RESUMEN
Narcolepsy type 1 (NT1) is a sleep disorder caused by a loss of orexinergic neurons. Narcolepsy type 2 (NT2) is heterogeneous; affected individuals typically have normal orexin levels. Following evaluation in mice, the effects of the orexin 2 receptor (OX2R)-selective agonist danavorexton were evaluated in single- and multiple-rising-dose studies in healthy adults, and in individuals with NT1 and NT2. In orexin/ataxin-3 narcolepsy mice, danavorexton reduced sleep/wakefulness fragmentation and cataplexy-like episodes during the active phase. In humans, danavorexton administered intravenously was well tolerated and was associated with marked improvements in sleep latency in both NT1 and NT2. In individuals with NT1, danavorexton dose-dependently increased sleep latency in the Maintenance of Wakefulness Test, up to the ceiling effect of 40 min, in both the single- and multiple-rising-dose studies. These findings indicate that OX2Rs remain functional despite long-term orexin loss in NT1. OX2R-selective agonists are a promising treatment for both NT1 and NT2.
Asunto(s)
Estimulantes del Sistema Nervioso Central , Narcolepsia , Receptores de Orexina , Adulto , Animales , Ataxina-3/genética , Ataxina-3/metabolismo , Cataplejía/tratamiento farmacológico , Cataplejía/genética , Estimulantes del Sistema Nervioso Central/farmacología , Estimulantes del Sistema Nervioso Central/uso terapéutico , Modelos Animales de Enfermedad , Humanos , Ratones , Narcolepsia/tratamiento farmacológico , Narcolepsia/genética , Neuronas/metabolismo , Receptores de Orexina/agonistas , Receptores de Orexina/genética , Receptores de Orexina/metabolismo , Receptores de Orexina/uso terapéutico , Orexinas/genética , Orexinas/metabolismo , Fenotipo , Vigilia/efectos de los fármacos , Vigilia/genéticaRESUMEN
The orexin 2 receptor-selective agonist danavorexton (TAK-925) has been shown to produce wake-promoting effects in wild-type mice, narcolepsy-model mice, and individuals with narcolepsy type 1 and type 2. Here, we report wake-promoting effects of danavorexton in non-human primates and healthy men during their sleep phase. Electroencephalogram analyses revealed that subcutaneous administration of danavorexton significantly increased wakefulness in common marmosets (p < 0.05 at 0.1 mgâ kg-1 , and p < 0.001 at 1 mgâ kg-1 and 10 mg kg-1 ) and cynomolgus monkeys (p ≤ 0.05 at 1 mgâ kg-1 and 3 mgâ kg-1 ). In a phase 1b crossover, randomized, double-blind, placebo-controlled and active-controlled study in sleep-deprived healthy participants (ClinicalTrials.gov identifier: NCT03522506), modafinil 300 mg (used to demonstrate assay sensitivity) and continuous infusion of danavorexton 44 mg and danavorexton 112 mg showed statistically superior wake-promoting effects to placebo (n = 18). Measured using the Maintenance of Wakefulness Test, mean (standard deviation) sleep latencies during infusion of danavorexton 44 mg, danavorexton 112 mg and placebo were 21.4 (8.9), 31.8 (3.2) and 9.2 (6.4) min, respectively. Least-squares mean difference from placebo in average sleep latency was 16.8 min with danavorexton 44 mg and 30.2 min with danavorexton 112 mg (both p < 0.001). Karolinska Sleepiness Scale scores were statistically significantly lower (indicating decreased sleepiness) for participants receiving danavorexton than for those receiving placebo during infusion (danavorexton 44 mg, p = 0.010; danavorexton 112 mg, p < 0.001). Together, these results indicate that an orexin 2 receptor agonist increases wakefulness in non-human primates and healthy individuals during their sleep phase.
Asunto(s)
Narcolepsia , Orexinas , Vigilia , Animales , Método Doble Ciego , Narcolepsia/tratamiento farmacológico , Orexinas/farmacología , Primates , Somnolencia , Resultado del Tratamiento , Vigilia/efectos de los fármacos , Humanos , MasculinoRESUMEN
Major mental illnesses such as schizophrenia (SZ) and bipolar disorder (BP) frequently accompany metabolic conditions, but their relationship is still unclear, in particular at the mechanistic level. We implemented an approach of "from population to neuron", combining population-based epidemiological analysis with neurobiological experiments using cell and animal models based on a hypothesis built from the epidemiological study. We characterized high-quality population data, olfactory neuronal cells biopsied from patients with SZ or BP, and healthy subjects, as well as mice genetically modified for insulin signaling. We accessed the Danish Registry and observed (1) a higher incidence of diabetes in people with SZ or BP and (2) higher incidence of major mental illnesses in people with diabetes in the same large cohort. These epidemiological data suggest the existence of common pathophysiological mediators in both diabetes and major mental illnesses. We hypothesized that molecules associated with insulin resistance might be such common mediators, and then validated the hypothesis by using two independent sets of olfactory neuronal cells biopsied from patients and healthy controls. In the first set, we confirmed an enrichment of insulin signaling-associated molecules among the genes that were significantly different between SZ patients and controls in unbiased expression profiling data. In the second set, olfactory neuronal cells from SZ and BP patients who were not pre-diabetic or diabetic showed reduced IRS2 tyrosine phosphorylation upon insulin stimulation, indicative of insulin resistance. These cells also displayed an upregulation of IRS1 protein phosphorylation at serine-312 at baseline (without insulin stimulation), further supporting the concept of insulin resistance in olfactory neuronal cells from SZ patients. Finally, Irs2 knockout mice showed an aberrant response to amphetamine, which is also observed in some patients with major mental illnesses. The bi-directional relationships between major mental illnesses and diabetes suggest that there may be common pathophysiological mediators associated with insulin resistance underlying these mental and physical conditions.
Asunto(s)
Trastorno Bipolar , Resistencia a la Insulina , Esquizofrenia , Animales , Trastorno Bipolar/genética , Humanos , Insulina , Ratones , Neuronas , Esquizofrenia/genéticaRESUMEN
Autophagy plays an essential role in intracellular degradation and maintenance of cellular homeostasis in all cells, including neurons. Although a recent study reported a copy number variation of Ulk2, a gene essential for initiating autophagy, associated with a case of schizophrenia (SZ), it remains to be studied whether Ulk2 dysfunction could underlie the pathophysiology of the disease. Here we show that Ulk2 heterozygous (Ulk2+/-) mice have upregulated expression of sequestosome-1/p62, an autophagy-associated stress response protein, predominantly in pyramidal neurons of the prefrontal cortex (PFC), and exhibit behavioral deficits associated with the PFC functions, including attenuated sensorimotor gating and impaired cognition. Ulk2+/- neurons showed imbalanced excitatory-inhibitory neurotransmission, due in part to selective down-modulation of gamma-aminobutyric acid (GABA)A receptor surface expression in pyramidal neurons. Genetically reducing p62 gene dosage or suppressing p62 protein levels with an autophagy-inducing agent restored the GABAA receptor surface expression and rescued the behavioral deficits in Ulk2+/- mice. Moreover, expressing a short peptide that specifically interferes with the interaction of p62 and GABAA receptor-associated protein, a protein that regulates endocytic trafficking of GABAA receptors, also restored the GABAA receptor surface expression and rescued the behavioral deficits in Ulk2+/- mice. Thus, the current study reveals a novel mechanism linking deregulated autophagy to functional disturbances of the nervous system relevant to SZ, through regulation of GABAA receptor surface presentation in pyramidal neurons.
Asunto(s)
Autofagia/genética , Proteínas Serina-Treonina Quinasas/genética , Esquizofrenia/genética , Proteína Sequestosoma-1/genética , Animales , Variaciones en el Número de Copia de ADN/genética , Regulación de la Expresión Génica/genética , Humanos , Ratones , Péptidos/genética , Corteza Prefrontal/metabolismo , Corteza Prefrontal/patología , Transporte de Proteínas/genética , Células Piramidales/metabolismo , Células Piramidales/patología , Receptores de GABA-A/genética , Esquizofrenia/fisiopatología , Transmisión Sináptica/genéticaRESUMEN
Chromosome 15q11.2-13.1 duplication (Dup15q) syndrome is one of the most common autism spectrum disorders (ASDs) associated with copy number variants (CNVs). For the analysis of CNV-relevant pathological cellular phenotypes, a CNV-corrected isogenic cell line is useful for excluding the influence of genetic background. Here, we devised a strategy to remove the isodicentric chromosome 15 by inserting a puro-ΔTK selection cassette into the extra chromosome using the CRISPR-Cas9 system, followed by a subsequent two-step drug selection. A series of assays, including qPCR-based copy number analysis and karyotype analysis, confirmed the elimination of the extra chromosome. Furthermore, cerebral organoids were generated from the parental Dup15q iPSCs and their isogenic iPSCs. scRNA-seq analysis revealed the alteration of expression levels in ion-channel-related genes and synapse-related genes in glutamatergic and GABAergic neurons in Dup15q organoids, respectively. The established isogenic cell line is a valuable resource for unraveling cellular and molecular alterations associated with Dup15q syndrome.
Asunto(s)
Cromosomas Humanos Par 15 , Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Cromosomas Humanos Par 15/genética , Cromosomas Humanos Par 15/metabolismo , Duplicación Cromosómica , Variaciones en el Número de Copia de ADN , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Organoides/metabolismo , Aberraciones Cromosómicas , Discapacidad IntelectualRESUMEN
NGLY1 deficiency is a genetic disease caused by biallelic mutations of the Ngly1 gene. Although epileptic seizure is one of the most severe symptoms in patients with NGLY1 deficiency, preclinical studies have not been conducted due to the lack of animal models for epileptic seizures in NGLY1 deficiency. Here, we observed the behaviors of male and female Ngly1-/- mice by video monitoring and found that these mice exhibit spontaneous seizure-like behaviors. Gene expression analyses and enzyme immunoassay revealed significant decreases in oxytocin, a well-known neuropeptide, in the hypothalamus of Ngly1-/- mice. Seizure-like behaviors in Ngly1-/- mice were transiently suppressed by a single intranasal administration of oxytocin. These findings suggest the therapeutic potential of oxytocin for epileptic seizure in patients with NGLY1 deficiency and contribute to the clarification of the disease mechanism.
Asunto(s)
Trastornos Congénitos de Glicosilación , Oxitocina , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Convulsiones , Animales , Femenino , Masculino , Ratones , Administración Intranasal , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Hipotálamo/metabolismo , Hipotálamo/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Noqueados , Oxitocina/administración & dosificación , Oxitocina/farmacología , Convulsiones/tratamiento farmacológico , Convulsiones/etiología , Trastornos Congénitos de Glicosilación/complicaciones , Trastornos Congénitos de Glicosilación/tratamiento farmacológico , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/deficienciaRESUMEN
Schizophrenia (SZ) and bipolar disorder (BP) are highly heritable major psychiatric disorders that share a substantial portion of genetic risk as well as their clinical manifestations. This raises a fundamental question of whether, and how, common neurobiological pathways translate their shared polygenic risks into shared clinical manifestations. This study shows the miR-124-3p-AMPAR pathway as a key common neurobiological mediator that connects polygenic risks with behavioral changes shared between these two psychotic disorders. We discovered the upregulation of miR-124-3p in neuronal cells and the postmortem prefrontal cortex from both SZ and BP patients. Intriguingly, the upregulation is associated with the polygenic risks shared between these two disorders. Seeking mechanistic dissection, we generated a mouse model that upregulates miR-124-3p in the medial prefrontal cortex. We demonstrated that the upregulation of miR-124-3p increases GRIA2-lacking calcium-permeable AMPARs and perturbs AMPAR-mediated excitatory synaptic transmission, leading to deficits in the behavioral dimensions shared between SZ and BP.
Asunto(s)
Trastorno Bipolar , MicroARNs , Esquizofrenia , Ratones , Animales , Esquizofrenia/genética , Esquizofrenia/metabolismo , Trastorno Bipolar/genética , Trastorno Bipolar/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Herencia Multifactorial , Corteza Prefrontal/metabolismoRESUMEN
TAK-925, a potent, selective, and brain-penetrant orexin 2 receptor (OX2R) agonist, [methyl (2R,3S)-3-((methylsulfonyl)amino)-2-(((cis-4-phenylcyclohexyl)oxy)methyl)piperidine-1-carboxylate, 16], was identified through the optimization of compound 2, which was discovered by a high throughput screening (HTS) campaign. Subcutaneous administration of compound 16 produced wake-promoting effects in mice during the sleep phase. Compound 16 (TAK-925) is being developed for the treatment of narcolepsy and other related disorders.
RESUMEN
Orexin-producing neurons in the lateral hypothalamus are a critical regulator of sleep/wake states, and their loss is associated with narcolepsy type 1 (NT1). Orexin peptides act on two G protein-coupled receptors: orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R). OX2R knockout (KO) mice, but not OX1R KO mice, showed clear narcolepsy-like phenotypes, including fragmented sleep-wake cycles. Moreover, OX2R-selective antagonists have been shown to induce sleepiness in mice, and activation of OX2R has been reported to increase wakefulness. In this study, we characterized in vitro and in vivo profiles of a novel, highly selective OX2R agonist, TAK-925 [methyl (2R,3S)-3-[(methylsulfonyl)amino]-2-{[(cis-4-phenylcyclohexyl)oxy]methyl}piperidine-1-carboxylate]. TAK-925 activated human recombinant OX2R with 50% effective concentration value of 5.5â¯nM, and showed >5,000-fold selectivity over OX1R in calcium mobilization assays. TAK-925 induced OX2R-downstream signals similar to those displayed by orexin peptides in Chinese hamster ovary cells stably expressing human OX2R. In an electrophysiological study, TAK-925 activated physiological OX2R on histaminergic neurons in the mouse tuberomammillary nucleus (TMN). Subcutaneous (SC) administration of TAK-925 also modulated neuronal activity in various brain regions, including TMN, as measured by an immunohistochemical analysis using an anti-c-fos antibody. TAK-925 (SC) increased wakefulness in wild-type mice, but not in OX2R KO mice, during their sleep phase, demonstrating that a highly selective OX2R agonist can increase wakefulness in mice via OX2R activation. TAK-925 may have therapeutic potential to reduce hypersomnia in multiple disorders including NT1.
Asunto(s)
Receptores de Orexina/agonistas , Sueño/efectos de los fármacos , Vigilia/efectos de los fármacos , Animales , Células CHO , Cricetulus , Humanos , Área Hipotalámica Lateral/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Narcolepsia/tratamiento farmacológico , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Receptores de Orexina/genética , Receptores de Orexina/metabolismo , Orexinas/metabolismo , Sueño/genética , Transfección , Vigilia/genéticaRESUMEN
Oxidative stress is involved in pathophysiology and pathological conditions of numerous human diseases. Thus, understanding the mechanisms underlying the redox homeostasis in cells and organs is valuable for discovery of therapeutic drugs for oxidative stress-related diseases. Recently, by applying chemical biology approach with an ARE activator, BTZO-1, we found macrophage migration inhibitory factor (MIF) as a new regulator of antioxidant response element- (ARE-) mediated gene transcription. BTZO-1 and its active derivatives bound to MIF and protected cells and organs from oxidative insults via ARE activation in animal models with oxidative stress such as ischemia/reperfusion injury, inflammatory bowel diseases, and septic shock. In this review, we briefly highlight key findings in understanding the MIF-ARE system.
Asunto(s)
Elementos de Respuesta Antioxidante/fisiología , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Estrés Oxidativo/fisiología , Animales , Regulación de la Expresión Génica/fisiología , HumanosRESUMEN
AMY-1 has been identified by us as a c-Myc-binding protein and was found to stimulate c-Myc transcription activity. AMY-1 was also found to be associated with AKAP84/149 in the mitochondria in somatic cells and sperm, suggesting that it plays a role in spermatogenesis. To access the molecular function of AMY-1, a two-hybrid screening of cDNAs encoding AMY-1-binding proteins was carried out with AMY-1 as a bait using a human testis cDNA library, and a clone encoding a novel protein, AMAP-1, was obtained. The amap-1 gene was mapped at human chromosome 17q21. AMY-1 was found to bind to and be colocalized with AMAP-1 in human 293T and HeLa cells. AMAP-1 was found to be specifically expressed in the testis and expressed post-meiotically in the testis, as was AMY-1. These results suggest that both AMAP-1 and AMY-1 play roles in spermatogenesis.
Asunto(s)
Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Proteínas de Unión al ADN/metabolismo , Espermatogénesis , Testículo/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromosomas Humanos Par 17 , Regulación de la Expresión Génica , Células HeLa , Humanos , Cinética , Masculino , Ratones , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , alfa-Amilasas Salivales , Testículo/crecimiento & desarrollo , Testículo/fisiología , Distribución Tisular , Transcripción GenéticaRESUMEN
We recently reported a unique antioxidant response element (ARE)-activator, BTZO-1, which induced expression of cytoprotective proteins such as heme oxygenase-1 (HO-1) and suppressed oxidative stress-induced cardiomyocyte apoptosis via binding to macrophage migration inhibitory factor (MIF). HO-1 induction and apoptosis inhibition have been reported to improve the outcomes following experimental sepsis by protecting the organs. Therefore, we investigated the potential of BTZO-2, an active BTZO-1 derivative, as a drug for sepsis. BTZO-2 significantly protected mice from the endotoxic shock induced by 5mg/kg lipopolysaccharide (LPS); survival rates increased from 42% to 100%. In contrast, BTZO-2 did not provide significant protection to mice from the shock induced by 10 µg/kg LPS together with d-galactosamine (d-GalN, hepatocyte-specific transcription inhibitor) (LPS/d-GalN). Hepatic HO-1 protein was up-regulated by BTZO-2 in mice injected with 5mg/kg LPS, but not in those injected with 10 µg/kg LPS/d-GalN. Interestingly, BTZO-2 showed little or no effect on LPS-induced up-regulation of plasma cytokine levels in mice. Thus, the organ protection mediated by HO-1 may have a pivotal role in the pharmacological effect of BTZO-2. These results suggest that BTZO-2 is a promising compound for a novel drug for sepsis.
Asunto(s)
Antioxidantes/metabolismo , Piridinas/farmacología , Elementos de Respuesta/efectos de los fármacos , Choque Séptico/prevención & control , Tiazinas/farmacología , Animales , Citocinas/sangre , Hemo-Oxigenasa 1/metabolismo , Lipopolisacáridos/efectos adversos , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos BALB C , Piridinas/sangre , Piridinas/farmacocinética , Choque Séptico/inducido químicamente , Choque Séptico/metabolismo , Tiazinas/sangre , Tiazinas/farmacocinéticaRESUMEN
Inflammatory bowel disease (IBD) is a group of chronic inflammatory disorders that are primarily represented by ulcerative colitis and Crohn's disease. The etiology of IBD is not well understood; however, oxidative stress is considered a potential etiological and/or triggering factor for IBD. We have recently reported the identification of BTZO-1, an activator of antioxidant response element (ARE)-mediated gene expression, which protects cardiomyocytes from oxidative stress-induced insults. Here we describe the potential of BTZO-15, an active BTZO-1 derivative for ARE-activation with a favorable ADME-Tox profile, for the treatment of IBD. BTZO-15 induced expression of heme oxygenase-1 (HO-1), an ARE-regulated cytoprotective protein, and inhibited NO-induced cell death in IEC-18 cells. Large intestine shortening, rectum weight gain, diarrhea, intestinal bleeding, and an increase in rectal myeloperoxidase (MPO) activity were observed in a dextran sulfate sodium (DSS)-induced colitis rat model. Oral administration of BTZO-15 induced HO-1 expression in the rectum and attenuated DSS-induced changes. Furthermore BTZO-15 reduced the ulcerated area and rectal MPO activity in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis rats without affecting rectal TNF-α levels. These results suggest that BTZO-15 is a promising compound for a novel IBD therapeutic drug with ARE activation properties.
Asunto(s)
Antioxidantes/metabolismo , Colitis/tratamiento farmacológico , Piridinas/uso terapéutico , Elementos de Respuesta/genética , Tiazinas/uso terapéutico , Animales , Muerte Celular/efectos de los fármacos , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Sulfato de Dextran , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Oxidorreductasas Intramoleculares/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Óxido Nítrico/farmacología , Unión Proteica/efectos de los fármacos , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Recto/efectos de los fármacos , Recto/metabolismo , Recto/patología , Tiazinas/química , Tiazinas/metabolismo , Tiazinas/farmacología , Transcripción Genética/efectos de los fármacos , Ácido TrinitrobencenosulfónicoRESUMEN
In a screening program to discover therapeutic drugs for heart diseases, we identified BTZO-1, a 1,3-benzothiazin-4-one derivative, which activated antioxidant response element (ARE)-mediated gene expression and suppressed oxidative stress-induced cardiomyocyte apoptosis in vitro. An active BTZO-1 derivative for ARE-activation protected heart tissue during ischemia/reperfusion injury in rats. Macrophage migration inhibitory factor (MIF), which is known to protect cells from oxidative insult, was identified as a specific BTZO-1-binding protein. BTZO-1 binds to MIF with a K(d) of 68.6 nM, and its binding required the intact N-terminal Pro1. MIF, in the presence of BTZO-1, activated the glutathione S-transferase Ya subunit (GST Ya) gene ARE, whereas reduction of cellular MIF protein levels by siRNA suppressed BTZO-1-induced GST Ya expression. These results suggest that BTZO-1 activates the GST Ya gene ARE by interacting with MIF.
Asunto(s)
Antioxidantes/metabolismo , Cardiotónicos/química , Regulación de la Expresión Génica , Factores Inhibidores de la Migración de Macrófagos/química , Piridinas/química , Tiazinas/química , Animales , Cardiotónicos/farmacología , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Factores Inhibidores de la Migración de Macrófagos/farmacología , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Miocitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Unión Proteica , Piridinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Elementos de Respuesta , Tiazinas/farmacologíaRESUMEN
ITZ-1 is a chondroprotective agent that inhibits interleukin-1beta-induced matrix metalloproteinase-13 (MMP-13) production and suppresses nitric oxide-induced chondrocyte death. Here we describe its mechanisms of action. Heat shock protein 90 (Hsp90) was identified as a specific ITZ-1-binding protein. Almost all known Hsp90 inhibitors have been reported to bind to the Hsp90 N-terminal ATP-binding site and to simultaneously induce degradation and activation of its multiple client proteins. However, within the Hsp90 client proteins, ITZ-1 strongly induces heat shock factor-1 (HSF1) activation and causes mild Raf-1 degradation, but scarcely induces degradation of a broad range of Hsp90 client proteins by binding to the Hsp90 C terminus. These results may explain ITZ-1's inhibition of MMP-13 production, its cytoprotective effect, and its lower cytotoxicity. These results suggest that ITZ-1 is a client-selective Hsp90 inhibitor.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Imidazoles/farmacología , Osteoartritis/tratamiento farmacológico , Sustancias Protectoras/farmacología , Tiazinas/farmacología , Factores de Transcripción/metabolismo , Adenosina Trifosfato/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/genética , Factores de Transcripción del Choque Térmico , Humanos , Interleucina-1beta/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-raf/metabolismoRESUMEN
In a screening program aimed at discovering anti-osteoarthritis (OA) drugs, we identified an imidazo[5,1-c][1,4]thiazine derivative, ITZ-1, that suppressed both interleukin-1beta (IL-1beta)-induced proteoglycan and collagen release from bovine nasal cartilage in vitro and suppressed intra-articular infusion of IL-1beta-induced cartilage proteoglycan degradation in rat knee joints. ITZ-1 did not inhibit enzyme activities of various matrix metalloproteinases (MMPs), which have pivotal roles in cartilage degradation, while it selectively inhibited IL-1beta-induced production of MMP-13 in human articular chondrocytes (HAC). IL-1beta-induced MMP production has been shown to be mediated by extracellular signal-regulated protein kinase (ERK), p38 kinase, and c-Jun N-terminal kinase (JNK) of the mitogen-activated protein kinase (MAPK) family signal transduction molecules. An ERK-MAPK pathway inhibitor (U0126), but not a p38 kinase inhibitor (SB203580) or a JNK inhibitor (SP600125), also selectively inhibited IL-1beta-induced MMP-13 production in HAC. Furthermore, ITZ-1 selectively inhibited IL-1beta-induced ERK activation without affecting p38 kinase and JNK activation, which may account for its selective inhibition of MMP-13 production. Inhibition of nitric oxide (NO)-induced chondrocyte apoptosis has been another area of interest as a therapeutic strategy for OA, and ITZ-1 also suppressed NO-induced death in HAC. These results suggest that ITZ-1 is a promising lead compound for a disease modifying anti-OA drug program.
Asunto(s)
Condrocitos/efectos de los fármacos , Imidazoles/farmacología , Interleucina-1beta/administración & dosificación , Metaloproteinasa 13 de la Matriz/efectos de los fármacos , Tiazinas/farmacología , Animales , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Bovinos , Muerte Celular/efectos de los fármacos , Condrocitos/metabolismo , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Humanos , Articulación de la Rodilla/efectos de los fármacos , Articulación de la Rodilla/metabolismo , Masculino , Metaloproteinasa 13 de la Matriz/metabolismo , Cartílagos Nasales/efectos de los fármacos , Cartílagos Nasales/metabolismo , Óxido Nítrico/administración & dosificación , Osteoartritis/tratamiento farmacológico , Osteoartritis/fisiopatología , Proteoglicanos/efectos de los fármacos , Proteoglicanos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
AMY-1 has been identified by us as a c-Myc-binding protein and was found to stimulate c-Myc transcription activity. AMY-1 was also found to be associated with protein kinase A anchor protein 84/149 (S-AKAP84/AKAP149) in the mitochondria in somatic cells and sperm, suggesting that it plays a role in spermatogenesis. To determine the molecular function of AMY-1, a two-hybrid screening of cDNAs encoding AMY-1-binding proteins was carried out with AMY-1 as a bait using a human testis cDNA library, and a clone encoding a novel protein, AAT-1, was obtained. Three isoforms of AAT-1, AAT-1alpha, -beta, and -gamma, were found to be derived from an alternative splicing of the transcripts of the aat-1 gene, which was mapped at human chromosome 3q13-3q21. AAT-1 was found to be specifically expressed in the testis during the course of spermatogenesis and also to be present in the spermatid and mature sperm, as was AMY-1. AAT-1alpha was found to bind to and be colocalized in mitochondria with AMY-1 in human HeLa and mouse GC-1 cells. Furthermore, AAT-1alpha was found to bind to the N-terminal half of S-AKAP84/AKAP149 in a quaternary complex with AMY-1 and a regulatory subunit (RII) of cAMP-dependent kinase (PKA), in which AAT-1alpha was associated with RII via S-AKAP84/AKAP149, in rat testis and HeLa cells. It was then found that AAT-1alpha weakly stimulated a phosphorylation activity of PKA and also that AAT-1 itself was phosphorylated by PKA in vivo and in vitro. These results suggest that both AAT-1 and AMY-1 play roles in spermatogenesis.