RESUMO
Citrullinemia type I (CTLN1) is a rare autosomal recessive disorder caused by mutations in the gene encoding argininosuccinate synthetase 1 (ASS1) that catalyzes the third step of the urea cycle. CTLN1 patients suffer from impaired elimination of nitrogen, which leads to neurotoxic levels of circulating ammonia and urea cycle byproducts that may cause severe metabolic encephalopathy, death or irreversible brain damage. Standard of care (SOC) of CTLN1 consists of daily nitrogen-scavenger administration, but patients remain at risk of life-threatening decompensations. We evaluated the therapeutic efficacy of a recombinant adeno-associated viral vector carrying the ASS1 gene under the control of a liver-specific promoter (VTX-804). When administered to three-week-old CTLN1 mice, all the animals receiving VTX-804 in combination with SOC gained body weight normally, presented with a normalization of ammonia and reduction of citrulline levels in circulation, and 100% survived for 7 months. Similar to what has been observed in CTLN1 patients, CTLN1 mice showed several behavioral abnormalities such as anxiety, reduced welfare and impairment of innate behavior. Importantly, all clinical alterations were notably improved after treatment with VTX-804. This study demonstrates the potential of VTX-804 gene therapy for future clinical translation to CTLN1 patients.
Assuntos
Amônia , Citrulinemia , Camundongos , Animais , Nitrogênio , Citrulinemia/genética , Citrulinemia/terapia , Argininossuccinato Sintase/genética , Argininossuccinato Sintase/metabolismo , Terapia Genética , Ureia/metabolismoRESUMO
Morphine and structurally-derived compounds are µ opioid receptor (µOR) agonists, and the most effective analgesic drugs. However, their usefulness is limited by serious side effects, including dependence and abuse potential. The N-substituent in morphinans plays an important role in opioid activities in vitro and in vivo. This study presents the synthesis and pharmacological evaluation of new N-phenethyl substituted 14-O-methylmorphinan-6-ones. Whereas substitution of the N-methyl substituent in morphine (1) and oxymorphone (2) by an N-phenethyl group enhances binding affinity, selectivity and agonist potency at the µOR of 1a and 2a, the N-phenethyl substitution in 14-methoxy-N-methylmorphinan-6-ones (3 and 4) converts selective µOR ligands into dual µ/δOR agonists (3a and 4a). Contrary to N-methylmorphinans 1-4, the N-phenethyl substituted morphinans 1a-4a produce effective and potent antinociception without motor impairment in mice. Using docking and molecular dynamics simulations with the µOR, we establish that N-methylmorphinans 1-4 and their N-phenethyl counterparts 1a-4a share several essential receptor-ligand interactions, but also interaction pattern differences related to specific structural features, thus providing a structural basis for their pharmacological profiles. The emerged structure-activity relationships in this class of morphinans provide important information for tuning in vitro and in vivo opioid activities towards discovery of effective and safer analgesics.
Assuntos
Analgésicos Opioides/farmacologia , Morfinanos/farmacologia , Receptores Opioides delta/agonistas , Receptores Opioides delta/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetulus , Humanos , Ligantes , Masculino , Camundongos , Morfina/farmacologia , Relação Estrutura-AtividadeRESUMO
Chronic pain is currently treated with opioids that offer unsatisfactory long-term analgesia and produce serious side effects. There is a clear need for alternative therapies. Herein, peptide-based hydrogels are used as extended-release drug delivery carriers. Two different formulations were developed: the drug is coformulated within the hydrogel; the drug is an integral part of the hydrogelator. Both strategies afford a prolonged and significant antinociception up to 72 h after subcutaneous administration in mice.
Assuntos
Portadores de Fármacos/química , Hidrogéis/química , Interações Hidrofóbicas e Hidrofílicas , Oligopeptídeos/química , Peptídeos Opioides/química , Peptídeos Opioides/farmacologia , Sequência de Aminoácidos , Animais , Portadores de Fármacos/metabolismo , Estabilidade de Medicamentos , Humanos , Camundongos , Oligopeptídeos/metabolismoRESUMO
We previously reported on a series of small molecules targeting the κ-opioid (KOP) receptor featuring a diphenethylamine scaffold and showed the promise of these ligands as effective analgesics with reduced liability for adverse effects. This study expands the structure-activity relationships on our original series by presenting several modifications in the lead compounds 1 (HS665) and 2 (HS666). A library of new diphenethylamines was designed, synthesized, and pharmacologically evaluated. In comparison with 1 and 2, the KOP receptor affinity, selectivity, and agonist activity were modulated by introducing bulkier N-substituents, a 2-fluoro substitution, and additional hydroxyl groups at positions 3' and 4'. Several analogues showed subnanomolar affinity and excellent KOP receptor selectivity acting as full or partial agonists, and one as an antagonist. The new diphenethylamines displayed antinociceptive efficacies with increased potencies than U50,488, 1 and 2 in the writhing assay and without inducing motor dysfunction after sc administration in mice.
Assuntos
Analgésicos/química , Analgésicos/farmacologia , Fenetilaminas/química , Fenetilaminas/farmacologia , Receptores Opioides kappa/metabolismo , Analgésicos/síntese química , Animais , Células CHO , Cricetulus , Humanos , Masculino , Camundongos , Fenetilaminas/síntese química , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
BACKGROUND AND PURPOSE: The κ receptor has a central role in modulating neurotransmission in central and peripheral neuronal circuits that subserve pain and other behavioural responses. Although κ receptor agonists do not produce euphoria or lead to respiratory suppression, they induce dysphoria and sedation. We hypothesized that brain-penetrant κ receptor ligands possessing biased agonism towards G protein signalling over ß-arrestin2 recruitment would produce robust antinociception with fewer associated liabilities. EXPERIMENTAL APPROACH: Two new diphenethylamines with high κ receptor selectivity, HS665 and HS666, were assessed following i.c.v. administration in mouse assays of antinociception with the 55°C warm-water tail withdrawal test, locomotor activity in the rotorod and conditioned place preference. The [35 S]-GTPγS binding and ß-arrestin2 recruitment in vitro assays were used to characterize biased agonism. KEY RESULTS: HS665 (κ receptor agonist) and HS666 (κ receptor partial agonist) demonstrated dose-dependent antinociception after i.c.v. administration mediated by the κ receptor. These highly selective κ receptor ligands displayed varying biased signalling towards G protein coupling in vitro, consistent with a reduced liability profile, reflected by reduced sedation and absence of conditioned place aversion for HS666. CONCLUSIONS AND IMPLICATIONS: HS665 and HS666 activate central κ receptors to produce potent antinociception, with HS666 displaying pharmacological characteristics of a κ receptor analgesic with reduced liability for aversive effects correlating with its low efficacy in the ß-arrestin2 signalling pathway. Our data provide further understanding of the contribution of central κ receptors in pain suppression, and the prospect of dissociating the antinociceptive effects of HS665 and HS666 from κ receptor-mediated adverse effects.
Assuntos
Analgésicos Opioides/farmacologia , Analgésicos/farmacologia , Fenetilaminas/farmacologia , Receptores Opioides kappa/agonistas , Analgésicos/administração & dosagem , Analgésicos/química , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/química , Animais , Células CHO , Células Cultivadas , Cricetulus , Relação Dose-Resposta a Droga , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Fenetilaminas/administração & dosagem , Fenetilaminas/química , Relação Estrutura-Atividade , Cauda/efeitos dos fármacosRESUMO
Glioblastoma is the most common and malignant brain cancer in adults. Current therapy consisting of surgery followed by radiation and temozolomide has a moderate success rate and the tumor reappears. Among the features that a cancer cell must have to survive the therapeutic treatment and reconstitute the tumor is the ability of self-renewal. Therefore, it is vital to identify the molecular mechanisms that regulate this activity. Sex-determining region Y (SRY)-box 2 (SOX2) is a transcription factor whose activity has been associated with the maintenance of the undifferentiated state of cancer stem cells in several tissues, including the brain. Several groups have detected increased SOX2 levels in biopsies of glioblastoma patients, with the highest levels associated with poor outcome. Therefore, SOX2 silencing might be a novel therapeutic approach to combat cancer and particularly brain tumors. In this review, we will summarize the current knowledge about SOX2 in glioblastoma and recapitulate several strategies that have recently been described targeting SOX2 in this malignancy.
RESUMO
The µ opioid receptor (MOR) is a prominent member of the G protein-coupled receptor family and the molecular target of morphine and other opioid drugs. Despite the long tradition of MOR-targeting drugs, still little is known about the ligand-receptor interactions and structure-function relationships underlying the distinct biological effects upon receptor activation or inhibition. With the resolved crystal structure of the ß-funaltrexamine-MOR complex, we aimed at the discovery of novel agonists and antagonists using virtual screening tools, i.e. docking, pharmacophore- and shape-based modeling. We suggest important molecular interactions, which active molecules share and distinguish agonists and antagonists. These results allowed for the generation of theoretically validated in silico workflows that were employed for prospective virtual screening. Out of 18 virtual hits evaluated in in vitro pharmacological assays, three displayed antagonist activity and the most active compound significantly inhibited morphine-induced antinociception. The new identified chemotypes hold promise for further development into neurochemical tools for studying the MOR or as potential therapeutic lead candidates.
Assuntos
Simulação de Acoplamento Molecular , Naltrexona/análogos & derivados , Antagonistas de Entorpecentes/química , Receptores Opioides mu/química , Cristalografia por Raios X , Humanos , Naltrexona/química , Receptores Opioides mu/metabolismoRESUMO
The α2δ-ligands pregabalin (PGB) and gabapentin (GBP) are used to treat neuropathic pain. We used whole cell recording to study their long-term effects on substantia gelatinosa and dorsal root ganglion (DRG) neurons. Spinal cord slices were prepared from embryonic day 13 rat embryos and maintained in organotypic culture for >5 wk (neuronal age equivalent to young adult rats). Exposure of similarly aged DRG neurons (dissociated and cultured from postnatal day 19 rats) to GBP or PGB for 5-6 days attenuated high-voltage-activated calcium channel currents (HVA ICa). Strong effects were seen in medium-sized and in small isolectin B4-negative (IB4-) DRG neurons, whereas large neurons and small neurons that bound isolectin B4 (IB4+) were hardly affected. GBP (100 µM) or PGB (10 µM) were less effective than 20 µM Mn(2+) in suppression of HVA ICa in small DRG neurons. By contrast, 5-6 days of exposure to these α2δ-ligands was more effective than 20 µM Mn(2+) in reducing spontaneous excitatory postsynaptic currents at synapses in substantia gelatinosa. Spinal actions of gabapentinoids cannot therefore be ascribed to decreased expression of HVA Ca(2+) channels in primary afferent nerve terminals. In substantia gelatinosa, 5-6 days of exposure to PGB was more effective in inhibiting excitatory synaptic drive to putative excitatory neurons than to putative inhibitory neurons. Although spontaneous inhibitory postsynaptic currents were also attenuated, the overall long-term effect of α2δ-ligands was to decrease network excitability as monitored by confocal Ca(2+) imaging. We suggest that selective actions of α2δ-ligands on populations of DRG neurons may predict their selective attenuation of excitatory transmission onto excitatory vs. inhibitory neurons in substantia gelatinosa.
Assuntos
Aminas/farmacologia , Analgésicos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Ácidos Cicloexanocarboxílicos/farmacologia , Gânglios Espinais/efeitos dos fármacos , Substância Gelatinosa/efeitos dos fármacos , Ácido gama-Aminobutírico/análogos & derivados , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Células Cultivadas , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Gabapentina , Gânglios Espinais/fisiologia , Imuno-Histoquímica , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Masculino , Microscopia Confocal , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Pregabalina , Ratos Sprague-Dawley , Substância Gelatinosa/fisiologia , Técnicas de Cultura de Tecidos , Ácido gama-Aminobutírico/farmacologiaRESUMO
Transforming growth factor-ß1 (TGF-ß1) protects against neuroinflammatory events underlying neuropathic pain. TGF-ß signaling enhancement is a phenotypic characteristic of mice lacking the TGF-ß pseudoreceptor BAMBI (BMP and activin membrane-bound inhibitor), which leads to an increased synaptic release of opioid peptides and to a naloxone-reversible hypoalgesic/antiallodynic phenotype. Herein, we investigated the following: (1) the effects of BAMBI deficiency on opioid receptor expression, functional efficacy, and analgesic responses to endogenous and exogenous opioids; and (2) the involvement of the opioid system in the antiallodynic effect of TGF-ß1. BAMBI-KO mice were subjected to neuropathic pain by sciatic nerve crash injury (SNI). Gene (PCR) and protein (Western blot) expressions of µ- and δ-opioid receptors were determined in the spinal cord. The inhibitory effects of agonists on the adenylyl cyclase pathway were investigated. Two weeks after SNI, wild-type mice developed mechanical allodynia and the functionality of µ-opioid receptors was reduced. By this time, BAMBI-KO mice were protected against allodynia and exhibited increased expression and function of opioid receptors. Four weeks after SNI, when mice of both genotypes had developed neuropathic pain, the analgesic responses induced by morphine and RB101 (an inhibitor of enkephalin-degrading enzymes, which increases the synaptic levels of enkephalins) were enhanced in BAMBI-KO mice. Similar results were obtained in the formalin-induced chemical-inflammatory pain model. Subcutaneous TGF-ß1 infusion prevented pain development after SNI. The antiallodynic effect of TGF-ß1 was naloxone-sensitive. In conclusion, modulation of the endogenous opioid system by TGF-ß signaling improves the analgesic effectiveness of exogenous and endogenous opioids under pathological pain conditions.
Assuntos
Analgésicos Opioides/farmacologia , Proteínas de Membrana/metabolismo , Morfina/farmacologia , Neuralgia/metabolismo , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Inibidores de Adenilil Ciclases , Analgesia , Analgésicos Opioides/uso terapêutico , Animais , Dissulfetos/farmacologia , Dissulfetos/uso terapêutico , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Infusões Subcutâneas , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Morfina/uso terapêutico , Naloxona/farmacologia , Neuralgia/tratamento farmacológico , Traumatismos dos Nervos Periféricos/metabolismo , Traumatismos dos Nervos Periféricos/patologia , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Fenilalanina/uso terapêutico , Receptores Opioides delta/genética , Receptores Opioides mu/genética , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Transdução de Sinais , Medula Espinal/metabolismo , Fator de Crescimento Transformador beta/administração & dosagem , Fator de Crescimento Transformador beta/uso terapêuticoRESUMO
Left ventricular (LV) pressure overload is a major cause of heart failure. Transforming growth factors-ß (TGF-ßs) promote LV remodeling under biomechanical stress. BAMBI (BMP and activin membrane-bound inhibitor) is a pseudoreceptor that negatively modulates TGF-ß signaling. The present study tests the hypothesis that BAMBI plays a protective role during the adverse LV remodeling under pressure overload. The subjects of the study were BAMBI knockout mice (BAMBI(-/-)) undergoing transverse aortic constriction (TAC) and patients with severe aortic stenosis (AS). We examined LV gene and protein expression of remodeling-related elements, histological fibrosis, and heart morphology and function. LV expression of BAMBI was increased in AS patients and TAC-mice and correlated directly with TGF-ß. BAMBI deletion led to a gain of myocardial TGF-ß signaling through canonical (Smads) and non-canonical (TAK1-p38 and TAK1-JNK) pathways. As a consequence, the remodeling response to pressure overload in BAMBI(-/-) mice was exacerbated in terms of hypertrophy, chamber dilation, deterioration of long-axis LV systolic function and diastolic dysfunction. Functional remodeling associated transcriptional activation of fibrosis-related TGF-ß targets, up-regulation of the profibrotic micro-RNA-21, histological fibrosis and increased metalloproteinase-2 activity. Histological remodeling in BAMBI(-/-) mice involved TGF-ßs. BAMBI deletion in primary cardiac fibroblasts exacerbated TGF-ß-induced profibrotic responses while BAMBI overexpression in NIH-3T3 fibroblasts attenuated them. Our findings identify BAMBI as a critical negative modulator of myocardial remodeling under pressure overload. We suggest that BAMBI is involved in negative feedback loops that restrain the TGF-ß remodeling signals to protect the pressure-overloaded myocardium from uncontrolled extracellular matrix deposition in humans and mice.
Assuntos
Coração/fisiologia , Proteínas de Membrana/fisiologia , Transdução de Sinais , Estresse Fisiológico , Fator de Crescimento Transformador beta/metabolismo , Animais , Hibridização Genômica Comparativa , Imunofluorescência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Transcrição GênicaRESUMO
Sustained administration of opioid antagonists to rodents results in an enhanced antinociceptive response to agonists. We investigated the changes in spinal µ-opioid receptor signalling underlying this phenomenon. Rats received naltrexone (120 µg/h; 7 days) via osmotic minipumps. The antinociceptive response to the µ-agonist sufentanil was tested 24 h after naltrexone withdrawal. In spinal cord samples, we determined the interaction of µ-receptors with Gα proteins (agonist-stimulated [(35)S]GTPγS binding and immunoprecipitation of [(35)S]GTPγS-labelled Gα subunits) as well as µ-opioid receptor-dependent inhibition of the adenylyl cyclase (AC) activity. Chronic naltrexone treatment augmented DAMGO-stimulated [(35)S]GTPγS binding, potentiated the inhibitory effect of DAMGO on the AC/cAMP pathway, and increased the inverse agonist effect of naltrexone on cAMP accumulation. In control rats, the inhibitory effect of DAMGO on cAMP production was antagonized by pertussis toxin (PTX) whereas, after chronic naltrexone, the effect became resistant to the toxin, suggesting a coupling of µ-receptors to PTX-insensitive Gα(z) subunits. Immunoprecipitation assays confirmed the transduction switch from Gα(i/o) to Gα(z) proteins. The consequence was an enhancement of the antinociceptive response to sufentanil that, in consonance with the neurochemical data, was prevented by Gα(z)-antisense oligodeoxyribonucleotides but not by PTX. Such changes in opioid receptor signalling can be a double-edged sword. On the one hand, they may have potential applicability to the optimisation of the analgesic effects of opioid drugs for the control of pain. On the other hand, they represent an important homeostatic dysregulation of the endogenous opioid system that might account for undesirable effects in patients chronically treated with opioid antagonists. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Receptores Opioides mu/metabolismo , Medula Espinal/efeitos dos fármacos , Animais , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Masculino , Medição da Dor/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores Opioides mu/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
The transforming growth factor-ß (TGF-ß) superfamily is a multifunctional, contextually acting family of cytokines that participate in the regulation of development, disease and tissue repair in the nervous system. The TGF-ß family is composed of several members, including TGF-ßs, bone morphogenetic proteins (BMPs) and activins. In this review, we discuss recent findings that suggest TGF-ß function as important pleiotropic modulators of nociceptive processing both physiologically and under pathological painful conditions. The strategy of increasing TGF-ß signaling by deleting "BMP and activin membrane-bound inhibitor" (BAMBI), a TGF-ß pseudoreceptor, has demonstrated the inhibitory role of TGF-ß signaling pathways in normal nociception and in inflammatory and neuropathic pain models. In particular, strong evidence suggests that TGF-ß1 is a relevant mediator of nociception and has protective effects against the development of chronic neuropathic pain by inhibiting the neuroimmune responses of neurons and glia and promoting the expression of endogenous opioids within the spinal cord. In the peripheral nervous system, activins and BMPs function as target-derived differentiation factors that determine and maintain the phenotypic identity and circuit assembly of peptidergic nociceptors. In this context, activin is involved in the complex events of neuroinflammation that modulate the expression of pain during wound healing. These findings have provided new insights into the physiopathology of nociception. Moreover, specific members of the TGF-ß family and their signaling effectors and modulator molecules may be promising molecular targets for novel therapeutic agents for pain management.
Assuntos
Neuralgia/metabolismo , Neuralgia/fisiopatologia , Nociceptores/metabolismo , Nociceptores/patologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Modelos Animais de Doenças , Humanos , Inflamação/metabolismo , Inflamação/patologia , Inflamação/fisiopatologia , Neuralgia/patologia , Nociceptores/fisiologiaRESUMO
Transforming growth factors-beta (TGF-betas) signal through type I and type II serine-threonine kinase receptor complexes. During ligand binding, type II receptors recruit and phosphorylate type I receptors, triggering downstream signaling. BAMBI [bone morphogenetic protein (BMP) and activin membrane-bound inhibitor] is a transmembrane pseudoreceptor structurally similar to type I receptors but lacks the intracellular kinase domain. BAMBI modulates negatively pan-TGF-beta family signaling; therefore, it can be used as an instrument for unraveling the roles of these cytokines in the adult CNS. BAMBI is expressed in regions of the CNS involved in pain transmission and modulation. The lack of BAMBI in mutant mice resulted in increased levels of TGF-beta signaling activity, which was associated with attenuation of acute pain behaviors, regardless of the modality of the stimuli (thermal, mechanical, chemical/inflammatory). The nociceptive hyposensitivity exhibited by BAMBI(-/-) mice was reversed by the opioid antagonist naloxone. Moreover, in a model of chronic neuropathic pain, the allodynic responses of BAMBI(-/-) mice also appeared attenuated through a mechanism involving delta-opioid receptor signaling. Basal mRNA and protein levels of precursor proteins of the endogenous opioid peptides proopiomelanocortin (POMC) and proenkephalin (PENK) appeared increased in the spinal cords of BAMBI(-/-). Transcript levels of TGF-betas and their intracellular effectors correlated directly with genes encoding opioid peptides, whereas BAMBI correlated inversely. Furthermore, incubation of spinal cord explants with activin A or BMP-7 increased POMC and/or PENK mRNA levels. Our findings identify TGF-beta family members as modulators of acute and chronic pain perception through the transcriptional regulation of genes encoding the endogenous opioids.