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(R)-3-((3S,4S)-3-fluoro-4-(4-hydroxyphenyl)piperidin-1-yl)-1-(4-methylbenzyl)pyrrolidin-2-one (BMS-986169) and the phosphate prodrug 4-((3S,4S)-3-fluoro-1-((R)-1-(4-methylbenzyl)-2-oxopyrrolidin-3-yl)piperidin-4-yl)phenyl dihydrogen phosphate (BMS-986163) were identified from a drug discovery effort focused on the development of novel, intravenous glutamate N-methyl-d-aspartate 2B receptor (GluN2B) negative allosteric modulators (NAMs) for treatment-resistant depression (TRD). BMS-986169 showed high binding affinity for the GluN2B subunit allosteric modulatory site (Ki = 4.03-6.3 nM) and selectively inhibited GluN2B receptor function in Xenopus oocytes expressing human N-methyl-d-aspartate receptor subtypes (IC50 = 24.1 nM). BMS-986169 weakly inhibited human ether-a-go-go-related gene channel activity (IC50 = 28.4 μM) and had negligible activity in an assay panel containing 40 additional pharmacological targets. Intravenous administration of BMS-986169 or BMS-986163 dose-dependently increased GluN2B receptor occupancy and inhibited in vivo [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([3H]MK-801) binding, confirming target engagement and effective cleavage of the prodrug. BMS-986169 reduced immobility in the mouse forced swim test, an effect similar to intravenous ketamine treatment. Decreased novelty suppressed feeding latency, and increased ex vivo hippocampal long-term potentiation was also seen 24 hours after acute BMS-986163 or BMS-986169 administration. BMS-986169 did not produce ketamine-like hyperlocomotion or abnormal behaviors in mice or cynomolgus monkeys but did produce a transient working memory impairment in monkeys that was closely related to plasma exposure. Finally, BMS-986163 produced robust changes in the quantitative electroencephalogram power band distribution, a translational measure that can be used to assess pharmacodynamic activity in healthy humans. Due to the poor aqueous solubility of BMS-986169, BMS-986163 was selected as the lead GluN2B NAM candidate for further evaluation as a novel intravenous agent for TRD.

The NaV1.7 voltage-gated sodium channel is implicated in human pain perception by genetics. Rare gain of function mutations in NaV1.7 lead to spontaneous pain in humans whereas loss of function mutations results in congenital insensitivity to pain. Hence, agents that specifically modulate the function of NaV1.7 have the potential to yield novel therapeutics to treat pain. The complexity of the channel and the challenges to generate recombinant cell lines with high NaV1.7 expression have led to a surrogate target strategy approach employing chimeras with the bacterial channel NaVAb. In this report we describe the design, synthesis, purification, and characterization of a chimera containing part of the voltage sensor domain 2 (VSD2) of NaV1.7. Importantly, this chimera, DII S1-S4, forms functional sodium channels and is potently inhibited by the NaV1.7 VSD2 targeted peptide toxin ProTx-II. Further, we show by [125I]ProTx-II binding and surface plasmon resonance that the purified DII S1-S4 protein retains high affinity ProTx-II binding in detergent. We employed the purified DII S1-S4 protein to create a scintillation proximity assay suitable for high-throughput screening. The creation of a NaV1.7-NaVAb chimera with the VSD2 toxin binding site provides an important tool for the identification of novel NaV1.7 inhibitors and for structural studies to understand the toxin-channel interaction.

To identify novel targets for neuropathic pain, 3097 mouse knockout lines were tested in acute and persistent pain behavior assays. One of the lines from this screen, which contained a null allele of the adapter protein-2 associated kinase 1 (AAK1) gene, had a normal response in acute pain assays (hot plate, phase I formalin), but a markedly reduced response to persistent pain in phase II formalin. AAK1 knockout mice also failed to develop tactile allodynia following the Chung procedure of spinal nerve ligation (SNL). Based on these findings, potent, small-molecule inhibitors of AAK1 were identified. Studies in mice showed that one such inhibitor, LP-935509, caused a reduced pain response in phase II formalin and reversed fully established pain behavior following the SNL procedure. Further studies showed that the inhibitor also reduced evoked pain responses in the rat chronic constriction injury (CCI) model and the rat streptozotocin model of diabetic peripheral neuropathy. Using a nonbrain-penetrant AAK1 inhibitor and local administration of an AAK1 inhibitor, the relevant pool of AAK1 for antineuropathic action was found to be in the spinal cord. Consistent with these results, AAK1 inhibitors dose-dependently reduced the increased spontaneous neural activity in the spinal cord caused by CCI and blocked the development of windup induced by repeated electrical stimulation of the paw. The mechanism of AAK1 antinociception was further investigated with inhibitors of α2 adrenergic and opioid receptors. These studies showed that α2 adrenergic receptor inhibitors, but not opioid receptor inhibitors, not only prevented AAK1 inhibitor antineuropathic action in behavioral assays, but also blocked the AAK1 inhibitor-induced reduction in spinal neural activity in the rat CCI model. Hence, AAK1 inhibitors are a novel therapeutic approach to neuropathic pain with activity in animal models that is mechanistically linked (behaviorally and electrophysiologically) to α2 adrenergic signaling, a pathway known to be antinociceptive in humans.

Background: A significant proportion of patients suffering from major depression fail to remit following treatment and develop treatment-resistant depression. Developing novel treatments requires animal models with good predictive validity. MRL/lpr mice, an established model of systemic lupus erythematosus, show depression–like behavior. Aims: We evaluated responses to classical antidepressants, and associated immunological and biochemical changes in MRL/lpr mice. Methods and results: MRL/lpr mice showed increased immobility in the forced swim test, decreased wheel running and sucrose preference when compared with the controls, MRL/MpJ mice. In MRL/lpr mice, acute fluoxetine (30 mg/kg, intraperitoneally (i.p.)), imipramine (10 mg/kg, i.p.) or duloxetine (10 mg/kg, i.p.) did not decrease the immobility time in the Forced Swim Test. Interestingly, acute administration of combinations of olanzapine (0.03 mg/kg, subcutaneously)+fluoxetine (30 mg/kg, i.p.) or bupropion (10 mg/kg, i.p.)+fluoxetine (30 mg/kg, i.p.) retained efficacy. A single dose of ketamine but not three weeks of imipramine (10 mg/kg, i.p.) or escitalopram (5 mg/kg, i.p.) treatment in MRL/lpr mice restored sucrose preference. Further, we evaluated inflammatory, immune-mediated and neuronal mechanisms. In MRL/lpr mice, there was an increase in autoantibodies’ titers, [3H]PK11195 binding and immune complex deposition. There was a significant infiltration of the brain by macrophages, neutrophils and T-lymphocytes. p11 mRNA expression was decreased in the prefrontal cortex. Further, there was an increase in the 5-HT2aR expression, plasma corticosterone and indoleamine 2,3-dioxygenase activity. Conclusion: In summary, the MRL/lpr mice could be a useful model for Treatment Resistant Depression associated with immune dysfunction with potential to expedite antidepressant drug discovery.

Introduction A radioligand for measuring the density of corticotropin-releasing factor subtype-1 receptors (CRF 1 receptors) in living animal and human brain with positron emission tomography (PET) would be a useful tool for neuropsychiatric investigations and the development of drugs intended to interact with this target. This study was aimed at discovery of such a radioligand from a group of CRF 1 receptor ligands based on a core 3-(phenylamino)‐pyrazin-2(1 H )-one scaffold. Methods CRF 1 receptor ligands were selected for development as possible PET radioligands based on their binding potency at CRF 1 receptors (displacement of [ 125 I]CRF from rat cortical membranes), measured lipophilicity, autoradiographic binding profile in rat and rhesus monkey brain sections, rat biodistribution, and suitability for radiolabeling with carbon-11 or fluorine-18. Two identified candidates (BMS-721313 and BMS-732098) were labeled with fluorine-18. A third candidate (BMS-709460) was labeled with carbon-11 and all three radioligands were evaluated in PET experiments in rhesus monkey. CRF 1 receptor density ( B max ) was assessed in rhesus brain cortical and cerebellum membranes with the CRF 1 receptor ligand, [ 3 H]BMS-728300. Results The three ligands selected for development showed high binding affinity ( IC 50 values, 0.3–8 nM) at CRF 1 receptors and moderate lipophilicity ( LogD , 2.8–4.4). [ 3 H]BMS-728300 and the two 18 F-labeled ligands showed region-specific binding in rat and rhesus monkey brain autoradiography, namely higher binding density in the frontal and limbic cortex, and cerebellum than in thalamus and brainstem. CRF 1 receptor B max in rhesus brain was found to be 50–120 fmol/mg protein across cortical regions and cerebellum. PET experiments in rhesus monkey showed that the radioligands [ 18 F]BMS-721313, [ 18 F]BMS-732098 and [ 11 C]BMS-709460 gave acceptably high brain radioactivity uptake but no indication of the specific binding as seen in vitro . Conclusions Candidate CRF 1 receptor PET radioligands were identified but none proved to be effective for imaging monkey brain CRF 1 receptors. Higher affinity radioligands are likely required for successful PET imaging of CRF 1 receptors.

The effect of γ-aminobutyric acid (GABA) on neuronal firing rate in rat suprachiasmatic nucleus (SCN) slices was examined using continuous recording methods. GABA inhibited neuronal discharge during both the subjective day and the subjective night in a concentration-dependent manner characterized by two apparent affinity states. The GABAA receptor agonist muscimol caused potent inhibition regardless of circadian time; repeated applications of the agonist did not reverse the direction of effect. The GABAA receptor antagonists bicuculline and picrotoxin increased excitability when applied during either subjective day or subjective night. A significant increase in GABAA receptor– mediated inhibition, as well as endogenous GABAergic tone, was observed on the second day after slice preparation. The GABAB receptor agonist baclofen inhibited cell firing during subjective day and night, but the GABAB antagonist phaclofen had no significant effect. These data provide additional strong support for a predominantly inhibitory role of GABA in the rat SCN, regardless of the time of application in relation to the circadian rhythm, and demonstrate an important level of plasticity of this system in vitro.

Three independent electrophysiological approaches in hypothalamic slices were used to test the hypothesis that [.gamma]-amino butyric acid (GABA)Areceptor activation excites suprachiasmatic nucleus (SCN) neurons during the subjective day, consistent with a recent report. First, multiple-unit recordings during either the subjective day or night showed that GABA or muscimol inhibited firing activity of the SCN population in a dose-dependent manner. Second, cell-attached recordings during the subjective day demonstrated an inhibitory effect of bath-or microapplied GABA on action currents of single SCN neurons. Third, gramicidin perforated-patch recordings showed that bicuculline increased the spontaneous firing rate during the subjective day. Therefore, electrophysiological data obtained by three different experimental methods provide evidence that GABA is inhibitory rather than excitatory during the subjective day.

The pineal hormone melatonin elicits two effects on the suprachiasmatic nuclei (SCN): acute neuronal inhibition and phase-shifting. Melatonin evokes its biological effects through G protein-coupled receptors. Since the Mel1a melatonin receptor may transduce the major neurobiological actions of melatonin in mammals, we examined whether it mediates both melatonin effects on SCN function by using mice with targeted disruption of the Mel1a receptor. The Mel1a receptor accounts for all detectable, high affinity melatonin binding in mouse brain. Functionally, this receptor is necessary for the acute inhibitory action of melatonin on the SCN. Melatonin-induced phase shifts, however, are only modestly altered in the receptor-deficient mice; pertussis toxin still blocks melatonin-induced phase shifts in Mel1a receptor-deficient mice. The other melatonin receptor subtype, the Mel1b receptor, is expressed in mouse SCN, implicating it in the phase-shifting response. The results provide a molecular basis for two distinct, mechanistically separable effects of melatonin on SCN physiology.

The hormone, melatonin, is produced rhythmically in the vertebrate pineal gland (13). Rhythmic melatonin production plays a critical role in the regulation of reproduction in seasonally breeding mammals (1, 24). In nonmammalian vertebrates, melatonin also plays a major role in the regulation of circadian rhythms (4, 46). While endogenous melatonin appears to play only a subtle role in the regulation of circadian rhythms in mammals, exogenous melatonin influences circadian rhythms in several rodent species and in humans (2, 3, 9, 21, 22, 39, 46, 47). While the magnitude of this phase shifting effect is small for adult mammals, it is nevertheless critical in some situations; e.g., daily melatonin administration appears to be useful for entrainment of non-24-h circadian cycles to the 24-hour day for blind individuals (22). Two high-affinity receptors for melatonin have been identified in mammals (for reviews, see references 33 and 43). These receptors, the Mel1a and Mel1b receptor subtypes, are members of the G protein-coupled receptor superfamily (29, 30). A melatonin receptor-related receptor (also called H9 and GPR50) has sequence homology to the Mel1a and Mel1b receptors but does not bind melatonin (7, 16, 32). In addition to these three members of the gene family in mammals, nonmammalian species have a third high-affinity melatonin receptor subtype, the Mel1c receptor (31). The mammalian Mel1a and Mel1b receptors are also called the MT1 and MT2 receptors, respectively [10]. The inability of this MT nomenclature system to accommodate the nonmammalian Mel1c receptor has resulted in persistence of dual nomenclature systems; here, we use the original nomenclature. The relative importance of the mammalian melatonin receptor subtypes in mediating circadian responses to the hormone is unclear. The circadian clocks of neonatal hamsters are very efficiently set by even a single injection of melatonin, and several physiological and in vitro responses of the suprachiasmatic nucleus (SCN) to melatonin have been observed in hamsters (25, 36, 39, 47). Remarkably, however, the Mel1b receptor gene of several hamster species, including those with robust circadian and reproductive responses to the hormone, does not encode a functional melatonin receptor (47; see also GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AY145849","term_id":"24251143","term_text":"AY145849"}}AY145849). Pharmacological studies with mice, however, suggest that the mouse Mel1b receptor mediates circadian responses (9, 19). Other data, derived from mice with targeted disruption of the Mel1a melatonin receptor, indicate that this subtype mediates an acute suppressive effect of melatonin on SCN neuronal firing, and they reveal apparent redundancy of receptor subtypes in mediating the phase shifting response to melatonin (23). To determine whether the mouse Mel1b receptor contributes to responses to the hormone, the mouse Mel1b receptor cDNA was isolated. Finding that the mouse Mel1b receptor cDNA encodes a high-affinity melatonin receptor, we then disrupted the Mel1b receptor gene. Analysis of the mutant mice indicates that the Mel1b receptor encodes a functionally relevant melatonin receptor. The Mel1b receptor appears to mediate a response to the hormone occurring at higher ligand concentrations in mice lacking the Mel1a melatonin receptor.

During ischemic stroke, neurons at risk are exposed to pathologically high levels of intracellular calcium (Ca++), initiating a fatal biochemical cascade. To protect these neurons, we have developed openers of large-conductance, Ca++-activated (maxi-K or BK) potassium channels, thereby augmenting an endogenous mechanism for regulating Ca++ entry and membrane potential. The novel fluoro-oxindoles BMS-204352 and racemic compound 1 are potent, effective and uniquely Ca++-sensitive openers of maxi-K channels. In rat models of permanent large-vessel stroke, BMS-204352 provided significant levels of cortical neuroprotection when administered two hours after the onset of occlusion, but had no effects on blood pressure or cerebral blood flow. This novel approach may restrict Ca++ entry in neurons at risk while having minimal side effects.

Neuropeptide Y (NPY) has been implicated in the phase shifting of circadian rhythms in the hypothalamic suprachiasmatic nucleus (SCN). Using long-term, multiple-neuron recordings, we examined the direct effects and phase-shifting properties of NPY application in rat SCN slicesin vitro(n= 453). Application of NPY and peptide YY to SCN slices at circadian time (CT) 7.5–8.5 produced concentration-dependent, reversible inhibition of cell firing and a subsequent significant phase advance. Several lines of evidence indicated that these two effects of NPY were mediated by different receptors. NPY-induced inhibition and phase shifting had different concentration–response relationships and very different phase–response relationships. NPY-induced phase advances, but not inhibition, were blocked by the GABAAantagonist bicuculline, suggesting that NPY-mediated modulation of GABA may be an underlying mechanism whereby NPY phase shifts the circadian clock. Application of the Y2 receptor agonists NPY 13–36 and (Cys2,8-aminooctanoic acid5,24,d-Cys27)-NPY advanced the peak of the circadian rhythm but did not inhibit cell firing. The Y1 and Y5 agonist [Leu31,Pro34]-NPY evoked a substantial inhibition of discharge but did not generate a phase shift. NPY-induced inhibition was not blocked by the specific Y1 antagonist BIBP-3226; the antagonist also had no effect on the timing of the peak of the circadian rhythm. Application of the Y5 agonist [d-Trp32]-NPY produced only direct neuronal inhibition. These are the first data to indicate that at least two functional populations of NPY receptors exist in the SCN, distinguishable on the basis of pharmacology, each mediating a different physiological response to NPY application.