RESUMEN
There is much interest in identifying novel pharmacotherapeutic targets that improve clinical outcomes for the treatment of alcohol use disorder (AUD). One promising target for therapeutic intervention is the relaxin family peptide 3 (RXFP3) receptor, a cognate receptor for neuropeptide relaxin-3, which has previously been implicated in regulating alcohol drinking behavior. Recently, we developed the first small-molecule RXFP3-selective negative allosteric modulator (NAM) RLX-33. Therefore, the goal of the present work was to characterize the impact of this novel NAM on affective-related behaviors and alcohol self-administration in rats. First, the effects of RLX-33 were tested on alcohol and sucrose self-administration in Wistar and alcohol-preferring P rats to determine the dose-response profile and specificity for alcohol. Then, we assessed the effects of systemic RLX-33 injection in Wistar rats in a battery of behavioral assays (open-field test, elevated zero maze, acoustic startle response test, and prepulse inhibition) and tested for alcohol clearance. We found that the lowest effective dose (5 mg/kg) reduced alcohol self-administration in both male and female Wistar rats, while in alcohol-preferring P rats, this effect was restricted to males, and there were no effects on sucrose self-administration or general locomotor activity. The characterization of affective and metabolic effects in Wistar rats generally found few locomotor, affective, or alcohol clearance changes, particularly at the 5 mg/kg dose. Overall, these findings are promising and suggest that RXFP3 NAM has potential as a pharmacological target for treating AUD.
Asunto(s)
Alcoholismo , Relaxina , Ratas , Masculino , Femenino , Animales , Ratas Wistar , Reflejo de Sobresalto , Relaxina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Etanol , Alcoholismo/tratamiento farmacológico , Alcoholismo/metabolismo , Sacarosa , Receptores de PéptidosRESUMEN
G protein-coupled receptor 3 (GPR3) is an orphan receptor potentially involved in many important physiological processes such as drug abuse, neuropathic pain, and anxiety and depression related disorders. Pharmacological studies of GPR3 have been limited due to the restricted number of known agonists and inverse agonists for this constitutively active receptor. In this medicinal chemistry study, we report the discovery of GPR3 agonists based off the diphenyleneiodonium (DPI) scaffold. The most potent full agonist was the 3-trifluoromethoxy analog (32) with an EC50 of 260 nM and 90% efficacy compared to DPI. Investigation of a homology model of GPR3 from multiple sequence alignment resulted in the finding of a binding site rich in potential π-π and π-cation interactions stabilizing DPI-scaffold agonists. MMGBSA free energy analysis showed a good correlation with trends in observed EC50s. DPI analogs retained the same high receptor selectivity for GPR3 over GPR6 and GPR12 as observed with DPI. Collectively, the DPI analog series shows that order of magnitude improvements in potency with the scaffold were attainable; however, attempts to replace the iodonium ion to make the scaffold more druggable failed.
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Agonismo Inverso de Drogas , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/agonistas , Compuestos Onio , Sitios de UniónRESUMEN
Partial agonists of peripheral cannabinoid receptors (CBRs) have potential therapeutic applications in several medical conditions. However, (-)-trans-Δ9-tetrahydrocannabinol (THC), the principal active component of marijuana, which is a partial agonist of CB1 and CB2 penetrates the central nervous system (CNS) and produces adverse effects. Peripherally restricted partial agonists of CBRs, particularly of CB1, can be used to treat illnesses safely and effectively with a better therapeutic index. Here, we report on our efforts to synthesize pyrazole partial CBR agonists with peripheral selectivity, resulting in lead compound 40. This compound is a potent partial agonist of CB1 with â¼ 5-fold higher plasma biodistribution over brain and represents an early lead for optimization.
Asunto(s)
Agonistas de Receptores de Cannabinoides , Dronabinol , Agonistas de Receptores de Cannabinoides/farmacología , Distribución Tisular , Pirazoles/farmacología , Receptores de Cannabinoides , Receptor Cannabinoide CB1 , Receptor Cannabinoide CB2RESUMEN
The neuropeptide relaxin-3/RXFP3 system is involved in many important physiological processes such as stress responses, appetite control, and motivation for reward. To date, pharmacological studies of RXFP3 have been limited to peptide ligands. In this study, we report the discovery of the first small-molecule antagonists of RXFP3 through a high-throughput screening campaign. Focused structure-activity relationship studies of the hit compound resulted in RLX-33 (33) that was able to inhibit relaxin-3 activity in a battery of functional assays. RLX-33 is selective for RXFP3 over RXFP1 and RXFP4, two related members in the relaxin/insulin superfamily, and has favorable pharmacokinetic properties for behavioral assessment. When administered to rats intraperitoneally, RLX-33 blocked food intake induced by the RXFP3-selective agonist R3/I5. Collectively, our findings demonstrated that RLX-33 represents a promising antagonist scaffold for the development of drugs targeting the relaxin-3/RXFP3 system.
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Relaxina , Animales , Insulina , Ligandos , Ratas , Receptores Acoplados a Proteínas G/química , Receptores de Péptidos , Relaxina/farmacologíaRESUMEN
Synthetic cathinones are used as stimulants of abuse. Many abused drugs, including stimulants, activate nuclear factor-κB (NF-κB) transcription leading to increases in NF-κB-regulated pro-inflammatory cytokines, and the level of inflammation appears to correlate with length of abuse. The purpose of this study was to measure the profile of IL-1α, IL-1ß, IL-6, CCL2 and TNF-α in brain and plasma to examine if drug exposure alters inflammatory markers. Male and female Sprague-Dawley rats were trained to self-administer α-pyrrolidinopentiophenone (α-PVP) (0.1 mg/kg/infusion), 4-methylmethcathinone (4MMC) (0.5 mg/kg/infusion), or saline through autoshaping, and then self-administered for 21 days during 1 h (short access; ShA) or 6 h (long access; LgA) sessions. Separate rats were assigned to a naïve control group. Cytokine levels were examined in amygdala, hippocampus, hypothalamus, prefrontal cortex, striatum, thalamus, and plasma. Rats acquired synthetic cathinone self-administration, and there were no sex differences in drug intake. Synthetic cathinone self-administration produced sex differences in IL-1α, IL-1ß, IL-6, CCL2 and TNF-α levels. There were widespread increases in inflammatory cytokines in the brains of male rats compared to females, particularly for 4MMC, whereas females were more likely to show increased inflammatory cytokines in plasma compared to saline groups than males. Furthermore, these sex differences in cytokine levels were more common after LgA access to synthetic cathinones than ShA. These results suggest that synthetic cathinone use likely produces sex-selective patterns of neuroinflammation during the transition from use to abuse. Consequently, treatment need may differ depending on the progression of synthetic cathinone abuse and based on sex.
Asunto(s)
Alcaloides/farmacología , Estimulantes del Sistema Nervioso Central/farmacología , Citocinas/análisis , Alcaloides/administración & dosificación , Animales , Química Encefálica/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/administración & dosificación , Quimiocina CCL2/análisis , Quimiocina CCL2/sangre , Citocinas/sangre , Femenino , Interleucina-1alfa/análisis , Interleucina-1alfa/sangre , Interleucina-1beta/análisis , Interleucina-1beta/sangre , Interleucina-6/análisis , Interleucina-6/sangre , Masculino , Enfermedades Neuroinflamatorias/inducido químicamente , Enfermedades Neuroinflamatorias/metabolismo , Ratas , Ratas Sprague-Dawley , Autoadministración , Factores Sexuales , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/sangreRESUMEN
The central relaxin-3/RXFP3 system plays important roles in stress responses, feeding, and motivation for reward. However, exploration of its therapeutic applications has been hampered by the lack of small molecule ligands and the cross-activation of RXFP1 in the brain and RXFP4 in the periphery. Herein, we report the first structure-activity relationship studies of a series of novel nonpeptide amidinohydrazone-based agonists, which were characterized by RXFP3 functional and radioligand binding assays. Several potent and efficacious RXFP3 agonists (e.g., 10d) were identified with EC50 values <10 nM. These compounds also had high potency at RXFP4 but no agonist activity at RXFP1, demonstrating > 100-fold selectivity for RXFP3/4 over RXFP1. In vitro ADME and pharmacokinetic assessments revealed that the amidinohydrazone derivatives may have limited brain permeability. Collectively, our findings provide the basis for further optimization of lead compounds to develop a suitable agonist to probe RXFP3 functions in the brain.
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Hidrazonas/farmacología , Indoles/química , Receptores Acoplados a Proteínas G/agonistas , Receptores de Péptidos/agonistas , Humanos , Hidrazonas/síntesis química , Hidrazonas/química , Modelos Moleculares , Ensayo de Unión Radioligante , Relación Estructura-ActividadRESUMEN
Activation of the neuropeptide S receptor (NPSR) system has been shown to produce anxiolytic-like actions, arousal, and enhance memory consolidation, whereas blockade of the NPSR has been shown to reduce relapse to substances of abuse and duration of anesthetics. We report here the discovery of a novel core scaffold (+) N-benzyl-3-(2-methylpropyl)-1-oxo-3-phenyl-1H,3H,4H,5H,6H,7H-furo[3,4-c]pyridine-5-carboxamide with potent NPSR antagonist activity in vitro. Pharmacokinetic parameters demonstrate that 14b reaches pharmacologically relevant levels in plasma and the brain following intraperitoneal (i.p.) administration, but is cleared rapidly from plasma. Compound 14b was able to block NPS (0.3 nmol)-stimulated locomotor activity in C57/Bl6 mice at 3 mg/kg (i.p.), indicating potent in vivo activity for the structural class. This suggests that 14b can serve as a useful tool for continued mapping of the pharmacological functions of the NPS receptor system.
RESUMEN
Metabolic syndrome (MetS) is a complex disorder that stems from the additive effects of multiple underlying causes such as obesity, insulin resistance, and chronic low-grade inflammation. The endocannabinoid system plays a central role in appetite regulation, energy balance, lipid metabolism, insulin sensitivity, and ß-cell function. The type 1 cannabinoid receptor (CB1R) antagonist SR141716A (rimonabant) showed promising antiobesity effects, but its use was discontinued due to adverse psychiatric events in some users. These adverse effects are due to antagonism of CB1R in the central nervous system (CNS). As such, CNS-sparing CB1R antagonists are presently being developed for various indications. In this study, we report that a recently described compound, 3-{1-[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-1-[6-(difluoromethoxy)pyridin-3-yl]urea (RTI1092769), a pyrazole based weak inverse agonist/antagonist of CB1 with very limited brain exposure, improves MetS related complications. Treatment with RTI1092769 inhibited weight gain and improved glucose utilization in obese mice maintained on a high fat diet. Hepatic triglyceride content and steatosis significantly improved with treatment. These phenotypes were supported by improvement in several biomarkers associated with nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). These results reinforce the idea that CB1 antagonists with limited brain exposure should be pursued for MetS and other important indications.
RESUMEN
Stimulant-induced neurochemical changes may occur at different times for different brain regions or neurotransmitter systems. This study sought to examine the behavioral and neurochemical effects of extended access to α-pyrrolidinopentiophenone (α-PVP) and 4-methylmethcathinone (4MMC). Male and female Sprague-Dawley rats were trained to self-administer α-PVP (0.1 mg/kg/infusion) or 4MMC (0.5 mg/kg/infusion) through autoshaping, and then self-administered for 21 days during 1 h (short access; ShA) or 6 h (long access; LgA) sessions. Separate rats were assigned to a naïve control group. Amygdala, hippocampus, hypothalamus, prefrontal cortex (PFC), striatum, and thalamus were extracted, and tissue was analyzed with electrochemical detection and liquid chromatography mass spectrometry. Rats acquired self-administration of α-PVP and 4MMC, and LgA rats showed more escalation of self-administration than ShA rats. Synthetic cathinone administration produced several effects on neurotransmitters. LgA self-administration of α-PVP increased 5-HIAA levels in all brain regions, compared to control. In contrast, both LgA and ShA 4MMC self-administration decreased 5-HT and 5-HIAA levels in most brain regions. LgA exposure to both synthetic cathinones increased DOPAC levels in hypothalamus and striatum, and increased HVA levels in striatum compared to control. LgA self-administration of either synthetic cathinone produced region-specific increases in NE levels, whereas ShA self-administration lowered NE levels in select locations compared to control. These alterations in neurotransmitter levels indicate that synthetic cathinone use may produce differential neurochemical changes during the transition from use to abuse, and that 21 days of self-administration only models the beginning stages of dysregulated drug intake.
Asunto(s)
Trastornos Relacionados con Anfetaminas/metabolismo , Encéfalo/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/administración & dosificación , Metanfetamina/análogos & derivados , Neurotransmisores/metabolismo , Pentanonas/administración & dosificación , Pirrolidinas/administración & dosificación , Ácido 3,4-Dihidroxifenilacético/metabolismo , Trastornos Relacionados con Anfetaminas/fisiopatología , Animales , Encéfalo/metabolismo , Encéfalo/fisiopatología , Femenino , Ácido Hidroxiindolacético/metabolismo , Masculino , Metanfetamina/administración & dosificación , Norepinefrina/metabolismo , Ratas Sprague-Dawley , Autoadministración , Serotonina/metabolismo , Factores Sexuales , Factores de TiempoRESUMEN
Organophosphates (OPs) induce acute and chronic neurotoxicity, primarily by inhibiting acetylcholinesterase (AChE) activity as well as by necrosis, and apoptosis. Butyrylcholinesterase (BuChE), an exogenous bioscavenger of OPs, can be used as a treatment for OP exposure. It is prerequisite to develop in vitro brain models that can study BuChE post-treatment for acute OP exposure. In this study, we developed a three-dimensional (3D) brain-on-chip platform with human induced pluripotent stem cell (iPSC)-derived neurons and astrocytes to simulate human brain behavior. The platform consists of two compartments: 1) a hydrogel embedded with human iPSC-derived GABAergic neurons and astrocytes and 2) a perfusion channel with dynamic medium flow. The brain tissue constructs were exposed to Malathion (MT) at various concentrations and then treated with BuChE after 20 minutes of MT exposure. Results show that the iPSC-derived neurons and astrocytes directly interacted and formed synapses in the 3D matrix, and that treatment with BuChE improved viability after MT exposure up to a concentration of 10-3 M. We conclude that the 3D brain-on-chip platform with human iPSC-derived brain cells is a suitable model to study the neurotoxicity of OP exposure and evaluate therapeutic compounds for treatment.
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Astrocitos/efectos de los fármacos , Encéfalo/efectos de los fármacos , Butirilcolinesterasa/farmacología , Inhibidores de la Colinesterasa/toxicidad , Neuronas GABAérgicas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/citología , Malatión/antagonistas & inhibidores , Astrocitos/citología , Astrocitos/metabolismo , Encéfalo/citología , Células Cultivadas , Neuronas GABAérgicas/citología , Neuronas GABAérgicas/metabolismo , Humanos , Malatión/toxicidadRESUMEN
We investigated a potential use of a 3D tetraculture brain microphysiological system (BMPS) for neurotoxic chemical agent screening. This platform consists of neuronal tissue with extracellular matrix (ECM)-embedded neuroblastoma cells, microglia, and astrocytes, and vascular tissue with dynamic flow and membrane-free culture of the endothelial layer. We tested the broader applicability of this model, focusing on organophosphates (OPs) Malathion (MT), Parathion (PT), and Chlorpyrifos (CPF), and chemicals that interact with GABA and/or opioid receptor systems, including Muscimol (MUS), Dextromethorphan (DXM), and Ethanol (EtOH). We validated the BMPS platform by measuring the neurotoxic effects on barrier integrity, acetylcholinesterase (AChE) inhibition, viability, and residual OP concentration. The results show that OPs penetrated the model blood brain barrier (BBB) and inhibited AChE activity. DXM, MUS, and EtOH also penetrated the BBB and induced moderate toxicity. The results correlate well with available in vivo data. In addition, simulation results from an in silico physiologically-based pharmacokinetic/pharmacodynamic (PBPK/PD) model that we generated show good agreement with in vivo and in vitro data. In conclusion, this paper demonstrates the potential utility of a membrane-free tetraculture BMPS that can recapitulate brain complexity as a cost-effective alternative to animal models.
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Encéfalo/fisiología , Imagenología Tridimensional , Neurotoxinas/toxicidad , Organofosfatos/toxicidad , Pruebas de Toxicidad , Acetilcolinesterasa/metabolismo , Animales , Encéfalo/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Ratones , Microfluídica , Neurotoxinas/farmacocinética , Organofosfatos/farmacocinética , Receptores de GABA/metabolismo , Receptores Opioides/metabolismo , Factores de TiempoRESUMEN
Synthetic cathinones are used for their stimulant-like properties. Stimulant-induced neurochemical changes are thought to occur at different times in different brain regions and neurotransmitter systems. This study sought to examine the behavioral and neurochemical effects of α-pyrrolidinopentiophenone (α-PVP) and mephedrone (4MMC) in female rats. Methods probed the chronology of effects of synthetic cathinone exposure. Female rats were trained to self-administer α-PVP, 4MMC, or saline. Drug exposure ceased after 7 days of autoshaping for half of each drug group; the other half self-administered for another 21 days. Amygdala, hippocampus, hypothalamus, PFC, striatum, and thalamus were extracted, and tissue was analyzed with electrochemical detection and liquid chromatography mass spectrometry. Responding was minimal during autoshaping; thus, most infusions were delivered noncontingently in the autoshaping phase. Rats acquired self-administration of α-PVP and 4MMC. Synthetic cathinone administration, and duration of exposure produced several effects on neurotransmitters. α-PVP primarily increased serotonin, 5-hydroxy-3-acetic acid (5-HIAA), norepinephrine, and glutamate in hypothalamus. In contrast, 4MMC decreased serotonin and 5-HIAA in several brain regions. Longer durations of exposure to both synthetic cathinones increased 5-HIAA, norepinephrine, and glutamate in multiple brain regions compared to the short exposure during autoshaping. Notably, both α-PVP and 4MMC produced minimal changes in dopamine levels, suggesting that the dopaminergic effects of these synthetic cathinones are transient. These alterations in neurotransmitter levels indicate that synthetic cathinone use may produce differential neurochemical changes during the transition from use to abuse.
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Alcaloides/farmacología , Conducta Adictiva/tratamiento farmacológico , Neurotransmisores/metabolismo , Alcaloides/metabolismo , Animales , Conducta Adictiva/metabolismo , Encéfalo/efectos de los fármacos , Estimulantes del Sistema Nervioso Central/farmacología , Dopamina/metabolismo , Femenino , Hipotálamo/efectos de los fármacos , Metanfetamina/análogos & derivados , Metanfetamina/farmacología , Neurotransmisores/farmacología , Pentanonas/farmacología , Pirrolidinas/farmacología , Ratas , Ratas Sprague-Dawley , Refuerzo en Psicología , Autoadministración , Serotonina/metabolismoRESUMEN
In Figure 3, in the amygdala panel (upper left panel), the HVA and NE levels are switched.
RESUMEN
BACKGROUND: The recent NIH mandate to consider sex as a biological variable in preclinical research has focused attention on delineation of sex differences in behavior. To investigate mechanisms underlying sex differences in Δ9-tetrahydrocannabinol (THC) effects, we examined the effects of sex and gonadal hormones on CB1 receptors in cerebellum, hippocampus, prefrontal cortex, and striatum. METHODS: Adult Sprague-Dawley rats underwent gonadectomy (GDX) or sham-GDX. Half of the GDX females and males received estradiol or testosterone replacement (GDX+H), respectively. All rats were injected with vehicle or 30 mg/kg THC twice daily for 1 week before brain collection. CP55,940-stimulated [35S]GTPγS and [3H]SR141716A saturation binding assays were performed. RESULTS: With exception of enhanced receptor activation in the hippocampi of female rats compared to males, vehicle-treated rats exhibited minimal sex differences in CB1 receptor densities or G-protein coupling. Repeated treatment with THC resulted in pronounced CB1 receptor desensitization and downregulation in both sexes in all brain regions with a greater magnitude of change in females. CONCLUSIONS: These results suggest that sex differences in the density and G-protein coupling of brain CB1 receptors may play a limited role in sex differences in acute THC effects not mediated by the hippocampus. In contrast, sex differences after repeated THC were common, with females (intact, GDX, and GDX+H) showing greater downregulation or desensitization in all four brain regions compared to the respective male groups. This result is consistent with a finding that women tend to progress to tolerance and dependence quicker than men after initiation of cannabis use.
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Encéfalo/metabolismo , Dronabinol/metabolismo , Hormonas Esteroides Gonadales/metabolismo , Receptor Cannabinoide CB1/metabolismo , Caracteres Sexuales , Animales , Encéfalo/efectos de los fármacos , Agonistas de Receptores de Cannabinoides/metabolismo , Agonistas de Receptores de Cannabinoides/farmacología , Antagonistas de Receptores de Cannabinoides/metabolismo , Antagonistas de Receptores de Cannabinoides/farmacología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Dronabinol/farmacología , Tolerancia a Medicamentos/fisiología , Estradiol/metabolismo , Femenino , Masculino , Ratas , Ratas Sprague-Dawley , Rimonabant/metabolismo , Rimonabant/farmacología , Testosterona/metabolismoRESUMEN
RATIONALE: Synthetic cathinones are used as stimulants of abuse. Different stimulants may induce distinct rates of disease progression, yielding neurochemical changes that may vary across brain regions or neurotransmitter systems. OBJECTIVES: This research sought to behaviorally and chemically differentiate stages of synthetic cathinone abuse through rodent self-administration and measurement of the neurotransmitter profile in multiple brain regions. METHODS: Male rats were trained to self-administer α-PVP, mephedrone (4MMC), or saline. Half of each drug group stopped self-administering after autoshaping; the other half self-administered for another 21 days. Brain tissue from amygdala, hippocampus, hypothalamus, PFC, striatum, and thalamus was profiled with electrochemical detection to assess neurotransmitter levels. RESULTS: During autoshaping, the majority of infusions were delivered noncontingently. In the self-administration phase, rats responded more for α-PVP and 4MMC than for saline, demonstrating that both synthetic cathinones were reinforcing. Longer durations of exposure elevated 5-HIAA in hypothalamus, PFC, and hippocampus, indicating that learning may produce changes in addiction-related brain regions. Both synthetic cathinones decreased norepinephrine in hippocampus, while α-PVP decreased glutamate in hippocampus and PFC, and 4MMC decreased glutamate in thalamus. Furthermore, α-PVP increased dopaminergic metabolites in striatum, whereas 4MMC decreased serotonin in the amygdala, hippocampus, and PFC. Interestingly, neither synthetic cathinone affected dopamine levels despite their functional effects on the dopaminergic system. CONCLUSIONS: In summary, the neurotransmitter changes observed here suggest that synthetic cathinone use likely produces sequential neurochemical changes during the transition from use to abuse. Consequently, treatment need may differ depending on the progression of synthetic cathinone abuse.
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Alcaloides/administración & dosificación , Conducta Adictiva/metabolismo , Encéfalo/metabolismo , Estimulantes del Sistema Nervioso Central/administración & dosificación , Neurotransmisores/metabolismo , Drogas Sintéticas/administración & dosificación , Alcaloides/química , Animales , Conducta Adictiva/psicología , Encéfalo/efectos de los fármacos , Masculino , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción , Refuerzo en Psicología , AutoadministraciónRESUMEN
G protein-gated inward rectifier K+ (GIRK) channels are members of the super-family of proteins known as inward rectifier K+ (Kir) channels and are expressed throughout the peripheral and central nervous systems. Neuronal GIRK channels are the downstream targets of a number of neuromodulators including opioids, somatostatin, dopamine and cannabinoids. Previous studies have demonstrated that the ATP-sensitive K+ channel, another member of the Kir channel family, is regulated by sulfonamide drugs. Therefore, to determine if sulfonamides also modulate GIRK channels, we screened a library of arylsulfonamide compounds using a GIRK channel fluorescent assay that utilized pituitary AtT20 cells expressing GIRK channels along with the somatostatin type-2 and -5 receptors. Enhancement of the GIRK channel fluorescent signal by one compound, N-(2-methoxyphenyl) benzenesulfonamide (MPBS), was dependent on the activation of the channel by somatostatin. In whole-cell patch clamp experiments, application of MPBS both shifted the somatostatin concentration-response curve (EC50 = 3.5nM [control] vs.1.0nM [MPBS]) for GIRK channel activation and increased the maximum GIRK current measured with 100nM somatostatin. However, GIRK channel activation was not observed when MPBS was applied to the cells in the absence of somatostatin. While the MPBS structural analog 4-fluoro-N-(2-methoxyphenyl) benzenesulfonamide also augmented the somatostatin-induced GIRK fluorescent signal, no increase in the signal was observed with the sulfonamides tolbutamide, sulfapyridine and celecoxib. In conclusion, MPBS represents a novel prototypic GPCR-dependent regulator of neuronal GIRK channels.
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Proteínas de Unión al GTP/metabolismo , Hidroxilaminas/farmacología , Activación del Canal Iónico/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Sulfonamidas/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , BencenosulfonamidasRESUMEN
The neuropeptide S system has been implicated in a number of centrally mediated behaviors including memory consolidation, anxiolysis, and increased locomotor activity. Characterization of these behaviors has been primarily accomplished using the endogenous 20AA peptide (NPS) that demonstrates relatively equal potency for the calcium mobilization and cAMP second messenger pathways at human and rodent NPS receptors. This study is the first to demonstrate that truncations of the NPS peptide provides small fragments that retain significant potency only at one of two single polymorphism variants known to alter NPSR function (NPSR-107I), yet demonstrate a strong level of bias for the calcium mobilization pathway over the cAMP pathway. We have also determined that the length of the truncated peptide correlates with the degree of bias for the calcium mobilization pathway. A modified tetrapeptide analog (4) has greatly attenuated hyperlocomotor stimulation in vivo but retains activity in assays that correlate with memory consolidation and anxiolytic activity. Analog 4 also has a bias for the calcium mobilization pathway, at the human and mouse receptor. This suggests that future agonist ligands for the NPS receptor having a bias for calcium mobilization over cAMP production will function as non-stimulatory anxiolytics that augment memory formation.
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Ansiolíticos/farmacología , Locomoción/efectos de los fármacos , Memoria/efectos de los fármacos , Neuropéptidos/farmacología , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Adaptación Ocular/efectos de los fármacos , Análisis de Varianza , Animales , Reacción de Prevención/efectos de los fármacos , Calcio/metabolismo , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Neuropéptidos/química , Receptores Acoplados a Proteínas G/genética , Transfección , alfa-Fetoproteínas/metabolismoRESUMEN
BACKGROUND: GPR88 is an orphan G protein-coupled receptor highly expressed in the striatum and is implicated in basal ganglia-associated disorders. However, the receptor functions of GPR88 are still largely unknown due to the lack of potent and selective ligands appropriate for central nervous system investigation. Development of a high-throughput screening assay for GPR88 should facilitate the discovery of novel ligands to probe GPR88 functions. METHODS: In this paper, we describe the development of a CHO-Gαqi5-GPR88 cell-based calcium mobilization assay. The assay takes advantage of functional coupling of GPR88 with the promiscuous Gαqi5 protein and consequent mobilization of intracellular calcium, which can be measured in a 384-well format with a Fluorescent Imaging Plate Reader. RESULTS: The CHO-Gαqi5-GPR88 cell-based calcium mobilization assay was validated by the structure-activity relationship study of known GPR88 agonist (1R,2R)-2-PCCA analogues. The assay was automated and miniaturized to a 384-well format, and was deemed robust and reproducible with a Z'-factor of 0.72 and tolerated dimethyl sulfoxide to a final concentration of 2%. Screening a pilot neurotransmitter library consisting of 228 compounds yielded 10 hits, but none of the hits were confirmed as GPR88 agonists in follow-up assays. CONCLUSIONS: We have developed a high-throughput calcium mobilization assay for the orphan receptor GPR88. This calcium mobilization assay can be used to identify several different types of GPR88 ligands including agonists, competitive and noncompetitive antagonists, inverse agonists, and allosteric modulators. These ligands will serve as valuable tools to probe signaling mechanisms and in vivo functions of GPR88, and could expedite development of novel therapies for diseases potentially mediated by GPR88.
Asunto(s)
Calcio/metabolismo , Cromanos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Receptores Acoplados a Proteínas G/agonistas , p-Cloroanfetamina/análogos & derivados , Animales , Células CHO , Cricetulus , Relación Estructura-Actividad , p-Cloroanfetamina/farmacologíaRESUMEN
Microelectrode arrays (MEAs) recording extracellular field potentials of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) provide a rich data set for functional assessment of drug response. The aim of this work is the development of a method for a systematic analysis of arrhythmia using MEAs, with emphasis on the development of six parameters accounting for different types of cardiomyocyte signal irregularities. We describe a software approach to carry out such analysis automatically including generation of a heat map that enables quick visualization of arrhythmic liability of compounds. We also implemented signal processing techniques for reliable extraction of the repolarization peak for field potential duration (FPD) measurement even from recordings with low signal to noise ratios. We measured hiPS-CM's on a 48 well MEA system with 5minute recordings at multiple time points (0.5, 1, 2 and 4h) after drug exposure. We evaluated concentration responses for seven compounds with a combination of hERG, QT and clinical proarrhythmia properties: Verapamil, Ranolazine, Flecainide, Amiodarone, Ouabain, Cisapride, and Terfenadine. The predictive utility of MEA parameters as surrogates of these clinical effects were examined. The beat rate and FPD results exhibited good correlations with previous MEA studies in stem cell derived cardiomyocytes and clinical data. The six-parameter arrhythmia assessment exhibited excellent predictive agreement with the known arrhythmogenic potential of the tested compounds, and holds promise as a new method to predict arrhythmic liability.
Asunto(s)
Arritmias Cardíacas/inducido químicamente , Ensayos Analíticos de Alto Rendimiento/instrumentación , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Microelectrodos , Miocitos Cardíacos/efectos de los fármacos , Pruebas de Toxicidad/instrumentación , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatología , Automatización de Laboratorios , Diferenciación Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Diseño de Equipo , Frecuencia Cardíaca/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Medición de Riesgo , Procesamiento de Señales Asistido por Computador , Relación Señal-Ruido , Programas Informáticos , Factores de Tiempo , Pruebas de Toxicidad/métodosRESUMEN
The recent discovery of allosteric modulators of the CB1 receptor including PSNCBAM-1 (4) has generated significant interest in CB1 receptor allosteric modulation. Here in the first SAR study on 4, we have designed and synthesized a series of analogs focusing on modifications at two positions. Pharmacological evaluation in calcium mobilization and binding assays revealed the importance of alkyl substitution at the 2-aminopyridine moiety and electron deficient aromatic groups at the 4-chlorophenyl position for activity at the CB1 receptor, resulting in several analogs with comparable potency to 4. These compounds increased the specific binding of [(3)H]CP55,940, in agreement with previous reports. Importantly, 4 and two analogs dose-dependently reduced the Emax of the agonist curve in the CB1 calcium mobilization assays, confirming their negative allosteric modulator characteristics. Given the side effects associated with CB1 receptor orthosteric antagonists, negative allosteric modulators provide an alternative approach to modulate the pharmacologically important CB1 receptor.