate excellent selectivity over the other mGlu receptor subtypes, while addition of larger C4-substituents (e.g., SPh) led to a loss of agonist potency and/or the appearance of weak mGlu2/3 receptor antagonist activity. Further characterization of the α-fluoro-substituted analogue (LY459477) in vivo revealed that this molecule possesses good oral bioavailability in rats and effectively suppresses phencyclidine-evoked locomotor activity at doses that do not impair neuromuscular coordination. This molecule therefore represents a valuable new addition to the arsenal of pharmacological tools competent to investigate mGlu2/3 receptor function both in vitro and in vivo.

The objective of the described research effort was to identify a novel serotonin and norepinephrine reuptake inhibitor (SNRI) with improved norepinephrine transporter activity and acceptable metabolic stability and exhibiting minimal drug-drug interaction. We describe herein the discovery of a series of 3-substituted pyrrolidines, exemplified by compound 1. Compound 1 is a selective SNRI in vitro and in vivo, has favorable ADME properties, and retains inhibitory activity in the formalin model of pain behavior. Compound 1 thus represents a potential new probe to explore utility of SNRIs in central nervous system disorders, including chronic pain conditions.

A novel series of selective negative allosteric modulators (NAMs) for metabotropic glutamate receptor 5 (mGlu5) was discovered from an isothiazole scaffold. One compound of this series, (1R,2R)-N-(4-(6-isopropylpyridin-2-yl)-3-(2-methyl-2H-indazol-5-yl)isothiazol-5-yl)-2-methylcyclopropanecarboxamide (24), demonstrated satisfactory pharmacokinetic properties and, following oral dosing in rats, produced dose-dependent and long-lasting mGlu5 receptor occupancy. Consistent with the hypothesis that blockade of mGlu5 receptors will produce analgesic effects in mammals, compound 24 produced a dose-dependent reduction in paw licking responses in the formalin model of persistent pain.

The group II metabotropic glutamate (mGlu) receptors comprised of the mGlu2 and mGlu3 receptor subtypes have gained recognition in recent years as potential targets for psychiatric disorders, including anxiety and schizophrenia. In addition to studies already indicating which subtype mediates the anxiolytic and anti-psychotic effects observed in disease models, studies to help further define the preferred properties of selective group II mGlu receptor ligands will be essential. Comparison of the in vitro properties of these ligands to their in vivo efficacy and tolerance profiles may help provide these additional insights. We have developed a relatively high-throughput native group II mGlu receptor functional assay to aid this characterisation. We have utilised dissociated primary cortical neuronal cultures, which after 7 days in vitro have formed functional synaptic connections and display periodic and spontaneous synchronised calcium (Ca2+) oscillations in response to intrinsic action potential bursts. We herein demonstrate that in addition to non-selective group II mGlu receptor agonists, (2R,4R)-APDC, LY379268 and DCG-IV, a selective mGlu2 agonist, LY541850, and mGlu2 positive allosteric modulators, BINA and CBiPES, inhibit the frequency of synchronised Ca2+ oscillations in primary cultures of rat and mouse cortical neurons. Use of cultures from wild-type, mGlu2−/−, mGlu3−/− and mGlu2/3−/− mice allowed us to further probe the contribution of mGlu2 and mGlu3, and revealed LY541850 to be a partial mGlu2 agonist and a full mGlu3 antagonist. Overnight pre-treatment of cultures with these ligands revealed a preferred desensitisation profile after treatment with a positive allosteric modulator. This article is part of a Special Issue entitled ‘Metabotropic Glutamate Receptors’.

The demonstrated functional interaction of metabotropic glutamate 5 (mGlu₅) receptors with N-methyl-d-aspartate (NMDA) receptors has prompted speculation that their activation may offer a potential treatment for aspects of schizophrenia. Development of selective mGlu₅ agonists has been difficult, but several different positive allosteric modulator (PAM) molecules have now been identified. This study describes two novel mGlu₅ PAMs, LSN2463359 (N-(1-methylethyl)-5-(pyridin-4-ylethynyl)pyridine-2-carboxamide) and LSN2814617 [(7S)-3-tert-butyl-7-[3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl]-5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-A]pyridine], which are useful tools for this field of research. Both compounds are potent and selective potentiators of human and rat mGlu₅ receptors in vitro, displaying curve shift ratios of two to three fold in the concentration-response relationship to glutamate or the glutamate receptor agonist, DHPG, with no detectable intrinsic agonist properties. Both compounds displaced the mGlu₅ receptor antagonist radioligand, [³H]MPEP in vitro and, following oral administration reached brain concentrations sufficient to occupy hippocampal mGlu₅ receptors as measured in vivo by dose-dependent displacement from the hippocampus of intravenously administered MPEPy. In vivo EEG studies demonstrated that these mGlu₅ PAMs have marked wake-promoting properties but little in the way of rebound hypersomnolence. In contrast, the previously described mGlu₅ PAMs CDPPB and ADX47273 showed relatively poor evidence of in vivo target engagement in either receptor occupancy assays or EEG disturbance. Wake-promoting doses of LSN2463359 and LSN2814617 attenuated deficits in performance induced by the competitive NMDA receptor antagonist SDZ 220,581 in two tests of operant behaviour: the variable interval 30 s task and the DMTP task. These effects were lost if the dose of either compound extended into the range which disrupted performance in the baseline DMTP task. However, the improvements in response accuracy induced by the mGlu₅ potentiators in SDZ 220,581-treated rats were not delay-dependent and, therefore, perhaps more likely reflected optimization of general arousal than specific beneficial effects on discrete cognitive processes. The systematic profiling of LSN2463359 and LSN2814617 alongside other previously described molecules will help determine more precisely how mGlu₅ potentiator pharmacology might provide therapeutic benefit. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

As part of our ongoing efforts to identify novel ligands for the metabotropic glutamate 2 and 3 (mGlu

Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a Kb of 26 nM. The maximum response to DETQ alone was ∼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was ∼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3-20 mg/kg orally) caused a robust (∼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30-240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists.

Transmembrane AMPA receptor regulatory proteins (TARPs) are a family of scaffolding proteins that regulate AMPA receptor trafficking and function. TARP γ-8 is one member of this family and is highly expressed within the hippocampus relative to the cerebellum. A selective TARP γ-8-dependent AMPA receptor antagonist (TDAA) is an innovative approach to modulate AMPA receptors in specific brain regions to potentially increase the therapeutic index relative to known non-TARP-dependent AMPA antagonists. We describe here, for the first time, the discovery of a noncompetitive AMPA receptor antagonist that is dependent on the presence of TARP γ-8. Three major iteration cycles were employed to improve upon potency, CYP1A2-dependent challenges, and in vivo clearance. An optimized molecule, compound (-)-25 (LY3130481), was fully protective against pentylenetetrazole-induced convulsions in rats without the motor impairment associated with non-TARP-dependent AMPA receptor antagonists. Compound (-)-25 could be utilized to provide proof of concept for antiepileptic efficacy with reduced motor side effects in patients.

Background and Purpose CGRP is a potent vasodilator and nociceptive neuropeptide linked to migraine. CGRP receptors are heterodimers of receptor activity modifying protein 1 (RAMP1) and either calcitonin receptor-like receptor (CLR; forms canonical CGRP receptor) or calcitonin receptor (CT receptor; forms AMY1 receptor). The goal of this study was to test whether transgenic mice globally expressing human RAMP1 have increased CGRP receptor activity and whether the receptors are sensitive to human selective antagonist telcagepant. Experimental Approach cAMP production was measured in primary cultures of aortic smooth muscle and trigeminal ganglia neurons from global hRAMP1 mice and non-transgenic littermates. Functional activity and inhibition were compared with clonal cell lines expressing combinations of CLR or CT receptors with RAMP1. Key Results Cultured smooth muscle from global hRAMP1 mice had a 10-fold greater CGRP-induced cAMP maximal response (Rmax) than non-transgenic littermates, with similar EC50s. In contrast, cultured trigeminal ganglia from global hRAMP1 mice had a 40-fold leftward shift of the EC50, with similar Rmax values as littermates. In both hRAMP1 cultures, telcagepant blocked CGRP-induced cAMP production, but was not effective in non-transgenic cultures. IC50 values were closer to those observed for CT receptor/hRAMP1 than CLR/hRAMP1 in clonal cell lines. Conclusions and Implications Overexpression of hRAMP1 increases CGRP signalling by changing the maximal response or ligand sensitivity, depending on tissue type. Furthermore, telcagepant inhibited transgenic hRAMP1 CGRP receptors, but the degree of inhibition suggests that the transgenic mice are only partially humanized or both canonical CGRP and AMY1 receptors are functional in trigeminal ganglia neurons and vascular smooth muscle.

The disclosed 3-phenyl-5-isothiazole carboxamides are potent allosteric antagonists of mGluR1 with generally good selectivity relative to the related group 1 receptor mGluR5. Pharmacokinetic properties of a member of this series (1R,2R)-N-(3-(4-methoxyphenyl)-4-methylisothiazol-5-yl)-2-methylcyclopropanecarboxamide (14) are good, showing acceptable plasma and brain exposure after oral dosing. Oral administration of isothiazole 14 gave robust activity in the formalin model of persistent pain which correlated with CNS receptor occupancy.

Dual orthosteric agonists of metabotropic glutamate 2 (mGlu2) and mGlu3 receptors are being developed as novel antipsychotic agents devoid of the adverse effects of conventional antipsychotics. Therefore, these drugs could be helpful for the treatment of psychotic symptoms associated with Alzheimer's disease (AD). In experimental animals, the antipsychotic activity of mGlu2/3 receptor agonists is largely mediated by the activation of mGlu2 receptors and is mimicked by selective positive allosteric modulators (PAMs) of mGlu2 receptors. We investigated the distinct influence of mGlu2 and mGlu3 receptors in mixed and pure neuronal cultures exposed to synthetic β-amyloid protein (Aβ) to model neurodegeneration occurring in AD. The mGlu2 receptor PAM, N-4'-cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride (LY566332), devoid of toxicity per se, amplified Aβ-induced neurodegeneration, and this effect was prevented by the mGlu2/3 receptor antagonist (2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl)glycine (LY341495). LY566332 potentiated Aβ toxicity regardless of the presence of glial mGlu3 receptors, but it was inactive when neurons lacked mGlu2 receptors. The dual mGlu2/3 receptor agonist, (-)-2-oxa-4-aminobicyclo[3.1.0]exhane-4,6-dicarboxylic acid (LY379268), was neuroprotective in mixed cultures via a paracrine mechanism mediated by transforming growth factor-β1. LY379268 lost its protective activity in neurons grown with astrocytes lacking mGlu3 receptors, indicating that protection against Aβ neurotoxicity was mediated entirely by glial mGlu3 receptors. The selective noncompetitive mGlu3 receptor antagonist, (3S)-1-(5-bromopyrimidin-2-yl)-N-(2,4-dichlorobenzyl)pyrrolidin-3-amine methanesulfonate hydrate (LY2389575), amplified Aβ toxicity on its own, and, interestingly, unmasked a neurotoxic activity of LY379268, which probably was mediated by the activation of mGlu2 receptors. These data indicate that selective potentiation of mGlu2 receptors enhances neuronal vulnerability to Aβ, whereas dual activation of mGlu2 and mGlu3 receptors is protective against Aβ-induced toxicity.

Sazetidine-A has been recently proposed to be a "silent desensitizer" of alpha4beta2 nicotinic acetylcholine receptors (nAChRs), implying that it desensitizes alpha4beta2 nAChRs without first activating them. This unusual pharmacological property of sazetidine-A makes it, potentially, an excellent research tool to distinguish between the role of activation and desensitization of alpha4beta2 nAChRs in mediating the central nervous system effects of nicotine itself, as well as those of new nicotinic drugs. We were surprised to find that sazetidine-A potently and efficaciously stimulated nAChR-mediated dopamine release from rat striatal slices, which is mediated by alpha4beta2(*) and alpha6beta2(*) subtypes of nAChR. The agonist effects on native striatal nAChRs prompted us to re-examine the effects of sazetidine-A on recombinant alpha4beta2 nAChRs in more detail. We expressed the two alternative stoichiometries of alpha4beta2 nAChR in Xenopus laevis oocytes and investigated the agonist properties of sazetidine-A on both alpha4(2)beta2(3) and alpha4(3)beta2(2) nAChRs. We found that sazetidine-A potently activated both stoichiometries of alpha4beta2 nAChR: it was a full agonist on alpha4(2)beta2(3) nAChRs, whereas it had an efficacy of only 6% on alpha4(3)beta2(2) nAChRs. In contrast to what has been published before, we therefore conclude that sazetidine-A is an agonist of native and recombinant alpha4beta2 nAChRs but shows differential efficacy on alpha4beta2 nAChRs subtypes.

Metabotropic glutamate

Identification of orthosteric mGlu(2/3) receptor agonists capable of discriminating between individual mGlu2 and mGlu3 subtypes has been highly challenging owing to the glutamate-site sequence homology between these proteins. Herein we detail the preparation and characterization of a series of molecules related to (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate 1 (LY354740) bearing C4-thiotriazole substituents. On the basis of second messenger responses in cells expressing other recombinant human mGlu2/3 subtypes, a number of high potency and efficacy mGlu2 receptor agonists exhibiting low potency mGlu3 partial agonist/antagonist activity were identified. From this, (1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 14a (LY2812223) was further characterized. Cocrystallization of 14a with the amino terminal domains of hmGlu2 and hmGlu3 combined with site-directed mutation studies has clarified the underlying molecular basis of this unique pharmacology. Evaluation of 14a in a rat model responsive to mGlu2 receptor activation coupled with a measure of central drug disposition provides evidence that this molecule engages and activates central mGlu2 receptors in vivo.

As part of our ongoing research to identify novel agents acting at metabotropic glutamate 2 (mGlu2) and 3 (mGlu3) receptors, we have previously reported the identification of the C4α-methyl analog of mGlu2/3 receptor agonist 1 (LY354740). This molecule, 1S,2S,4R,5R,6S-2-amino-4-methylbicyclo[3.1.0]hexane-2,6-dicarboxylate 2 (LY541850), exhibited an unexpected mGlu2 agonist/mGlu3 antagonist pharmacological profile, whereas the C4β-methyl diastereomer (3) possessed dual mGlu2/3 receptor agonist activity. We have now further explored this structure-activity relationship through the preparation of cyclic and acyclic C4-disubstituted analogs of 1, leading to the identification of C4-spirocyclopropane 5 (LY2934747), a novel, potent, and systemically bioavailable mGlu2/3 receptor agonist which exhibits both antipsychotic and analgesic properties in vivo. In addition, through the combined use of protein-ligand X-ray crystallography employing recombinant human mGlu2/3 receptor amino terminal domains, molecular modeling, and site-directed mutagenesis, a molecular basis for the observed pharmacological profile of compound 2 is proposed.

We describe herein the design, syntheses and evaluation of a number of bicycloproline P2 bearing HCV protease inhibitors endowed with impressive enzyme potency, enzyme selectivity, cellular activity and favorable ADME profiles.

The inability for the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) to be readily studied in the context of viral replication has been circumvented by constructing chimeras such as with poliovirus (PV), in which translation of the genome polyprotein is under control of the HCV IRES. During our attempts to configure the PV/HCV chimera for our drug discovery efforts, we discovered that an adenine- (A) to-guanine (G) change at nt 350 in domain IV of the HCV IRES resulted in a nonviable phenotype. Similarly, a mengovirus (MV)/HCV chimera using the same configuration with a G at nt 350 (G-350) was found to be nonviable. In contrast, a bovine viral diarrhea virus (BVDV)/HCV chimera remained viable with G-350 in the HCV IRES insert. Second-site, resuscitating mutations were identified from the G-350 PV/HCV and MV/HCV viruses after blind passaging. For both viruses, the resuscitating mutations involved destabilization of domain IV in the HCV IRES. The nonviability of G-350 in the picornavirus/HCV chimeric background might be linked to translation efficiency as indicated by analyses with dual reporter and PV/HCV replicon constructs.

We describe herein the synthesis and biological evaluation of a series of tripeptidyl α-ketoamides as human rhinovirus (HRV) 3C protease inhibitors. The most potent inhibitor discussed in this manuscript, 4I, exhibited impressive enzyme inhibitory activity as well as antiviral activity against HRV-14.

The base of knowledge concerning RNA structure and function has been expanding rapidly in recent years. Simultaneously, an increasing awareness of the pivotal role RNA plays in viral diseases has prompted many researchers to apply new technologies in high-throughput screening and molecular modelling to the design of antiviral drugs that target RNA. While the two RNA viruses with the greatest unmet medical need, HIV and HCV, have been most actively pursued, the approaches discussed in this review are relevant to all virus infections. Both traditional small-molecule and large-molecule therapeutics, such as antisense, ribozymes and interfering dsRNAs have been described, and several molecules are under development for commercialization. The purpose of this review is to summarize the current state of the art in this field and to postulate new directions in the future.

Hepatitis C virus (HCV) induces microtubule aggregates in infected hepatocytes. To determine if cytoskeletal elements are important for HCV RNA synthesis, we examined the effect of cytoskeleton inhibitors on HCV replicon transcription in Huh7 cells. The data demonstrate that HCV replication complex-mediated RNA synthesis requires microtubule and actin polymerization.

The RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV), nonstructural protein 5B (NS5B), has recently been shown to direct de novo initiation using a number of complex RNA templates. In this study, we analyzed the features in simple RNA templates that are required to direct de novo initiation of RNA synthesis by HCV NS5B. NS5B was found to protect RNA fragments of 8 to 10 nucleotides (nt) from RNase digestion. However, NS5B could not direct RNA synthesis unless the template contained a stable secondary structure and a single-stranded sequence that contained at least one 3′ cytidylate. The structure of a 25-nt template, named SLD3, was determined by nuclear magnetic resonance spectroscopy to contain an 8-bp stem and a 6-nt single-stranded sequence. Systematic analysis of changes in SLD3 revealed which features in the stem, loop, and 3′ single-stranded sequence were required for efficient RNA synthesis. Also, chimeric molecules composed of DNA and RNA demonstrated that a DNA molecule containing a 3′-terminal ribocytidylate was able to direct RNA synthesis as efficiently as a sequence composed entirely of RNA. These results define the template sequence and structure sufficient to direct the de novo initiation of RNA synthesis by HCV RdRp.

Previous studies in rats and humans demonstrated poor oral bioavailability of potent in vitro 2‐aminobenzimidazole inhibitors of rhinovirus replication due to significant first‐pass elimination and possibly also to poor aqueous solubility. Estimations of aqueous solubility, as well as measurements of caco‐2 permeability and NADPH dependent compound loss in rat liver microsomal incubations were employed alongside traditional in vivo experiments in rats to guide subsequent chemistry efforts. Retention of activity upon replacement of the metabolically labile vinyl oxime in the lead molecule with a vinyl carboxamide was a major breakthrough; however, oral bioavail‐ability among the latter compounds was variable. Based on the ability to independently measure solubility, permeability, and metabolic stability of new compounds, variable solubility across the series (ranging from approximately 1 to 10 µg/mL) was identified as the cause of the inconsistent performance. Subsequent efforts to improve solubility led to the discovery of highly soluble (>10 mg/mL) and potent dessulfonyl vinyl carboxamide benzimidazoles. Determination of the metabolic stability of these compounds as a surrogate of the extent of their first‐pass elimination supported a prediction of excellent oral bioavail‐ability. In comparison to the sulfonyl‐containing vinyl carboxamides, caco‐2 permeabilities were reduced 5 to 10‐fold; however, these were considered to be in the range of well‐absorbed compounds based on comparison to a series of reference compounds of known percentage absorption in humans. Subsequent experiments in the rat verified the oral bioavailability of these N‐alkyl compounds, with one compound (368177) having an absolute oral bioavailability of 89.4%. The application of solubility and caco‐2 permeability as surrogates for oral absorption potential, in conjunction with the use of microsomal incubations as a surrogate for first‐pass metabolism, was shown to augment a rational chemistry approach to discover orally bioavailable inhibitors of rhinovirus replication. Future expanded use of these surrogates is planned.

Curcumin, the major constituent of the spice tumeric produces a plethora of biological actions that have translated in vivo into behavioral and neurochemical effects in rodents that are also produced by clinically-used antidepressants. The present study was designed to provide a systematic replication of prior behavioral, pharmacological, and neurochemical experiments. In particular, the ability of curcumin to engender anti-immobility effects in the mouse forced-swim assay was established. Although prior work had shown curcumin to function as an inhibitor of the monoamine metabolizing enzyme, monoamine oxidase (MAO), neither MAOA nor MAOB was inhibitied by curcumin in the present study. Curcumin had also been reported previously to function as a cannabinoid CB1 receptor inverse agonist/antagonist. However, in our hands, curcumin did not potently alter GTP-γ.-35S binding indicative of functional CB1 antagonism (Kb = 2080 nM). Moreover, curcumin was not able to prevent the hypothermic effects of the cannabinoid receptor agonist (-)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP 55,940). Nonetheless, the anti-immobility effects of curcumin did not occur in CB1 -/- mice. Finally, a broad array of protein receptors and enzymes were evaluated in vitro for their potential interaction with and/or functional engagement with curcumin. Of the more than 100 targets screened, curcumin had very low potency in most. Of those targets with appreciable activity, curcumin had affinities for the human cloned muscarinic receptor subtypes (Ki = 1.3-3.1 uM). Moreover, the plasma and brain levels of curcumin at behaviorally-active doses were below quantitative limits. Given these findings, it is concluded that the prominent antidepressant-like behavioral effects of curcumin, replicated here and in multiple acute and chronic rodent models detailed in the literature, are the result of as yet undisclosed mechanisms of action. The scientific and patient communities await the full scale clinical evaluation of a sufficiently bioavailable curcumin analog in major depressive disorder.

We have developed a simple in vitro translation method to analyze compounds that inhibit the rhinovirus 3C protease in peptide substrate assays but demonstrate no antiviral activity. This complementary assay, which provides both qualitative and quantitative results, detects the inhibition of the 3CD protease in the native polyprotein form.

Peptide aldehydes have been synthesized and evaluated as inhibitors of human rhinovirus 3C protease. Those inhibitors containing a C-terminal glutamine aldehyde were prepared using newly developed methodology involving reduction of the corresponding glutarimide, which can be easily prepared from N-protected glutamine. Low molecular weight (

Two allosteric modulators of the group I metabotropic glutamate receptors (mGluR1 and mGluR5) were evaluated as positron emission tomography (PET) radioligands for mGluR1.LY2428703, a full mGluR1 antagonist (IC(50) 8.9 nM) and partial mGluR5 antagonist (IC(50) 118 nM), and LSN2606428, a full mGluR1 and mGluR5 antagonist (IC(50) 35.3 nM and 10.2 nM, respectively) were successfully labeled with (11)C and evaluated as radioligands for mGluR1. The pharmacology of LY2428703 was comprehensively assessed in vitro and in vivo, and its biodistribution was investigated by liquid chromatography-mass spectrometry/mass spectrometry, and by PET imaging in the rat. In contrast, LSN2606428 was only evaluated in vitro; further evaluation was stopped due to its unfavorable pharmacological properties and binding affinity.(11)C-LY2428703 showed promising characteristics, including: (1) high potency for binding to human mGluR1 (IC(50) 8.9 nM) with no significant affinity for other human mGlu receptors (mGluR2 through mGluR8); (2) binding to brain displaceable by administration of an mGluR1 antagonist; (3) only one major radiometabolite in both plasma and brain, with a negligible brain concentration (with 3.5 % of the total radioactivity in cerebellum) and no receptor affinity; (4) a large specific and displaceable signal in the mGluR1-rich cerebellum with no significant in vivo affinity for mGluR5, as shown by PET studies in rats; and (5) lack of substrate behavior for efflux transporters at the blood-brain barrier, as shown by PET studies conducted in wild-type and knockout mice.(11)C-LY2428703, a new PET radioligand for mGluR1 quantification, displayed promising characteristics both in vitro and in vivo in rodents.

An assay to detect the on-target effects of mGlu2/3 receptor antagonists in vivo would be valuable in guiding dosing regimens for the exploration of biological effects of potential therapeutic import. Multiple approaches involving blockade of mGlu2/3 receptor agoinist-driven behavioral effects in mice and rats were investigated. Most of these methods failed to provide a useful method of detection of antagonists in vivo (e.g., locomotor activity). In contrast, the mGlu2/3 receptor agonist LY379268 produced dose-dependent increases in body temperature of mice. The hyperthermic effects of LY379268 was abolished in mGlu2 and in mGlu2/3 receptor null mice but not in mGlu3 null mice. Hyperthermia was not produced by an mGlu8 receptor agonist. Agonist-induced hyperthermia was prevented in a dose-dependent manner by structurally-distinct mGlu2/3 receptor antagonists. The blockade was stereo-specific. Moreover, this biological readout was responsive to both orthosteric and to negative allosteric modulators of mGlu2/3 receptors. Antagonism of agonist-induced hyperthermia predicted antidepressant-like efficacy in the mouse forced swim test. As with the hyperthermic response, the antidepressant-like effects of mGlu2/3 receptor antagonists were shown to be due to mGlu2 and not to mGlu3 or mGlu8 receptors through the use of receptor knock-out mice. The ability to rapidly assess on-target activity of mGlu2/3 receptor antagonists enables determination of parameters for setting efficacy doses in vivo. In turn, efficacy-related data in the preclinical laboratory can help to set expectations of therapeutic potential and dosing in humans.