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1.
J Neurochem ; 168(9): 2722-2735, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38783749

RESUMEN

The dorsal striatum is composed of the caudate nucleus and the putamen in human and non-human primates. These two regions receive different cortical projections and are functionally distinct. The caudate is involved in the control of goal-directed behaviors, while the putamen is implicated in habit learning and formation. Previous reports indicate that ethanol differentially influences neurotransmission in these two regions. Because neurotransmitters primarily signal through G protein-coupled receptors (GPCRs) to modulate neuronal activity, the present study aimed to determine whether ethanol had a region-dependent impact on the expression of proteins that are involved in the trafficking and function of GPCRs, including G protein subunits and their effectors, protein kinases, and elements of the cytoskeleton. Western blotting was performed to examine protein levels in the caudate and the putamen of male cynomolgus macaques that self-administered ethanol for 1 year under free access conditions, along with control animals that self-administered an isocaloric sweetened solution under identical operant conditions. Among the 18 proteins studied, we found that the levels of one protein (PKCß) were increased, and 13 proteins (Gαi1/3, Gαi2, Gαo, Gß1γ, PKCα, PKCε, CaMKII, GSK3ß, ß-actin, cofilin, α-tubulin, and tubulin polymerization promoting protein) were reduced in the caudate of alcohol-drinking macaques. However, ethanol did not alter the expression of any proteins examined in the putamen. These observations underscore the unique vulnerability of the caudate nucleus to changes in protein expression induced by chronic ethanol exposure. Whether these alterations are associated with ethanol-induced dysregulation of GPCR function and neurotransmission warrants future investigation.


Asunto(s)
Núcleo Caudado , Etanol , Macaca fascicularis , Putamen , Receptores Acoplados a Proteínas G , Animales , Masculino , Putamen/metabolismo , Putamen/efectos de los fármacos , Núcleo Caudado/metabolismo , Núcleo Caudado/efectos de los fármacos , Etanol/farmacología , Etanol/administración & dosificación , Receptores Acoplados a Proteínas G/metabolismo , Consumo de Bebidas Alcohólicas/metabolismo , Depresores del Sistema Nervioso Central/farmacología , Depresores del Sistema Nervioso Central/administración & dosificación , Autoadministración
2.
Bioorg Med Chem Lett ; 112: 129942, 2024 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-39218405

RESUMEN

COVID-19 has caused severe consequences in terms of public health and economy worldwide since its outbreak in December 2019. SARS-CoV-2 3C-like protease (3CLpro), crucial for the viral replications, is an attractive target for the development of antiviral drugs. In this study, several kinds of Michael acceptor warheads were utilized to hunt for potent covalent inhibitors against 3CLpro. Meanwhile, novel 3CLpro inhibitors with the P3-3,5-dichloro-4-(2-(dimethylamino)ethoxy)phenyl moiety were designed and synthesized which may form salt bridge with residue Glu166. Among them, two compounds 12b and 12c exhibited high inhibitory activities against SARS-CoV-2 3CLpro. Further investigations suggested that 12b with an acrylate warhead displayed potent activity against HCoV-OC43 (EC50 = 97 nM) and SARS-CoV-2 replicon (EC50 = 45 nM) and low cytotoxicity (CC50 > 10 µM) in Huh7 cells. Taken together, this study devised two series of 3CLpro inhibitors and provided the potent SARS-CoV-2 3CLpro inhibitor (12b) which may be used for treating coronavirus infections.


Asunto(s)
Acrilatos , Antivirales , Proteasas 3C de Coronavirus , SARS-CoV-2 , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , SARS-CoV-2/efectos de los fármacos , Humanos , Antivirales/farmacología , Antivirales/síntesis química , Antivirales/química , Acrilatos/farmacología , Acrilatos/química , Acrilatos/síntesis química , Relación Estructura-Actividad , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/síntesis química , Descubrimiento de Drogas , COVID-19/virología , Estructura Molecular
3.
Bioorg Med Chem Lett ; 98: 129590, 2024 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-38092072

RESUMEN

Natural product cantharidin can inhibit multiple myeloma cell growth in vitro, while serious adverse effects limited its clinical application. Therefore, the structural modification of cantharidin is needed. Herein, inspired by the structural similarity of the aliphatic endocyclic moiety in cantharidin and TRIP13 inhibitor DCZ0415, we designed and synthesized DCZ5418 and its nineteen derivatives. The molecular docking study indicated that DCZ5418 had a similar binding mode to TRIP13 protein as DCZ0415 while with a stronger docking score. Moreover, the bioassay studies of the MM-cells viability inhibition, TRIP13 protein binding affinity and enzyme inhibiting activity showed that DCZ5418 had good anti-MM activity in vitro and definite interaction with TRIP13 protein. The acute toxicity test of DCZ5418 showed less toxicity in vivo than cantharidin. Furthermore, DCZ5418 showed good anti-MM effects in vivo with a lower dose administration than DCZ0415 (15 mg/kg vs 25 mg/kg) on the tumor xenograft models. Thus, we obtained a new TRIP13 inhibitor DCZ5418 with improved safety and good activity in vivo, which provides a new example of lead optimization by using the structural fragments of natural products.


Asunto(s)
Cantaridina , Mieloma Múltiple , Humanos , ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , Cantaridina/farmacología , Cantaridina/uso terapéutico , Cantaridina/química , Proteínas de Ciclo Celular , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Simulación del Acoplamiento Molecular , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología
4.
Bioorg Med Chem ; 111: 117843, 2024 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-39083980

RESUMEN

This study reported the design and synthesis of novel 1-amido-2-one-4-thio-deoxypyranose as inhibitors of potential drug target TRIP13 for developing new mechanism-based therapeutic agents in the treatment of multiple myeloma (MM). In comparison with the positive control DCZ0415, the most active compounds C16, C18, C20 and C32 exhibited strong anti-proliferative activity against human MM cell lines (ARP-1 and NCI-H929) with IC50 values of 1 âˆ¼ 2 µM. While the surface plasmon resonance (SPR) and ATPase activity assays demonstrated that the representative compound C20 is a potent inhibitor of TRIP13, C20 also showed good antitumor activity in vivo on BALB/c nude mice xenografted with MM tumor cells. An initial structure-activity study showed that the carbonyl group is crucial for anticancer activity. Overall, this study provided novel 1-amido-2-one-4-thio-deoxypyranoses, which are entirely different from previously reported potent inhibitor structures of TRIP13, and thus would aid the development of carbohydrate-based novel agents in MM pharmacotherapy.


Asunto(s)
Antineoplásicos , Proliferación Celular , Diseño de Fármacos , Ratones Endogámicos BALB C , Ratones Desnudos , Mieloma Múltiple , Humanos , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Animales , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Mieloma Múltiple/metabolismo , Relación Estructura-Actividad , Ratones , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Estructura Molecular , Relación Dosis-Respuesta a Droga , Tanquirasas
5.
J Neurochem ; 160(4): 469-481, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34928513

RESUMEN

Alcohol exposure alters the signaling of the serotoninergic system, which is involved in alcohol consumption, reward, and dependence. In particular, dysregulation of serotonin receptor type 1A (5-HT1AR) is associated with alcohol intake and withdrawal-induced anxiety-like behavior in rodents. However, how ethanol regulates 5-HT1AR activity and cell surface availability remains elusive. Using neuroblastoma 2a cells stably expressing human 5-HT1ARs tagged with hemagglutinin at the N-terminus, we found that prolonged ethanol exposure (18 h) reduced the basal surface levels of 5-HT1ARs in a concentration-dependent manner. This reduction is attributed to both enhanced receptor internalization and attenuated receptor recycling. Moreover, constitutive 5-HT1AR internalization in ethanol naïve cells was blocked by concanavalin A (ConA) but not nystatin, suggesting clathrin-dependent 5-HT1AR internalization. In contrast, constitutive 5-HT1AR internalization in ethanol-treated cells was blocked by nystatin but not by ConA, indicating that constitutive 5-HT1AR internalization switched from a clathrin- to a caveolin-dependent pathway. Dynasore, an inhibitor of dynamin, blocked 5-HT1AR internalization in both vehicle- and ethanol-treated cells. Furthermore, ethanol exposure enhanced the activity of dynamin I via dephosphorylation and reduced myosin Va levels, which may contribute to increased internalization and reduced recycling of 5-HT1ARs, respectively. Our findings suggest that prolonged ethanol exposure not only alters the endocytic trafficking of 5-HT1ARs but also the mechanism by which constitutive 5-HT1AR internalization occurs.


Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Receptor de Serotonina 5-HT1A/efectos de los fármacos , Receptor de Serotonina 5-HT1A/metabolismo , Línea Celular , Clatrina/metabolismo , Concanavalina A/farmacología , Relación Dosis-Respuesta a Droga , Dinaminas/metabolismo , Endocitosis , Humanos , Hidrazonas/farmacología , Nistatina/farmacología , Antagonistas del Receptor de Serotonina 5-HT1/farmacología , Proteínas de Unión al GTP rab/metabolismo
6.
Synapse ; 75(4): e22190, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33025628

RESUMEN

Metabotropic glutamate (mGlu) receptors are regulators of glutamate release and targets for development of therapies for hyperactive glutamatergic signaling. However, the effects of long-term stimulation of mGlu receptors on cellular signaling in the brain have not been described. This study investigated the effects of 2-day and 14-day osmotic mini-pump administration of the mGlu2,3 agonist LY379268 (3.0 mg kg-1  day-1 ) to rats on receptor-mediated G-protein activation and signaling in mesocorticolimbic regions in rat brain sections. A significant reduction in LY379268-stimulated [35 S]GTPγS binding was observed in the 14-day group in some cortical regions, prefrontal cortex, nucleus accumbens, and ventral pallidum. The 14-day LY379268 treatment group exhibited mGlu2 mRNA levels significantly lower in hippocampus, nucleus accumbens, caudate, and ventral pallidum. In both 2-day and 14-day treatment groups immunodetectable phosphorylated cAMP Response Element-Binding protein (CREB) was significantly reduced across all brain regions. In the 2-day group, we observed significantly lower immunodetectable CREB protein across all brain regions, which was subsequently increased in the 14-day group but failed to achieve control values. Neither immunodetectable extracellular signal-regulated kinase (ERK) protein nor phosphorylated ERK from 2-day or 14-day treatment groups differed significantly from control across all brain regions. However, the ratio of phosphorylated ERK to total ERK protein was significantly greater in the 14-day treatment group compared with the control. These results identify compensatory changes to mGlu2,3 signal transduction in rat brains after chronic systemic administration of agonist, which could be predictive of the mechanism of action in human pharmacotherapies.


Asunto(s)
Ácido Glutámico , Receptores de Glutamato Metabotrópico , Animales , Encéfalo/metabolismo , Proteínas de Unión al GTP/metabolismo , Ratas , Receptores de Glutamato Metabotrópico/agonistas , Transducción de Señal
7.
J Biol Chem ; 294(38): 14068-14080, 2019 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-31366729

RESUMEN

Acute alcohol exposure alters the trafficking and function of many G-protein-coupled receptors (GPCRs) that are associated with aberrant behavioral responses to alcohol. However, the molecular mechanisms underlying alcohol-induced changes in GPCR function remain unclear. ß-Arrestin is a key player involved in the regulation of GPCR internalization and thus controls the magnitude and duration of GPCR signaling. Although ß-arrestin levels are influenced by various drugs of abuse, the effect of alcohol exposure on ß-arrestin expression and ß-arrestin-mediated GPCR trafficking is poorly understood. Here, we found that acute ethanol exposure increases ß-arrestin2 degradation via its increased ubiquitination in neuroblastoma-2a (N2A) cells and rat prefrontal cortex (PFC). ß-Arrestin2 ubiquitination was likely mediated by the E3 ligase MDM2 homolog (MDM2), indicated by an increased coupling between ß-arrestin2 and MDM2 in response to acute ethanol exposure in both N2A cells and rat PFC homogenates. Importantly, ethanol-induced ß-arrestin2 reduction was reversed by siRNA-mediated MDM2 knockdown or proteasome inhibition in N2A cells, suggesting ß-arrestin2 degradation is mediated by MDM2 through the proteasomal pathway. Using serotonin 5-HT1A receptors (5-HT1ARs) as a model receptor system, we found that ethanol dose-dependently inhibits 5-HT1AR internalization and that MDM2 knockdown reverses this effect. Moreover, ethanol both reduced ß-arrestin2 levels and delayed agonist-induced ß-arrestin2 recruitment to the membrane. We conclude that ß-arrestin2 dysregulation by ethanol impairs 5-HT1AR trafficking. Our findings reveal a critical molecular mechanism underlying ethanol-induced alterations in GPCR internalization and implicate ß-arrestin as a potential player mediating behavioral responses to acute alcohol exposure.


Asunto(s)
Endocitosis , Etanol/farmacología , Receptor de Serotonina 5-HT1A/metabolismo , Ubiquitinación/efectos de los fármacos , Arrestina beta 2/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular , Masculino , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT1A/química , Agonistas del Receptor de Serotonina 5-HT1/farmacología , Arrestina beta 2/antagonistas & inhibidores , Arrestina beta 2/genética
8.
Anesthesiology ; 129(3): 544-556, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29912007

RESUMEN

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Recovery from pain after surgery is faster after cesarean delivery than after other abdominal procedures. The authors hypothesized that recovery in rats after surgery could be reversed by antagonism of spinal oxytocin or vasopressin receptors, that there may be a sex difference, and that spinal oxytocin innervation could change after surgery. METHODS: Male and female rats underwent partial spinal nerve ligation surgery. Effects of nonselective and selective oxytocin and vasopressin 1A receptor antagonists on mechanical hypersensitivity during partial recovery were assessed (n = 8 to 14/group). Oxytocin immunoreactivity in the dorsal horn of the spinal cord (n = 7 to 8/group) and messenger RNA (mRNA) expression for oxytocin-binding receptors in dorsal root ganglia and spinal cord (n = 8/group) were measured. RESULTS: Intrathecal injection of oxytocin and vasopressin receptor antagonists were similarly effective at reducing withdrawal threshold (in all experiments from 22 [19, 26] median [first quartile, third quartile]) g to 8.3 [6.4, 12] g after injection) in both sexes, while having no or minimal effects in animals without surgery. Oxytocin fiber immunoreactivity was 3- to 5-fold greater in lumbar than other regions of the spinal cord and was increased more than 2-fold in lumbar cord ipsilateral to surgery. Injury was also associated with a 6.5-fold increase in oxytocin receptor and a 2-fold increase in vasopressin 1A receptor messenger RNA expression in the L4 dorsal root ganglion ipsilateral to surgery. CONCLUSIONS: These findings suggest that the capacity for oxytocin signaling in the spinal cord increases after surgery and that spinal oxytocin signaling plays ongoing roles in both sexes in recovery from mechanical hypersensitivity after surgery with known nerve injury.


Asunto(s)
Receptores de Oxitocina/fisiología , Receptores de Vasopresinas/fisiología , Recuperación de la Función/fisiología , Transducción de Señal/fisiología , Nervios Espinales/lesiones , Nervios Espinales/cirugía , Animales , Antagonistas de los Receptores de Hormonas Antidiuréticas/farmacología , Femenino , Hiperalgesia/etiología , Hiperalgesia/prevención & control , Inyecciones Espinales , Ligadura , Masculino , Oxitocina/antagonistas & inhibidores , Oxitocina/fisiología , Dolor Postoperatorio/etiología , Dolor Postoperatorio/prevención & control , Ratas , Ratas Sprague-Dawley , Receptores de Oxitocina/antagonistas & inhibidores , Recuperación de la Función/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Nervios Espinales/efectos de los fármacos
9.
Eur J Med Chem ; 265: 116048, 2024 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-38150961

RESUMEN

Cannabinoid CB2R agonists have gained considerable attention as potential novel therapies for psychiatric disorders due to their non-psychoactive nature, in contrast to CB1R agonists. In this study, we employed molecular docking to design and synthesize 23 derivatives of cannabidiol (CBD) with the aim of discovering potent CB2R agonists rather than CB2R antagonists or inverse agonists. Structure-activity relationship (SAR) investigations highlighted the critical importance of the amide group at the C-3' site and the cycloalkyl group at the C-4' site for CB2R activation. Interestingly, three CBD derivatives, namely 2o, 6g, and 6h, exhibited substantial partial agonistic activity towards the CB2 receptor, in contrast to the inverse agonistic property of CBD. Among these, 2o acted as a CB2R and 5-HT1AR dual agonist, albeit with some undesired antagonist activity for CB1R. It demonstrated significant CB2R partial agonism while maintaining a level of 5-HT1AR agonistic and CB1R antagonistic activity similar to CBD. Pharmacokinetic experiments confirmed that 2o possesses favorable pharmacokinetic properties. Behavioral studies further revealed that 2o elicits significant antidepressant-like and anxiolytic-like effects while maintaining a good safety profile.


Asunto(s)
Cannabidiol , Receptor de Serotonina 5-HT1A , Humanos , Simulación del Acoplamiento Molecular , Serotonina , Depresión/tratamiento farmacológico , Agonismo Inverso de Drogas , Agonistas de Receptores de Cannabinoides , Cannabidiol/farmacología , Cannabidiol/uso terapéutico , Agonistas de Receptores de Serotonina , Ansiedad , Receptor Cannabinoide CB2 , Receptor Cannabinoide CB1
10.
Commun Chem ; 7(1): 93, 2024 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-38678046

RESUMEN

Amides are important intermediates in organic chemistry and the pharmaceutical industry, but their low reactivity requires catalysts and/or severe reaction conditions for esterification. Here, a novel approach was devised to convert amides into esters without the use of transition metals. The method effectively overcomes the inherent low reactivity of amides by employing dimethylsulfate-mediated reaction to activate the C-N bonds. To confirm the proposed reaction mechanism, control experiments and density functional theory (DFT) calculations were conducted. The method demonstrates a wide array of substrates, including amides with typical H/alkyl/aryl substitutions, N,N-disubstituted amides, amides derived from alkyl, aryl, or vinyl carboxylic acids, and even amino acid substrates with stereocentres. Furthermore, we have shown the effectiveness of dimethylsulfate in removing acyl protective groups in amino derivatives. This study presents a method that offers efficiency and cost-effectiveness in broadening the esterification capabilities of amides, thereby facilitating their increased utilization as synthetic compounds in diverse transformations.

11.
Carcinogenesis ; 34(1): 176-82, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23066085

RESUMEN

Fatty acid metabolism impacts multiple intracellular signaling pathways in many cell types, but its role in prostate cancer cells is still unclear. Our previous studies have shown that the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) induces apoptosis in human prostate cancer cells by a syndecan-1 (SDC-1)-dependent mechanism. Here, we examined the contribution of lipoxygenase (LOX)- and cyclooxygenase (COX)-mediated DHA metabolism to this effect. Pan-LOX inhibitor (nordihydroguaiaretic acid), 15-LOX inhibitor (luteolin) or 15/12-LOX inhibitor (baicalein) blocked the induced effect of DHA on SDC-1 expression and apoptosis in human prostate cancer cells, whereas 5-LOX inhibitor, AA861, was ineffective. Human prostate cancer cells lines (PC3, LNCaP and DU145 cells) expressed two 15-LOX isoforms, 15-LOX-1 and 15-LOX-2, with higher 15-LOX-1 and lower 15-LOX-2 expressions compared with human epithelial prostate cells. Knockdown of 15-LOX-1 blocked the effect of DHA on SDC-1 expression and caspase-3 activity, whereas silencing 15-LOX-2, 5-LOX, COX-1, COX-2 or 12-LOX had no effect. Moreover, the ability of DHA to inhibit the activity of the PDK/Akt (T308) signaling pathway was abrogated by silencing 15-LOX-1. These findings demonstrate that 15-LOX-1-mediated metabolism of DHA is required for it to upregulate SDC-1 and trigger the signaling pathway that elicits apoptosis in prostate cancer cells.


Asunto(s)
Apoptosis , Araquidonato 15-Lipooxigenasa/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Neoplasias de la Próstata/patología , Transducción de Señal , Sindecano-1/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/metabolismo
12.
J Neurochem ; 125(5): 663-72, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23458603

RESUMEN

The strength and duration of extracellular dopamine concentrations are regulated by the presynaptic dopamine transporter (DAT) and dopamine D2 autoreceptors (D2autoRs). There is a functional interaction between these two proteins. Activation of D2autoRs increases DAT trafficking to the surface whereas disruption of this interaction compromises activities of both proteins and alters dopaminergic transmission. Previously we reported that DAT expression and activity are subject to modulation by protein kinase Cß (PKCß). Here, we further demonstrate that PKCß is integral for the interaction between DAT and D2autoR. Inhibition or absence of PKCß abolished the communication between DAT and D2autoR. In mouse striatal synaptosomes and transfected N2A cells, the D2autoR-stimulated membrane insertion of DAT was abolished by PKCß inhibition. Moreover, D2autoR-stimulated DAT trafficking is mediated by a PKCß-extracellular signal-regulated kinase signaling cascade where PKCß is upstream of extracellular signal-regulated kinase. The increased surface DAT expression upon D2autoR activation resulted from enhanced DAT recycling as opposed to reduced internalization. Further, PKCß promoted accelerated DAT recycling. Our study demonstrates that PKCß critically regulates D2autoR-activated DAT trafficking and dopaminergic signaling. PKCß is a potential drug target for correcting abnormal extracellular dopamine levels in diseases such as drug addiction and schizophrenia.


Asunto(s)
Autorreceptores/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Proteína Quinasa C/fisiología , Receptores de Dopamina D2/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Cuerpo Estriado/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa C beta , Transporte de Proteínas/fisiología
13.
IBRO Neurosci Rep ; 14: 129-137, 2023 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-36748012

RESUMEN

Prior studies examining the effects of cocaine on the dynorphin/kappa opioid receptor (Dyn/KOR) system primarily focus on non-contingent cocaine exposure, but the effects of self-administration, which more closely reflects human drug-taking behaviors, are not well studied. In this study we characterized the effects of escalated intravenous cocaine self-administration on the functional state of the Dyn/KOR system and its interaction with mesolimbic dopamine signaling. Rats self-administered cocaine in an extended access, limited intake cocaine procedure, in which animals obtained 40 infusions per day (1.5 mg/kg/inf) for 5 consecutive days to ensure comparable consumption levels. Following single day tests of cue reactivity and progressive ratio responding, quantitative real-time polymerase chain reaction was used to measure levels of Oprk and Pdyn transcripts in the ventral tegmental area and nucleus accumbens. Additionally, after self-administration, ex vivo fast-scan cyclic voltammetry in the NAc was used to examine the ability of the KOR agonist U50,488 to inhibit dopamine release. We found that KOR-induced inhibition of dopamine release was enhanced in animals that self-administered cocaine compared to controls, suggesting upregulated Dyn/KOR activity after cocaine self-administration. Furthermore, expression levels of Pdyn in the nucleus accumbens and ventral tegmental area, and Oprk in the nucleus accumbens, were elevated in cocaine animals compared to controls. Additionally, Pdyn expression in the nucleus accumbens was negatively correlated with progressive ratio breakpoints, a measure of motivation to self-administer cocaine. Overall, these data suggest that cocaine self-administration elevates KOR/Dyn system activity in the mesolimbic dopamine pathway.

14.
Neuropharmacology ; 225: 109387, 2023 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-36567004

RESUMEN

The function of the dopamine transporter (DAT) is regulated by membrane cholesterol content. A direct, acute removal of membrane cholesterol by methyl-ß-cyclodextrin (MßCD) has been shown to reduce dopamine (DA) uptake and release mediated by the DAT. This is of particular interest because a few widely prescribed statins that lower peripheral cholesterol levels are blood-brain barrier (BBB) penetrants, and therefore could alter DAT function through brain cholesterol modulation. The goal of this study was to investigate the effects of prolonged atorvastatin treatment (24 h) on DAT function in neuroblastoma 2A cells stably expressing DAT. We found that atorvastatin treatment effectively lowered membrane cholesterol content in a concentration-dependent manner. Moreover, atorvastatin treatment markedly reduced DA uptake and abolished cocaine inhibition of DA uptake, independent of surface DAT levels. These deficits induced by atorvastatin treatment were reversed by cholesterol replenishment. However, atorvastatin treatment did not change amphetamine (AMPH)-induced DA efflux. This is in contrast to a small but significant reduction in DA efflux induced by acute depletion of membrane cholesterol using MßCD. This discrepancy may involve differential changes in membrane lipid composition resulting from chronic and acute cholesterol depletion. Our data suggest that the outward-facing conformation of DAT, which favors the binding of DAT blockers such as cocaine, is more sensitive to atorvastatin-induced cholesterol depletion than the inward-facing conformation, which favors the binding of DAT substrates such as AMPH. Our study on statin-DAT interactions may have clinical implications in our understanding of neurological side effects associated with chronic use of BBB penetrant statins.


Asunto(s)
Cocaína , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Anfetamina/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Cocaína/farmacología , Dopamina/metabolismo , Atorvastatina/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Colesterol/metabolismo
15.
eNeuro ; 10(7)2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37364995

RESUMEN

Here we describe the generation and characterization of a Cre knock-in mouse line that harbors a Cre insertion in the 3'UTR of the κ opioid receptor gene (Oprk1) locus and provides genetic access to populations of κ opioid receptor (KOR)-expressing neurons throughout the brain. Using a combination of techniques including RNA in situ hybridization and immunohistochemistry, we report that Cre is expressed with high fidelity in KOR-expressing cells throughout the brain in this mouse line. We also provide evidence that Cre insertion does not alter basal KOR function. Baseline anxiety-like behaviors and nociceptive thresholds are unaltered in Oprk1-Cre mice. Chemogenetic activation of KOR-expressing cells in the basolateral amygdala (BLAKOR cells) resulted in several sex-specific effects on anxiety-like and aversive behaviors. Activation led to decreased anxiety-like behavior on the elevated plus maze and increased sociability in female but not in male Oprk1-Cre mice. Activation of BLAKOR cells also attenuated KOR agonist-induced conditioned place aversion (CPA) in male Oprk1-Cre mice. Overall, these results suggest a potential role for BLAKOR cells in regulating anxiety-like behaviors and KOR-agonist mediated CPA. In summary, these results provide evidence for the utility of the newly generated Oprk1-Cre mice in assessing localization, anatomy, and function of KOR circuits throughout the brain.


Asunto(s)
Integrasas , Receptores Opioides kappa , Ratones , Masculino , Femenino , Animales , Receptores Opioides kappa/genética , Receptores Opioides kappa/metabolismo , Integrasas/genética , Encéfalo/metabolismo , Reacción de Prevención/fisiología
16.
J Med Chem ; 65(17): 11840-11853, 2022 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-36073068

RESUMEN

Site-selective lysine modification of peptides and proteins in aqueous solutions or in living cells is still a big challenge today. Here, we report a novel strategy to selectively quinolylate lysine residues of peptides and proteins under native conditions without any catalysts using our newly developed water-soluble zoliniums. The zoliniums could site-selectively quinolylate K350 of bovine serum albumin and inactivate SARS-CoV-2 3CLpro via covalently modifying two highly conserved lysine residues (K5 and K61). In living HepG2 cells, it was demonstrated that the simple zoliniums (5b and 5B) could quinolylate protein lysine residues mainly in the nucleus, cytosol, and cytoplasm, while the zolinium-fluorophore hybrid (8) showed specific lysosome-imaging ability. The specific chemoselectivity of the zoliniums for lysine was validated by a mixture of eight different amino acids, different peptides bearing potential reactive residues, and quantum chemistry calculations. This study offers a new way to design and develop lysine-targeted covalent ligands for specific application.


Asunto(s)
Lisina , Péptidos , Proteasas 3C de Coronavirus/química , Lisina/química , Péptidos/química , SARS-CoV-2/enzimología , Albúmina Sérica Bovina/química , Agua/química
17.
Carcinogenesis ; 32(10): 1518-24, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21771724

RESUMEN

Human epidemiological studies have shown that diets enriched in n-3 polyunsaturated fatty acids (n-3 PUFA) are associated with a lower incidence of cancers including breast cancer. Our previous studies showed that the n-3 PUFA, docosahexaenoic acid (DHA), upregulated syndecan-1 (SDC-1) expression to induce apoptosis in the human breast cancer cell line MCF-7. We now present evidence of a signaling pathway that is impacted by SDC-1 in these cells and in mouse mammary tissues to result in apoptosis. In MCF-7 cells and SK-BR-3 cells, DHA and a SDC-1 ectodomain impaired signaling of the p44/42 mitogen-activated protein kinase (MAPK) pathway by inhibiting the phosphorylation of MAPK/Erk (MEK)/extracellular signal-regulated kinase (Erk) and Bad to induce apoptosis. SDC-1 siRNA significantly enhanced phosphorylation of these signal molecules and blocked the inhibitory effects of DHA on their phosphorylation. SDC-1 siRNA diminished apoptosis of MCF-7 cells, an effect that was markedly blocked by MEK inhibitor, PD98059. In vivo studies used (i) Fat-1 mice, a genetic model able to convert n-6 to n-3 PUFA to result in higher SDC-1 levels in Fat-1 mammary tissue compared with that of wild-type (wt) mice. Phosphorylation of MEK, Erk and Bad was lower in the Fat-1 versus wt tissue and (ii) SDC-1(-/-) mice that demonstrated markedly higher levels of phosphorylated MEK, Erk and Bad in mammary gland tissue compared with those of SDC(+/+) mice. These data elucidate a pathway whereby SDC-1, upregulated by DHA, induces apoptosis in breast cancer cells through inhibition of MEK/Erk/Bad signaling.


Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Ácidos Docosahexaenoicos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glándulas Mamarias Animales/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Sindecano-1/fisiología , Animales , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Cadherinas/fisiología , Femenino , Humanos , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sindecano-1/antagonistas & inhibidores , Células Tumorales Cultivadas , Proteína Letal Asociada a bcl/metabolismo
18.
Eur J Med Chem ; 222: 113583, 2021 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-34119832

RESUMEN

Herein we disclosed the novel nucleophilic addition reactions of the thiophenols and oxazolinium (DCZ0358) to produce N-2'-aryletheryl-1'-alkoxy-ethyl substituted arylisoquinolones. After evaluating the anti-inflammatory activity in vitro, 2d was found having significant anti-TNFα activity. Through the amplified synthesis of 2d, four monomers (3a-b and 4a-d) were obtained by chiral separation of the product. The reaction mechanism was proposed and explored by the control experiments. However, only the R-stereoisomers 3b and 4b have significant anti-TNFα activity in vitro (IC50 = 56 and 14 nM, respectively). Moreover, 4b exerts potent therapeutic effects on ulcerative colitis in vivo (30 mg/kg bw, qd, i. g.). The subsequent bio-target exploration of compound 4bvia molecular docking and the experimental validation disclosed that 4b has 3-fold selectivity of binding activity on estrogen receptor (ER) beta (ß) (Ki = 760.86 nM) vs. alpha (α) (Ki = 2320.58 nM). Thus, it provides a novel type of non-steroidal leads for developing anti-inflammatory drugs.


Asunto(s)
Antiinflamatorios/farmacología , Colitis Ulcerosa/tratamiento farmacológico , Descubrimiento de Drogas , Oxazoles/farmacología , Fenoles/farmacología , Quinolonas/farmacología , Compuestos de Sulfhidrilo/farmacología , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/química , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/metabolismo , Sulfato de Dextran , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Oxazoles/química , Fenoles/química , Quinolonas/síntesis química , Quinolonas/química , Células RAW 264.7 , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/química , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo
19.
Neuroscience ; 443: 131-139, 2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-32730947

RESUMEN

The brain opioid system plays an important role in cocaine reward. Altered signaling in the opioid system by chronic cocaine exposure contributes to cocaine-seeking and taking behavior. The current study investigated concurrent changes in the gene expression of multiple components in rat brain opioid system following cocaine self-administration. Animals were limited to 40 infusions (1.5 mg/kg/infusion) within 6 h per day for five consecutive days. We then examined the mRNA levels of opioid receptors including mu (Oprm), delta (Oprd), and kappa (Oprk), and their endogenous opioid peptide precursors including proopiomelanocortin (Pomc), proenkephalin (Penk), prodynorphin (Pdyn) in the dorsal striatum (CPu) and the prefrontal cortex (PFC) 18 h after the last cocaine infusion. We found that cocaine self-administration significantly increased the mRNA levels of Oprm and Oprd in both the CPu and PFC, but had no effect on Oprk mRNA levels in either brain region. Moreover, cocaine had a greater influence on the mRNA levels of opioid peptide precursors in rat CPu than in the PFC. In the CPu, cocaine self-administration significantly increased the mRNA levels of Penk and Pdyn and abolished the mRNA levels of Pomc. In the PFC, cocaine self-administration only increased Pdyn mRNA levels without changing the mRNA levels of Pomc and Penk. These data suggest that cocaine self-administration influences the expression of multiple genes in the brain opioid system, and the concurrent changes in these targets may underlie cocaine-induced reward and habitual drug-seeking behavior.


Asunto(s)
Cocaína , Receptores Opioides , Animales , Encefalinas/genética , Encefalinas/metabolismo , Péptidos Opioides , Corteza Prefrontal/metabolismo , Putamen/metabolismo , Ratas , Receptores Opioides/metabolismo , Receptores Opioides mu
20.
Signal Transduct Target Ther ; 5(1): 31, 2020 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-32296013

RESUMEN

Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous malignant tumor characterized by diffuse growth. DCZ0858 is a novel small molecule with excellent antitumor effects in DLBCL. This study explored in depth the inhibitory effect of DCZ0858 on DLBCL cell lines. Cell Counting Kit-8 (CCK-8) and plate colony formation assays were used to evaluate cell proliferation levels. Flow cytometry was employed to analyze apoptosis and the cell cycle, and western blotting was used to quantify the expression of cell cycle regulators. The results indicated that DCZ0858 inhibited cell growth in a concentration-dependent and time-dependent manner while inducing no significant toxicity in normal cells. Moreover, DCZ0858 initiated cell apoptosis via both internal and external apoptotic pathways. DCZ0858 also induced cell cycle arrest in the G0/G1 phase, thereby controlling cell proliferation. Further investigation of the molecular mechanism showed that the JAK2/STAT3 pathway was involved in the DCZ0858-mediated antitumor effects and that JAK2 was the key target for DCZ0858 treatment. Knockdown of JAK2 partly weakened the DCZ0858-mediated antitumor effect in DLBCL cells, while JAK2 overexpression strengthened the effect of DCZ0858 in DLBCL cells. Moreover, a similar antitumor effect was observed for DCZ0858 and the JAK2 inhibitor ruxolitinib, and combining the two could significantly enhance cancer-suppressive signaling. Tumor xenograft models showed that DCZ0858 inhibited tumor growth in vivo and had low toxicity in important organs, findings that were consistent with the in vitro data. In summary, DCZ0858 is a promising drug for the treatment of DLBCL.


Asunto(s)
Janus Quinasa 2/genética , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Factor de Transcripción STAT3/genética , Siliconas/farmacología , Apoptosis/efectos de los fármacos , Recuento de Células , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Transducción de Señal/efectos de los fármacos
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