Quantitative proteomics in laser capture microdissected sleep nuclei from rat brain.

The combination of stable isotope labeling of amino acids in mammals (SILAM) and laser capture microdissection (LCM) for selective proteomic analysis of the targeted tissues holds tremendous potential for refined characterization of proteome changes within complex tissues such as the brain. The authors have applied this approach to measure changes in relative protein abundance in ventral tegmental area (VTA) of the rat brain that correlate to pharmacological perturbations. Enriched (13)C6(15)N2-lysine was introduced in vivo via diet. These animals were sacrificed during the middle of the 12-hour light period to extract isotopically "heavy" proteins, which were then used as a reference for extracts from dosed, unlabeled rats. Animals were administered an orexin peptide (Ox-B), an orexin receptor antagonist (ORA), or a mixture of both (Ox-B + ORA). All samples were obtained at same phase of the sleep cycle. Labeled-pair identification and differential quantitation provided protein identification and expression ratio data. Five proteins were found to exhibit decreased relative abundance after administration of an ORA, including α-synuclein and rat myelin basic protein. Conversely, six proteins showed increased relative abundance upon antagonist treatment, including 2',3'-cyclic nucleotide 3'-phosphodiesterase.

Discovery of 2,5-diarylnicotinamides as selective orexin-2 receptor antagonists (2-SORAs).

The orexin (or hypocretin) system has been identified as a novel target for the treatment of insomnia due to the wealth of biological and genetic data discovered over the past decade. Recently, clinical proof-of-concept was achieved for the treatment of primary insomnia using dual (OX1R/OX2R) orexin receptor antagonists. However, elucidation of the pharmacology associated with selective orexin-2 receptor antagonists (2-SORAs) has been hampered by the lack of orally bioavailable, highly selective small molecule probes. Herein, the discovery and optimization of a novel series of 2,5-diarylnicotinamides as potent and orally bioavailable orexin-2 receptor selective antagonists is described. A compound from this series demonstrated potent sleep promotion when dosed orally to EEG telemetrized rats.

Promotion of sleep by suvorexant-a novel dual orexin receptor antagonist.

Orexins/hypocretins are key neuropeptides responsible for regulating central arousal and reward circuits. Two receptors respond to orexin signaling, orexin 1 receptor (OX(1)R) and orexin 2 receptor (OX(2)R) with partially overlapping nervous system distributions. Genetic studies suggest orexin receptor antagonists could be therapeutic for insomnia and other disorders with disruptions of sleep and wake. Suvorexant (MK-4305) is a potent, selective, and orally bioavailable antagonist of OX(1)R and OX(2)R currently under clinical investigation as a novel therapy for insomnia. Examination of Suvorexant in radioligand binding assays using tissue from transgenic rats expressing the human OX(2)R found nearly full receptor occupancy (>90%) at plasma exposures of 1.1 µM. Dosed orally Suvorexant significantly and dose-dependently reduced locomotor activity and promoted sleep in rats (10, 30, and 100 mg/kg), dogs (1 and 3 mg/kg), and rhesus monkeys (10 mg/kg). Consistent cross-species sleep/wake architecture changes produced by Suvorexant highlight a unique opportunity to develop dual orexin antagonists as a novel therapy for insomnia.

Indazole derivatives as novel bradykinin B1 receptor antagonists.

A new class of indazole-derived bradykinin B(1) antagonists and their structure-activity relationships (SAR) is reported. A number of compounds were found to have low-nanomolar affinity for the human B(1) receptor and possess acceptable P-gp and pharmacokinetics properties.

Design and synthesis of conformationally constrained N,N-disubstituted 1,4-diazepanes as potent orexin receptor antagonists.

Orexins are neuropeptides that regulate wakefulness and arousal. Small molecule antagonists of orexin receptors may provide a novel therapy for the treatment of insomnia and other sleep disorders. In this Letter we describe the design and synthesis of conformationally constrained N,N-disubstituted 1,4-diazepanes as orexin receptor antagonists. The design of these constrained analogs was guided by an understanding of the preferred solution and solid state conformation of the diazepane central ring.

Discovery of 3,9-diazabicyclo[4.2.1]nonanes as potent dual orexin receptor antagonists with sleep-promoting activity in the rat.

Orexins are excitatory neuropeptides that regulate arousal and sleep. Orexin receptor antagonists promote sleep and offer potential as a new therapy for the treatment of insomnia. In this Letter, we describe the synthesis of constrained diazepanes having a 3,9 diazabicyclo[4.2.1]nonane bicyclic core with good oral bioavailability and sleep-promoting activity in a rat EEG model.

Synthesis and evaluation of a new series of Neuropeptide S receptor antagonists.

Administration of Neuropeptide S (NPS) has been shown to produce arousal, that is, independent of novelty and to induce wakefulness by suppressing all stages of sleep, as demonstrated by EEG recordings in rat. Medicinal chemistry efforts have identified a quinolinone class of potent NPSR antagonists that readily cross the blood-brain barrier. We detail here optimization efforts resulting in the identification of a potent NPSR antagonist which dose-dependently and specifically inhibited (125)I-NPS binding in the CNS when administered to rats.

A new class of bradykinin B1 receptor antagonists with high oral bioavailability and minimal PXR activity.

The design and synthesis of a novel class of human bradykinin B1 antagonists featuring difluoroethyl ether and isoxazole carboxamide moieties are disclosed. Compound 7g displayed excellent pharmacokinetic properties, efficient ex vivo receptor occupancy, and low potential for P450 induction via PXR activation.

Bradykinin B1 receptor antagonists: an alpha-hydroxy amide with an improved metabolism profile.

A series of carbo- and heterocyclic alpha-hydroxy amide-derived bradykinin B1 antagonists was prepared and evaluated. A 4,4-difluorocyclohexyl alpha-hydroxy amide was incorporated along with a 2-methyl tetrazole in lieu of an oxadiazole to afford a suitable compound with good pharmacokinetic properties, CNS penetration, and clearance by multiple metabolic pathways.

Proline bis-amides as potent dual orexin receptor antagonists.

A series of OX(2)R/OX(1)R dual orexin antagonists was prepared based on a proline bis-amide identified as a screening lead. Through a combination of classical and library synthesis, potency enhancing replacements for both amide portions were discovered. N-methylation of the benzimidazole moiety within the lead structure significantly reduced P-gp susceptibility while increasing potency, giving rise to good brain penetration. A compound from this series has demonstrated in vivo central activity when dosed peripherally in a pharmacodynamic model of orexin activity.

Development of orally bioavailable and CNS penetrant biphenylaminocyclopropane carboxamide bradykinin B1 receptor antagonists.

A series of biphenylaminocyclopropane carboxamide based bradykinin B1 receptor antagonists has been developed that possesses good pharmacokinetic properties and is CNS penetrant. Discovery that the replacement of the trifluoropropionamide in the lead structure with polyhaloacetamides, particularly a trifluoroacetamide, significantly reduced P-glycoprotein mediated efflux for the series proved essential. One of these novel bradykinin B1 antagonists (13b) also exhibited suitable pharmacokinetic properties and efficient ex vivo receptor occupancy for further development as a novel approach for the treatment of pain and inflammation.

Pharmacological characterization and radioligand binding properties of a high-affinity, nonpeptide, bradykinin B1 receptor antagonist.

Compound A (N-[2-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]ethyl]-2-[(2R)-1-(2-napthylsulfonyl)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetamide) is a member of a new class of aryl sulfonamide dihydroquinoxalinone bradykinin B1 receptor antagonists that should be useful pharmacological tools. Here we report on some of the pharmacological properties of compound A as well as the characterization of [35S]compound A as the first nonpeptide bradykinin B1 receptor radioligand. Compound A inhibited tritiated peptide ligand binding to the cloned human, rabbit, dog, and rat bradykinin B1 receptors expressed in CHO cells with Ki values of 0.016, 0.050, 0.56, and 29 nM, respectively. It was inactive at 10 microM in binding assays with the cloned human bradykinin B2 receptor. In functional antagonist assays with the cloned bradykinin B1 receptors, compound A inhibited agonist-induced signaling with activities consistent with the competition binding results, but had no antagonist activity at the bradykinin B2 receptor. Compound A was also found to be a potent antagonist in a rabbit aorta tissue bath preparation and to effectively block des-Arg9 bradykinin depressor responses in lipopolysaccharide-treated rabbit following intravenous administration. The binding of [35S]compound A was evaluated with the cloned bradykinin B1 receptors. In assays with human, rabbit, and dog receptors, [35S]compound A labeled a single site with Kd values of 0.012, 0.064, and 0.37 nM, respectively, and with binding site densities equivalent to those obtained using the conventional tritiated peptide ligands. Binding assays with the cloned rat bradykinin B1 receptor were not successful, presumably due to the low affinity of the ligand for this species receptor. There was no specific binding of the ligand detected in CHO cells expressing the human bradykinin B2 receptor. In assays with the cloned human bradykinin B1 receptor, the pharmacologies of the binding of [35S]compound A and [3H][Leu9]des-Arg10-kallidin were the same. The high signal-to-noise ratio obtained with [35S]compound A will allow this ligand to be a very useful tool for future investigations of the bradykinin B1 receptor.

Dual orexin receptor antagonists show distinct effects on locomotor performance, ethanol interaction and sleep architecture relative to gamma-aminobutyric acid-A receptor modulators.

Dual orexin receptor antagonists (DORAs) are a potential treatment for insomnia that function by blocking both the orexin 1 and orexin 2 receptors. The objective of the current study was to further confirm the impact of therapeutic mechanisms targeting insomnia on locomotor coordination and ethanol interaction using DORAs and gamma-aminobutyric acid (GABA)-A receptor modulators of distinct chemical structure and pharmacological properties in the context of sleep-promoting potential. The current study compared rat motor co-ordination after administration of DORAs, DORA-12 and almorexant, and GABA-A receptor modulators, zolpidem, eszopiclone, and diazepam, alone or each in combination with ethanol. Motor performance was assessed by measuring time spent walking on a rotarod apparatus. Zolpidem, eszopiclone and diazepam [0.3-30 mg/kg administered orally (PO)] impaired rotarod performance in a dose-dependent manner. Furthermore, all three GABA-A receptor modulators potentiated ethanol- (0.25-1.5 g/kg) induced impairment on the rotarod. By contrast, neither DORA-12 (10-100 mg/kg, PO) nor almorexant (30-300 mg/kg, PO) impaired motor performance alone or in combination with ethanol. In addition, distinct differences in sleep architecture were observed between ethanol, GABA-A receptor modulators (zolpidem, eszopiclone, and diazepam) and DORA-12 in electroencephalogram studies in rats. These findings provide further evidence that orexin receptor antagonists have an improved motor side-effect profile compared with currently available sleep-promoting agents based on preclinical data and strengthen the rationale for further evaluation of these agents in clinical development.

Discovery of a pharmacologically active antagonist of the two-pore-domain potassium channel K2P9.1 (TASK-3).

TWIK-related acid-sensitive K(+) (K(2P) 9.1, TASK-3) ion channels have the capacity to regulate the activity of neuronal pathways by influencing the resting membrane potential of neurons on which they are expressed. The central nervous system (CNS) expression of these channels suggests potential roles in neurologic disorders, and it is believed that the development of TASK-3 antagonists could lead to the therapeutic treatment of a number of neurological conditions. While a therapeutic potential for TASK-3 channel modulation exists, there are only a few documented examples of potent and selective small-molecule channel blockers. Herein, we describe the discovery and lead optimization efforts for a novel series of TASK-3 channel antagonists based on a 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine high-throughput screening lead from which a subseries of potent and selective inhibitors were identified. One compound was profiled in detail with respect to its physical properties and demonstrated pharmacological target engagement as indicated by its ability to modulate sleep architecture in rodent electroencephalogram (EEG) telemetry models.

Pharmacological characterization of MK-6096 - a dual orexin receptor antagonist for insomnia.

Orexin (hypocretin) neuropeptides promote wakefulness by signaling through two G-protein coupled receptors, Orexin 1 Receptor (OX(1)R) and Orexin 2 Receptor (OX(2)R). MK-6096 is an orally bioavailable potent and selective reversible antagonist of OX(1)R and OX(2)R currently in clinical development for insomnia. In radioligand binding and functional cell based assays MK-6096 demonstrated potent binding and antagonism of both human OX(1)R and OX(2)R (<3 nM in binding, 11 nM in FLIPR), with no significant off-target activities against a panel of >170 receptors and enzymes. MK-6096 occupies 90% of human OX(2)Rs expressed in transgenic rats at a plasma concentration of 142 nM, and dose-dependently reduced locomotor activity and significantly increased sleep in rats (3-30 mg/kg) and dogs (0.25 and 0.5 mg/kg). DORA-22, an analog of MK-6096, exhibits similar sleep promoting properties that are absent OX(1/2)R double knockouts, demonstrating the mechanism of action and specificity of these effects. These findings with a novel, structurally distinct class of OxR antagonists provide further validation of the orexin pathway as an effective target to promote normal sleep. Comparative analysis of the biochemical and pharmacokinetic properties of these compounds relative to other OXR antagonists provides a basis for understanding the attributes critical for in vivo efficacy. This mechanism is distinct from current standard of care such that MK-6096 represents a novel and selective therapeutic for the treatment of insomnia. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

Discovery of [(2R,5R)-5-{[(5-fluoropyridin-2-yl)oxy]methyl}-2-methylpiperidin-1-yl][5-methyl-2-(pyrimidin-2-yl)phenyl]methanone (MK-6096): a dual orexin receptor antagonist with potent sleep-promoting properties.

Insomnia is a common disorder that can be comorbid with other physical and psychological illnesses. Traditional management of insomnia relies on general central nervous system (CNS) suppression using GABA modulators. Many of these agents fail to meet patient needs with respect to sleep onset, maintenance, and next-day residual effects and have issues related to tolerance, memory disturbances, and balance. Orexin neuropeptides are central regulators of wakefulness, and orexin antagonism has been identified as a novel mechanism for treating insomnia with clinical proof of concept. Herein we describe the discovery of a series of α-methylpiperidine carboxamide dual orexin 1 and orexin 2 receptor (OX(1) R/OX(2) R) antagonists (DORAs). The design of these molecules was inspired by earlier work from this laboratory in understanding preferred conformational properties for potent orexin receptor binding. Minimization of 1,3-allylic strain interactions was used as a design principle to synthesize 2,5-disubstituted piperidine carboxamides with axially oriented substituents including DORA 28. DORA 28 (MK-6096) has exceptional in vivo activity in preclinical sleep models, and has advanced into phase II clinical trials for the treatment of insomnia.

TASK-3 as a potential antidepressant target.

Modulation of TASK-3 (Kcnk9) potassium channels affect neurotransmitter release in thalamocortical centers and other sleep-related nuclei having the capacity to regulate arousal cycles and REM sleep changes associated with mood disorders and antidepressant action. Circumstantial evidence from this and previous studies suggest the potential for TASK-3 to be a novel antidepressant therapeutic target; TASK-3 knock-out mice display augmented circadian amplitude and exhibit sleep architecture characterized by suppressed REM activity. Detailed analysis of locomotor activity indicates that the amplitudes of activity bout duration and bout number are augmented in TASK-3 mutants well beyond that seen in wildtypes, findings substantiated by amplitude increases in body temperature and EEG recordings of sleep stage bouts. Polysomnographic analysis of TASK-3 mutants reveals increases in nocturnal active wake and suppressed REM sleep time while increased slow wave sleep typifies the inactive phase, findings that have implications for the cognitive impact of reduced TASK-3 activity. In direct measures of their resistance to despair behavior, TASK-3 knock-outs displayed significant decreases in immobility relative to wildtype controls in both tail suspension and forced swim tests. Treatment of wildtype animals with the antidepressant Fluoxetine markedly reduced REM sleep, while leaving active wake and slow wave sleep relatively intact. Remarkably, these effects were absent in TASK-3 mutants indicating that TASK-3 is either directly involved in the mechanism of this drug's action, or participates in parallel pathways that achieve the same effect. Together, these results support the TASK-3 channel to act as a therapeutic target for antidepressant action.

Discovery of 3-substituted aminocyclopentanes as potent and orally bioavailable NR2B subtype-selective NMDA antagonists.

A series of 3-substituted aminocyclopentanes has been identified as highly potent and selective NR2B receptor antagonists. Incorporation of a 1,2,4-oxadiazole linker and substitution of the pendant phenyl ring led to the discovery of orally bioavailable analogues that showed efficient NR2B receptor occupancy in rats. Unlike nonselective NMDA antagonists, the NR2B-selective antagonist 22 showed no adverse affects on motor coordination in the rotarod assay at high dose. Compound 22 was efficacious following oral administration in a spinal nerve ligation model of neuropathic pain and in an acute model of Parkinson's disease in a dose dependent manner.

Orexin receptor antagonism prevents transcriptional and behavioral plasticity resulting from stimulant exposure.

Orexin is a key neurotransmitter of central arousal and reward circuits in the CNS. Two receptors respond to orexin signaling, Orexin 1 Receptor (OX1R) and Orexin 2 Receptor (OX2R) with partially overlapping brain distributions. Genetic and pharmacological studies suggest orexin receptor antagonists could provide therapeutic benefit for insomnia and other disorders in which sleep/wake cycles are disrupted. Preclinical data has also emerged showing that the orexin system is involved in the behavioral and neurological effects of drugs of abuse (Aston-Jones et al., 2009; Harris et al., 2005). Here we report sleep promoting effects of a recently described small molecule dual orexin receptor OX1R and OX2R antagonist. This dual orexin receptor antagonist (DORA) also inhibits the ability of subchronic amphetamine to produce behavioral sensitization measured 10 days following pre-treatment. Transcriptional profiling of isolated reward and arousal circuits from brains of behaviorally sensitized animals showed that the DORA blocked the significant alteration of gene expression levels in response to amphetamine exposure, particularly those associated with synaptic plasticity in the VTA. Further, DORA attenuates the ability of nicotine to induce reinstatement of extinguished responding for a reinforcer, demonstrating selectivity of the effect to reward pathways and not to food intake. In summary, these data demonstrate efficacy of a dual orexin receptor antagonist for promotion of sleep and suggest that pharmacological inhibition of the orexin system may play a role in both prevention of drug-induced plasticity and drug-relapse.

Discovery of the dual orexin receptor antagonist [(7R)-4-(5-chloro-1,3-benzoxazol-2-yl)-7-methyl-1,4-diazepan-1-yl][5-methyl-2-(2H-1,2,3-triazol-2-yl)phenyl]methanone (MK-4305) for the treatment of insomnia.

Despite increased understanding of the biological basis for sleep control in the brain, few novel mechanisms for the treatment of insomnia have been identified in recent years. One notable exception is inhibition of the excitatory neuropeptides orexins A and B by design of orexin receptor antagonists. Herein, we describe how efforts to understand the origin of poor oral pharmacokinetics in a leading HTS-derived diazepane orexin receptor antagonist led to the identification of compound 10 with a 7-methyl substitution on the diazepane core. Though 10 displayed good potency, improved pharmacokinetics, and excellent in vivo efficacy, it formed reactive metabolites in microsomal incubations. A mechanistic hypothesis coupled with an in vitro assay to assess bioactivation led to replacement of the fluoroquinazoline ring of 10 with a chlorobenzoxazole to provide 3 (MK-4305), a potent dual orexin receptor antagonist that is currently being tested in phase III clinical trials for the treatment of primary insomnia.