RESUMEN
Orexins are a family of neuropeptides that regulate various physiological events, such as sleep/wakefulness as well as emotional and feeding behavior, and that act on two G-protein-coupled receptors, i.e., orexin 1 (OX1R) and orexin 2 receptors (OX2R). Since the discovery that dysfunction of the orexin/OX2R system causes the sleep disorder narcolepsy, several OX2R-selective and OX1/2R dual agonists have been disclosed. However, an OX1R-selective agonist has not yet been reported, despite the importance of the biological function of OX1R. Herein, we report the discovery of a potent OX1R-selective agonist, (R,E)-3-(4-methoxy-3-(N-(8-(2-(3-methoxyphenyl)-N-methylacetamido)-5,6,7,8-tetrahydronaphthalen-2-yl)sulfamoyl)phenyl)-N-(pyridin-4-yl)acrylamide [(R)-YNT-3708; EC50 = 7.48 nM for OX1R; OX2R/OX1R EC50 ratio = 22.5]. The OX1R-selective agonist (R)-YNT-3708 exhibited antinociceptive and reinforcing effects through the activation of OX1R in mice.
Asunto(s)
Neuropéptidos , Receptores Acoplados a Proteínas G , Ratones , Animales , Orexinas , Receptores de Orexina/agonistas , SueñoRESUMEN
Facilitation of fear extinction is a desirable action for the drugs to treat fear-related diseases, such as posttraumatic stress disorder (PTSD). We previously reported that a selective agonist of the δ-opioid receptor (DOP), KNT-127, facilitates contextual fear extinction in mice. However, its site of action in the brain and the underlying molecular mechanism remains unknown. Here, we investigated brain regions and cellular signaling pathways that may mediate the action of KNT-127 on fear extinction. Twenty-four hours after the fear conditioning, mice were reexposed to the conditioning chamber for 6 min as extinction training (reexposure 1). KNT-127 was microinjected into either the basolateral nucleus of the amygdala (BLA), hippocampus (HPC), prelimbic (PL), or infralimbic (IL) subregions of the medial prefrontal cortex, 30 min before reexposure 1. Next day, mice were reexposed to the chamber for 6 min as memory testing (reexposure 2). KNT-127 that infused into the BLA and IL, but not HPC or PL, significantly reduced the freezing response in reexposure 2 compared with those of control. The effect of KNT-127 administered into the BLA and IL was antagonized by pretreatment with a selective DOP antagonist. Further, the effect of KNT-127 was abolished by local administration of MEK/ERK inhibitor into the BLA, and PI3K/Akt inhibitor into the IL, respectively. These results suggested that the effect of KNT-127 was mediated by MEK/ERK signaling in the BLA, PI3K/Akt signaling in the IL, and DOPs in both brain regions. Here, we propose that DOPs play a role in fear extinction via distinct signaling pathways in the BLA and IL.
RESUMEN
A novel series of 1-amino-tetralin derivatives were designed and synthesized based on the putative binding mode of the naphthalene-type orexin receptor agonist 5 and their agonist activities against orexin receptors were evaluated. The introduction of N-methyl-(3-methoxyphenyl)acetamide unit onto the 1-amino-tetralin skeleton remarkably enhanced the potency of the agonist. The asymmetric synthesis of 6 revealed that (-)-6 having a (S)-1-amino-tetralin skeleton showed a OX2R selective agonist activity (EC50 = 2.69 nM for OX2R, OX1R/OX2R = 461) yet its enantiomer (R)-(+)-6 showed a potent OX1/2R dual agonist activity (EC50 = 13.5 nM for OX1R, 0.579 nM for OX2R, OX1R/OX2R = 23.3). These results suggested that upward orientation of the amide side chain against the tetralin scaffold (S-configuration) would be selective for OX2R activation, and the downward orientation (R-configuration) would be significant for dual agonist activity. To our best knowledge, there have been no reports thus far that the stereochemistry of one carbon center on the agonist structure regulates the orexin receptor selectivity. Our results would provide important information for the development of OX1R selective agonists.
Asunto(s)
Descubrimiento de Drogas , Receptores de Orexina/agonistas , Tetrahidronaftalenos/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Relación Estructura-Actividad , Tetrahidronaftalenos/síntesis química , Tetrahidronaftalenos/químicaRESUMEN
Ultrasonic vocalization (USVs) is a promising tool to measure behavioral anxiety in rodents as USV recording is noninvasive, behaviorally relevant, ethological, and reproducible. Studies reporting the effects of stress-induced USVs in adult mice remain limited and debated. We investigated the conditions under which mice emit aversive USVs and evaluated the effects of psychiatric drugs on stress-induced USVs. Male C57BL/6J mice were used. USVs during entire stress sessions were recorded according to their frequency. To investigate the effect of psychiatric drugs on USVs, the number of USVs under cold-restraint stress conditions before and after drug administration was compared. Immediately after stress exposure, blood samples were collected and plasma corticosterone levels were measured. The combination of cold and restraint stress conditions significantly increased the USV numbers and plasma corticosterone levels compared with each stress alone. A benzodiazepine anxiolytic (midazolam) and δ-opioid receptor agonist putative anxiolytic (KNT-127) significantly reduced the stress-induced USV number and plasma corticosterone levels; however, a monoaminergic antidepressant (duloxetine) and N-methyl-D-aspartic acid receptor antagonist antidepressant (ketamine) did not reduce the USV numbers. No changes were noted in the USV numbers after repeated exposure to cold-restraint stress on days 1 and 3. The suppressive effect of midazolam on day 3 was comparable to that on day 1. Stress-induced USV may be used as a quantitative measure of anxiety to systematically assess the effects of anxiolytics. Therefore, cold-restraint stress-induced USVs may be used as a novel tool to measure rodent anxiety and as a useful anxiolytic-screening system.
Asunto(s)
Ansiolíticos , Vocalización Animal , Animales , Ansiolíticos/farmacología , Ansiolíticos/uso terapéutico , Ansiedad/tratamiento farmacológico , Ansiedad/etiología , Ansiedad/psicología , Masculino , Ratones , Ratones Endogámicos C57BL , UltrasonidoRESUMEN
Mas-related G protein-coupled receptor X2 (MRGPRX2) mediates the itch response in neurons and is involved in atopic dermatitis (AD)-associated inflammation and itch. Potent and MRGPRX2-selective ligands are essential to an understanding of the detailed function of the receptor and to develop new therapeutic agents for its related diseases. (+)-TAN-67 (1), the enantiomer of the δ-opioid receptor (DOR) selective ligand (-)-TAN-67 (1), has been reported to activate MRGPRX2, although (+)-1 also interacts with DOR, which prevents investigators from interrogating the function of MRGPRX2. Here, we have succeeded in developing a novel unnatural morphinan compound (+)-2a by a transformation based on the structure of (+)-1, which removes the DOR binding affinity. (+)-2a activated both human MRGPRX2 and the mouse orthologue Mrgprb2 in in vitro experiments and induced itch-like behaviors in mice to the same extent as (+)-1. The (+)-2a-induced itch response in mice was suppressed by administration of the tripeptide QWF, an MRGPRX2/Mrgprb2 antagonist, or the antipruritic drug nalfurafine. Together, (+)-2a serves as a useful tool to elucidate the itch-related function/action of MRGPRX2 and its mouse orthologue Mrgprb2.
Asunto(s)
Conducta Animal/efectos de los fármacos , Desarrollo de Medicamentos , Morfinanos/efectos adversos , Proteínas del Tejido Nervioso/metabolismo , Prurito/inducido químicamente , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/metabolismo , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Ligandos , Ratones , Estructura Molecular , Morfinanos/síntesis química , Morfinanos/química , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores de Neuropéptido/antagonistas & inhibidores , Receptores Opioides delta , Relación Estructura-ActividadRESUMEN
The medial prefrontal cortex (mPFC) plays a vital role in the processing of emotional events. It has been shown that activation of the glutamatergic transmission in prelimbic subregion of the mPFC (PL-PFC) evoked anxiety-like behavior in rodents. We previously reported that local perfusion of a selective agonist to delta-opioid receptor (DOP), KNT-127, attenuated the veratrine-induced elevation of extracellular glutamate in the PL-PFC and anxiety-like behavior in mice. These results suggested the possibility that KNT-127 suppresses glutamate release from the presynaptic site in the PL-PFC. To examine this possibility directly, we performed whole-cell patch-clamp recording from principal neurons in the PL-PFC and examined the spontaneous and electrically-evoked excitatory postsynaptic currents (EPSC)s. We found that bath application of KNT-127 significantly decreased the frequency of spontaneous and miniature EPSCs. Conversely, amplitude, rise time, and decay time of spontaneous and miniature EPSCs were not affected by bath application of KNT-127. Also, KNT-127 increased paired-pulse ratios of electrically-evoked EPSCs in the PL-PFC principal neurons tested. Further, we analyzed the firing properties of pyramidal neurons in the PL-PFC and found that KNT-127 treatment significantly reduced the number of action potentials and firing threshold. These results suggested that KNT-127 suppresses glutamatergic synaptic transmission by inhibiting glutamate release from the presynaptic site and reduces neuronal excitability in the mouse PL-PFC. We propose the possibility that these suppressing effects of KNT-127 on PL-PFC activity are part of the underlying mechanisms of its anxiolytic-like effects.
Asunto(s)
Morfinanos/farmacología , Neuronas/efectos de los fármacos , Corteza Prefrontal/fisiología , Receptores Opioides delta/agonistas , Transmisión Sináptica/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones Endogámicos C57BL , Neuronas/fisiologíaRESUMEN
Although delta opioid receptors (DOP) are now known to play a major role in modulating chronic pain and controlling emotional processes, unfortunately, some DOP agonists, such as SNC80, reportedly produced convulsive-like behaviors manifesting as tremor-like behaviors in a preclinical study. Therefore, these induced convulsions limit the progress of the clinical development of DOP agonists. However, mechanisms underlying DOP-induced convulsant activity remain unclarified. Thus, the study aimed to elucidate mechanisms that could cause tremor-like behaviors of SNC80. These drugs were microinjected into the ventral hippocampus CA3 (vCA3), amygdala (AMY), and insular cortex (IC) of mice. In addition, we examined the extracellular glutamate levels after DOP agonist local treatment. Microinjection of SNC80 into the vCA3 increased the number of tremor-like behaviors and extracellular glutamate levels but did not cause tremor-like behaviors in mice when microinjected into IC and AMY. Pretreatment with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainite receptor antagonist CNQX into vCA3 totally inhibited the SNC80-induced increases in tremor-like behaviors. In contrast, another DOP agonist, KNT-127, did not cause tremor-like behaviors in any of the tested brain areas. Further, the extracellular glutamate levels in the hippocampus were significantly lower in the KNT-127-treated mice than in the SNC80-treated mice. Our results showed that the administration of SNC80, but not KNT-127, into vCA3 induced tremor-like behaviors by activating glutamatergic neurons in mice. We propose that KNT-127 should be further studied clinically as a DOP agonist that is expected to have a low risk for convulsions than those resulting in antinociceptive and antidepressant effects.
Asunto(s)
Analgésicos Opioides/farmacología , Conducta Animal/efectos de los fármacos , Benzamidas/farmacocinética , Hipocampo/efectos de los fármacos , Morfinanos/farmacología , Piperazinas/farmacocinética , Animales , Ansiolíticos/farmacología , Antidepresivos/farmacología , Ansiedad/tratamiento farmacológico , Corteza Insular/efectos de los fármacos , Ratones , Actividad Motora/efectos de los fármacos , Naltrexona/farmacología , Receptores Opioides delta/efectos de los fármacos , Receptores Opioides delta/metabolismoRESUMEN
The D-nor-nalfurafine derivatives, which were synthesized by contraction of the six-membered D-ring in nalfurafine (1), had no affinity for orexin 1 receptors (OX1Rs). The 17N-lone electron pair in 1 oriented toward the axial direction, while that of D-nor-derivatives was directed in the equatorial configuration. The axial lone electron pair can form a hydrogen bond with the 14-hydroxy group, which could push the 6-amide side chain toward the downward direction with respect to the C-ring. The resulting conformation would be an active conformation for binding with OX1R. The dual affinities of 1 for OX1R and κ opioid receptor (KOR) led us to elucidate the mechanism by which only 1 showed no aversion but U-50488H. Actually, 1 selectively induced severe aversion in OX1R knockout mice, but not in wild-type mice. These results well support that OX1R suppresses the aversion of 1. This is the elucidation of long period puzzle which 1 showed no aversion in KOR.
Asunto(s)
Morfinanos/química , Antagonistas de los Receptores de Orexina/síntesis química , Receptores de Orexina/metabolismo , Compuestos de Espiro/química , Animales , Reacción de Prevención/efectos de los fármacos , Sitios de Unión , Ratones , Ratones Noqueados , Conformación Molecular , Simulación del Acoplamiento Molecular , Morfinanos/metabolismo , Morfinanos/farmacología , Antagonistas de los Receptores de Orexina/metabolismo , Antagonistas de los Receptores de Orexina/farmacología , Receptores de Orexina/química , Receptores de Orexina/genética , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Compuestos de Espiro/metabolismo , Compuestos de Espiro/farmacologíaRESUMEN
Elucidation of the binding mode of protein-ligand interactions provides insights for the design of new pharmacological tools and drug leads. Specific labeling of target proteins with chemical probes, in which the ligands are conjugated with reacting and detecting groups, can establish the binding positions of ligands. Label-assisted laser desorption/ionization mass spectrometry (LA-LDI MS) is a promising detection method to selectively detect labeled molecules. However, previous LDI MS tags, such as nitrogen-substituted pyrenes, had problems with low sensitivity and stability. Here we show 6-N,N-dimethylaminopyrene (dmpy) as a versatile mass tag, which was detected at an amount of 0.1 fmol by LA-LDI MS and applicable for MS/MS analysis. By using ligand-dissociation-type dmpy probes and affinity purification with a polystyrene gel, we demonstrated that dmpy-labeled peptides were predominantly detected by MALDI MS. Our dmpy-probe-labeling method might be highly useful for determining the target biomacromolecules of various ligands and their binding sites.
RESUMEN
To readily analyze the binding mode of protein-ligand interactions, we developed ligand-bound-type and ligand-dissociation-type probes having 6-amidopyrene (apy) as a detecting group. Matrix- and label-assisted laser desorption/ionization mass spectrometry (MALDI and LA-LDI MS) analyses and a covalent docking simulation using these probes precisely determined the binding position of the ligand biotin on the target protein avidin (RMSD = 0.786 and 0.332 Å). Our apy-probe-labeling method may be useful for determining the unknown ligand-binding sites of various target proteins.