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Herein, we report structure-activity relationship (SAR) studies to develop novel tricyclic M 4 PAM scaffolds with improved pharmacological properties. This endeavor involved a "tie-back" strategy to replace a 5-amino-2,4-dimethylthieno[2,3- d ]pyrimidine-6-carboxamide core, which led to the discovery of two novel tricyclic cores. While both tricyclic cores displayed low nanomolar potency against both human and rat M 4 and were highly brain-penetrant, the 2,4-dimethylpyrido[4',3':4,5]thieno[2,3- d ]pyrimidine tricycle core provided lead compound, VU6016235 , with an overall superior pharmacological and drug metabolism and pharmacokinetics (DMPK) profile, as well as efficacy in a preclinical antipsychotic animal model.

While the muscarinic acetylcholine receptor mAChR subtype 5 (M 5 ) has been studied over decades, recent findings suggest that more in-depth research is required to elucidate a thorough understanding of its physiological function related to neurological and psychiatric disorders. Our efforts to identify potent, selective, and pharmaceutically favorable next-generation M 5 antagonist tool compounds have led to the discovery of a novel triazolopyridine-based series. In particular, VU6036864 ( 45 ) showed exquisite potency (human M 5 IC 50 = 20 nM), good subtype selectivity (>500 fold selectivity against human M 1-4 ), desirable brain exposure ( K p = 0.68, K p,uu = 0.65), and high oral bioavailability (% F ) and its close analogues will support further studies of M VU6036864 as advanced antagonist tool compounds and play an important role in the emerging biology of M 45 ) and its close analogues will support further studies of M 5 as advanced antagonist tool compounds and play an important role in the emerging biology of M 5 .

The muscarinic acetylcholine receptor (mAChR) subtype 5 (M 5 ) represents a novel potential target for the treatment of multiple addictive disorders, including opioid use disorder. Through chemical optimization of several functional high-throughput screening hits, VU6019650 ( 27b ) was identified as a novel M 5 orthosteric antagonist with high potency (human M 5 IC 50 = 36 nM), M 5 subtype selectivity (>100-fold selectivity against human M 1-4 ) and favorable physicochemical properties for systemic dosing in preclinical addiction models. In acute brain slice electrophysiology studies, 27b blocked the nonselective muscarinic agonist oxotremorine-M-induced increases in neuronal firing rates of midbrain dopamine neurons in the ventral tegmental area, a part of the mesolimbic dopaminergic reward circuitry. Moreover, 27b also inhibited oxycodone self-administration in male Sprague-Dawley rats within a dose range that did not impair general motor output.

Opioid use disorder (OUD) is a debilitating neuropsychiatric condition characterized by compulsive opioid use, dependence, and repeated relapse after periods of abstinence. Given the high risk of developing OUD following prescription opioid use, the continued need for opioid-induced analgesia, and the limitations of current OUD treatments, it is necessary to develop novel, non-opioid-based treatments for OUD and decrease abuse potential of prescription opioids. Recent evidence suggests that negative allosteric modulation (NAM) of the M 5 muscarinic acetylcholine receptor (M 5 mAChR) may provide an alternative therapeutic approach for the treatment of OUD. Previous studies demonstrated localization of M 5 mAChR expression within the mesocorticolimbic reward circuitry and that the selective M 5 NAM ML375 attenuates both cocaine and alcohol self-administration in rats. In the present study, the effects of ML375 were evaluated in rats self-administering the μ-opioid agonists oxycodone or remifentanil on a progressive ratio (PR) schedule or on cue reactivity (a rodent model of relapse) in the absence of oxycodone following 72 h of abstinence. ML375 reduced the PR break point for oxycodone and remifentanil self-administration and attenuated cue-elicited responding. Importantly, ML375 did not affect sucrose pellet-maintained responding on a PR schedule or opioid-induced antinociception using the hot-plate and tail-flick assays. We also confirm expression of M 5 mAChR mRNA in the ventral tegmental area and show that this is primarily on dopamine (tyrosine hydroxylase mRNA-positive) neurons. Taken together, these findings suggest that selective functional antagonism of the M 5 mAChR may represent a novel, non-opioid-based treatment for OUD.

G protein-gated inwardly rectifying potassium (GIRK) channels are potassium-selective ion channels. As their name suggests, GIRK channels are effectors of G i/o G protein-couple receptors whereby activation of these GPCRs leads to increased GIRK channel activity resulting in decreased cellular excitability. In this way, GIRK channels play diverse roles in physiology as effectors of G i/o -coupled GPCRs: peacemaking in the heart rate, modulation of hormone secretion in endocrine tissues, as well as numerous CNS functions including learning, memory, and addiction/reward. Notably, GIRK channels are widely expressed along the spinothalamic tract and are positioned to play roles in both ascending and descending pain pathways. More notably, GIRK channel knockout and knock-down studies have found that GIRK channels play a major role in the action of opioid analgesics which act predominantly through G i/o -coupled, opioid-activated GPCRs (e.g., μ-opioid receptors). Recent advances in GIRK channel pharmacology have led to the development of small molecules that directly and selectively activate GIRK channels. Based on research implicating the involvement of GIRK channels in pain pathways and as effectors of opioid analgesics, we conducted a study to determine whether direct pharmacological activation of GIRK channels could produce analgesic efficacy and/or augment the analgesic efficacy morphine, an opioid receptor agonist capable of activating μ-opioid receptors as well as other opioid receptor subtypes. In the present study, we demonstrate that the small-molecule GIRK activator, VU0466551, has analgesic effects when dosed alone or in combination with submaximally effective doses of morphine.

Herein, we report the structure-activity relationships within a series of mGlu 7 PAMs based on a pyrazolo[1,5- a ]pyrimidine core with excellent CNS penetration ( K p s > 1 and K p,uu s > 1). Analogues in this series proved to display a range of Group III mGlu receptor selectivity, but VU6005649 emerged as the first dual mGlu 7/8 PAM, filling a void in the Group III mGlu receptor PAM toolbox and demonstrating in vivo efficacy in a mouse contextual fear conditioning model.

Herein, we detail the optimization of the mGlu 3 NAM, VU0650786, via a reductionist approach to afford a novel, simplified mGlu 3 NAM scaffold that engenders potent and selective mGlu 3 inhibition (mGlu 3 IC 50 = 245 nM, mGlu 2 IC 50 > 30 μM) with excellent central nervous system penetration (rat brain/plasma K p = 1.2, K p,uu = 0.40). Moreover, this new chemotype, exemplified by VU6010572, requires only four synthetic steps and displays improved physiochemical properties and in vivo efficacy in a mouse tail suspension test (MED = 3 mg/kg i.p.).

Herein, we detail the optimization of the mGlu 2 negative allosteric modulator (NAM), VU6001192, by a reductionist approach to afford a novel, simplified mGlu 2 NAM scaffold. This new chemotype not only affords potent and selective mGlu 2 inhibition, as exemplified by VU6001966 (mGlu 2 IC 50 = 78 nM, mGlu 3 IC 50 > 30 μM), but also excellent central nervous system (CNS) penetration ( K p = 1.9, K p,uu = 0.78), a feature devoid in all previously disclosed mGlu 2 NAMs ( K p s ≈ 0.3, K p,uu s ≈ 0.1). Moreover, this series, based on overall properties, represents an exciting lead series for potential mGlu 2 PET tracer development.

Abnormalities in the signaling of the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) within cortical and limbic brain regions are thought to underlie many of the complex cognitive and affective symptoms observed in individuals with schizophrenia. The M 1 muscarinic acetylcholine receptor (mAChR) subtype is a closely coupled signaling partner of the NMDAR. Accumulating evidence suggests that development of selective positive allosteric modulators (PAMs) of the M 1 receptor represent an important treatment strategy for the potential normalization of disruptions in NMDAR signaling in patients with schizophrenia. In the present studies, we evaluated the effects of the novel and highly potent M 1 PAM, VU6004256, in ameliorating selective prefrontal cortical (PFC)-mediated physiologic and cognitive abnormalities in a genetic mouse model of global reduction in the NR1 subunit of the NMDAR (NR1 knockdown [KD]). Using slice-based extracellular field potential recordings, deficits in muscarinic agonist-induced long-term depression (LTD) in layer V of the PFC in the NR1 KD mice were normalized with bath application of VU6004256. Systemic administration of VU6004256 also reduced excessive pyramidal neuron firing in layer V PFC neurons in awake, freely moving NR1 KD mice. Moreover, selective potentiation of M 1 by VU6004256 reversed the performance impairments of NR1 KD mice observed in two preclinical models of PFC-mediated learning, specifically the novel object recognition and cue-mediated fear conditioning tasks. VU6004256 also produced a robust, dose-dependent reduction in the hyperlocomotor activity of NR1 KD mice. Taken together, the current findings provide further support for M 1 PAMs as a novel therapeutic approach for the PFC-mediated impairments in schizophrenia., Competing Interests: The authors declare the following competing financial interest(s): Over the past year, C.W.L. consulted for Abbott. M.B., T.M.B., C.W.L., P.J.C., and C.K.J. received research/salary support from AstraZeneca and/or Bristol Myers Squibb. C.M.N., C.W.L., and P.J.C. are inventors on multiple composition of matter patents protecting allosteric modulators of GPCRs. The remaining authors declare no competing financial interests.

Herein, we report the structure-activity relationships within a series of potent, selective, and orally bioavailable muscarinic acetylcholine receptor 4 (M 4 ) positive allosteric modulators (PAMs). Compound 6c (VU0467485) possesses robust in vitro M 4 PAM potency across species and in vivo efficacy in preclinical models of schizophrenia. Coupled with an attractive DMPK profile and suitable predicted human PK, 6c (VU0467485) was evaluated as a preclinical development candidate., Competing Interests: The authors declare the following competing financial interest(s): The authors are developing M4 PAMs for the treatment of schizophrenia and hold patents on the same.

Metabotropic glutamate receptor 4 (mGlu4) is emerging as a potential therapeutic target for numerous central nervous system indications, including Parkinson's disease (PD). As the glutamate binding sites among the eight mGlu receptors are highly conserved, modulation of receptor activity via allosteric sites within the receptor transmembrane domains using positive and negative allosteric modulators (PAMs and NAMs, respectively) has become a common strategy. We and others have used PAMs targeting mGlu4 to show that potentiation of receptor signaling induces antiparkinsonian activity in a variety of PD animal models, including haloperidol-induced catalepsy and 6-hydroxydopamine-induced lesion. Recently, mGlu4 has been reported to form heteromeric complexes with other mGlu receptor subtypes, such as mGlu2, and the resulting heteromer exhibits a distinct pharmacological profile in response to allosteric modulators. For example, some mGlu4 PAMs do not appear to potentiate glutamate activity when mGlu2 and mGlu4 are coexpressed, whereas other compounds potentiate mGlu4 responses regardless of mGlu2 coexpression. We report here the discovery and characterization of VU0418506, a novel mGlu4 PAM with activity in rodent PD models. Using pharmacological approaches and Complemented Donor-Acceptor resonance energy transfer (CODA-RET) technology, we find that VU0418506 does not potentiate agonist-induced activity when mGlu2 and mGlu4 are heterodimerized, suggesting that the antiparkinsonian action of mGlu4 PAMs can be induced by compounds without activity at mGlu2/4 heteromers.

The M1 muscarinic acetylcholine receptor (mAChR) subtype has been implicated in the underlying mechanisms of learning and memory and represents an important potential pharmacotherapeutic target for the cognitive impairments observed in neuropsychiatric disorders such as schizophrenia. Patients with schizophrenia show impairments in top-down processing involving conflict between sensory-driven and goal-oriented processes that can be modeled in preclinical studies using touchscreen-based cognition tasks. The present studies used a touchscreen visual pairwise discrimination task in which mice discriminated between a less salient and a more salient stimulus to assess the influence of the M1 mAChR on top-down processing. M1 mAChR knockout (M1 KO) mice showed a slower rate of learning, evidenced by slower increases in accuracy over 12 consecutive days, and required more days to acquire (achieve 80% accuracy) this discrimination task compared to wild-type mice. In addition, the M1 positive allosteric modulator BQCA enhanced the rate of learning this discrimination in wild-type, but not in M1 KO, mice when BQCA was administered daily prior to testing over 12 consecutive days. Importantly, in discriminations between stimuli of equal salience, M1 KO mice did not show impaired acquisition and BQCA did not affect the rate of learning or acquisition in wild-type mice. These studies are the first to demonstrate performance deficits in M1 KO mice using touchscreen cognitive assessments and enhanced rate of learning and acquisition in wild-type mice through M1 mAChR potentiation when the touchscreen discrimination task involves top-down processing. Taken together, these findings provide further support for M1 potentiation as a potential treatment for the cognitive symptoms associated with schizophrenia.

Hyperarousal and sleep disturbances are common, debilitating symptoms of post-traumatic stress disorder (PTSD). PTSD patients also exhibit abnormalities in quantitative electroencephalography (qEEG) power spectra during wake as well as rapid eye movement (REM) and non-REM (NREM) sleep. Selective serotonin reuptake inhibitors (SSRIs), the first-line pharmacological treatment for PTSD, provide modest remediation of the hyperarousal symptoms in PTSD patients, but have little to no effect on the sleep-wake architecture deficits. Development of novel therapeutics for these sleep-wake architecture deficits is limited by a lack of relevant animal models. Thus, the present study investigated whether single prolonged stress (SPS), a rodent model of traumatic stress, induces PTSD-like sleep-wake and qEEG spectral power abnormalities that correlate with changes in central serotonin (5-HT) and neuropeptide Y (NPY) signaling in rats. Rats were implanted with telemetric recording devices to continuously measure EEG before and after SPS treatment. A second cohort of rats was used to measure SPS-induced changes in plasma corticosterone, 5-HT utilization, and NPY expression in brain regions that comprise the neural fear circuitry. SPS caused sustained dysregulation of NREM and REM sleep, accompanied by state-dependent alterations in qEEG power spectra indicative of cortical hyperarousal. These changes corresponded with acute induction of the corticosterone receptor co-chaperone FK506-binding protein 51 and delayed reductions in 5-HT utilization and NPY expression in the amygdala. SPS represents a preclinical model of PTSD-related sleep-wake and qEEG disturbances with underlying alterations in neurotransmitter systems known to modulate both sleep-wake architecture and the neural fear circuitry.

Schizophrenia is a complex and highly heterogeneous psychiatric disorder whose precise etiology remains elusive. While genome-wide association studies (GWAS) have identified risk genes, they have failed to determine if rare coding single nucleotide polymorphisms (nsSNPs) contribute in schizophrenia. Recently, two independent studies identified 12 rare, deleterious nsSNPS in the GRM1 gene, which encodes the metabotropic glutamate receptor subtype 1 (mGlu1), in schizophrenic patients. Here, we generated stable cell lines expressing the mGlu1 mutant receptors and assessed their pharmacology. Using both the endogenous agonist glutamate and the synthetic agonist DHPG, we found that several of the mutant mGlu1 receptors displayed a loss of function that was not due to a loss in plasma membrane expression. Due to a lack of mGlu1 positive allosteric modulators (PAM) tool compounds active at human mGlu1, we optimized a known mGlu4 PAM/mGlu1 NAM chemotype into a series of potent and selective mGlu1 PAMs by virtue of a double "molecular switch". Employing mGlu1 PAMs from multiple chemotypes, we demonstrate that the mutant receptors can be potentiated by small molecules and in some cases efficacy restored to that comparable to wild type mGlu1 receptors, suggesting deficits in patients with schizophrenia due to these mutations may be amenable to intervention with an mGlu1 PAM. However, in wild type animals, mGlu1 negative allosteric modulators (NAMs) are efficacious in classic models predictive of antipsychotic activity, whereas we show that mGlu1 PAMs have no effect to slight potentiation in these models. These data further highlight the heterogeneity of schizophrenia and the critical role of patient selection strategies in psychiatric clinical trials to match genotype with therapeutic mechanism.

Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a novel approach for the treatment of psychotic symptoms associated with schizophrenia and other neuropsychiatric disorders. We recently reported that the selective M4 PAM VU0152100 produced an antipsychotic drug-like profile in rodents after amphetamine challenge. Previous studies suggest that enhanced cholinergic activity may also improve cognitive function and reverse deficits observed with reduced signaling through the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) in the central nervous system. Prior to this study, the M1 mAChR subtype was viewed as the primary candidate for these actions relative to the other mAChR subtypes. Here we describe the discovery of a novel M4 PAM, VU0467154, with enhanced in vitro potency and improved pharmacokinetic properties relative to other M4 PAMs, enabling a more extensive characterization of M4 actions in rodent models. We used VU0467154 to test the hypothesis that selective potentiation of M4 receptor signaling could ameliorate the behavioral, cognitive, and neurochemical impairments induced by the noncompetitive NMDAR antagonist MK-801. VU0467154 produced a robust dose-dependent reversal of MK-801-induced hyperlocomotion and deficits in preclinical models of associative learning and memory functions, including the touchscreen pairwise visual discrimination task in wild-type mice, but failed to reverse these stimulant-induced deficits in M4 KO mice. VU0467154 also enhanced the acquisition of both contextual and cue-mediated fear conditioning when administered alone in wild-type mice. These novel findings suggest that M4 PAMs may provide a strategy for addressing the more complex affective and cognitive disruptions associated with schizophrenia and other neuropsychiatric disorders.

Herein, we report the structure-activity relationship of a chiral morpholine-based scaffold, which led to the identification of a potent and selective dopamine 4 (D4) receptor antagonist. The 4-chlorobenzyl moiety was identified, and the compound was designated an MLPCN probe molecule, ML398. ML398 is potent against the D4 receptor with IC50 = 130 nM and K i = 36 nM and shows no activity against the other dopamine receptors tested (>20 μM against D1, D2S, D2L, D3, and D5). Further in vivo studies showed that ML398 reversed cocaine-induced hyperlocomotion at 10 mg/kg.