RESUMO
Type 1 diabetes (T1D) is an autoimmune disease in which pathogenic lymphocytes target autoantigens expressed in pancreatic islets, leading to the destruction of insulin-producing ß-cells. Zinc transporter 8 (ZnT8) is a major autoantigen abundantly present on the ß-cell surface. This unique molecular target offers the potential to shield ß-cells against autoimmune attacks in T1D. Our previous work showed that a monoclonal antibody (mAb43) against cell-surface ZnT8 could home in on pancreatic islets and prevent autoantibodies from recognizing ß-cells. This study demonstrates that mAb43 binds to exocytotic sites on the ß-cell surface, masking the antigenic exposure of ZnT8 and insulin after glucose-stimulated insulin secretion. In vivo administration of mAb43 to NOD mice selectively increased the proportion of regulatory T cells in the islet, resulting in complete and sustained protection against T1D onset as well as reversal of new-onset diabetes. The mAb43-induced self-tolerance was reversible after treatment cessation, and no adverse effects were exhibited during long-term monitoring. Our findings suggest that mAb43 masking of the antigenic exposure of ß-cells suppresses the immunological cascade from B-cell antigen presentation to T cell-mediated ß-cell destruction, providing a novel islet-targeted and antigen-specific immunotherapy to prevent and reverse clinical T1D.
Assuntos
Diabetes Mellitus Tipo 1 , Ilhotas Pancreáticas , Camundongos , Animais , Diabetes Mellitus Tipo 1/metabolismo , Camundongos Endogâmicos NOD , Ilhotas Pancreáticas/metabolismo , Autoantígenos , InsulinaRESUMO
Defects in insulin processing and granule maturation are linked to pancreatic beta-cell failure during type 2 diabetes (T2D). Phosphatidylinositol transfer protein alpha (PITPNA) stimulates activity of phosphatidylinositol (PtdIns) 4-OH kinase to produce sufficient PtdIns-4-phosphate (PtdIns-4-P) in the trans-Golgi network to promote insulin granule maturation. PITPNA in beta-cells of T2D human subjects is markedly reduced suggesting its depletion accompanies beta-cell dysfunction. Conditional deletion of Pitpna in the beta-cells of Ins-Cre, Pitpnaflox/flox mice leads to hyperglycemia resulting from decreasing glucose-stimulated insulin secretion (GSIS) and reducing pancreatic beta-cell mass. Furthermore, PITPNA silencing in human islets confirms its role in PtdIns-4-P synthesis and leads to impaired insulin granule maturation and docking, GSIS, and proinsulin processing with evidence of ER stress. Restoration of PITPNA in islets of T2D human subjects reverses these beta-cell defects and identify PITPNA as a critical target linked to beta-cell failure in T2D.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Animais , Humanos , Camundongos , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Proinsulina/metabolismoRESUMO
BACKGROUNDPathophysiology of type 1 diabetes (T1D) is illustrated by pancreatic islet infiltration of inflammatory lymphocytes, including CD8+ T cells; however, the molecular factors mediating their recruitment remain unknown. We hypothesized that single-cell RNA-sequencing (scRNA-Seq) analysis of immune cell populations isolated from islets of NOD mice captured gene expression dynamics providing critical insight into autoimmune diabetes pathogenesis.METHODSPancreatic sections from human donors were investigated, including individuals with T1D, autoantibody-positive (aAb+) individuals, and individuals without diabetes who served as controls. IHC was performed to assess islet hormones and both novel and canonical immune cell markers that were identified from unbiased, state-of-the-art workflows after reanalyzing murine scRNA-Seq data sets.RESULTSComputational workflows identified cell adhesion molecule 1-mediated (Cadm1-mediated) homotypic binding among the most important intercellular interactions among all cell clusters, as well as Cadm1 enrichment in macrophages and DCs from pancreata of NOD mice. Immunostaining of human pancreata revealed an increased number of CADM1+glucagon+ cells adjacent to CD8+ T cells in sections from T1D and aAb+ donors compared with individuals without diabetes. Numbers of CADM1+CD68+ peri-islet myeloid cells adjacent to CD8+ T cells were also increased in pancreatic sections from both T1D and aAb+ donors compared with individuals without diabetes.CONCLUSIONIncreased detection of CADM1+ cells adjacent to CD8+ T cells in pancreatic sections of individuals with T1D and those who were aAb+ validated workflows and indicated CADM1-mediated intercellular contact may facilitate islet infiltration of cytotoxic T lymphocytes and serve as a potential therapeutic target for preventing T1D pathogenesis.FUNDINGThe Johns Hopkins All Children's Foundation Institutional Research Grant Program, the National Natural Science Foundation of China (grant 82071326), and the Deutsche Forschungsgemeinschaft (grants 431549029-SFB1451, EXC2030-390661388, and 411422114-GRK2550).
Assuntos
Molécula 1 de Adesão Celular , Diabetes Mellitus Tipo 1 , Ilhotas Pancreáticas , Animais , Molécula 1 de Adesão Celular/metabolismo , Comunicação Celular , Células Secretoras de Glucagon/metabolismo , Humanos , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos NODRESUMO
The histamine 3 receptor (H3R) is a presynaptic receptor, which modulates several neurotransmitters including histamine and various essential physiological processes, such as feeding, arousal, cognition, and pain. The H3R is considered as a drug target for the treatment of several central nervous system disorders. We have synthesized and identified a novel series of 4-aryl-6-methyl-5,6,7,8-tetrahydroquinazolinamines that act as selective H3R antagonists. Among all the synthesized compounds, in vitro and docking studies suggested that the 4-methoxy-phenyl-substituted tetrahydroquinazolinamine compound 4c has potent and selective H3R antagonist activity (IC50 < 0.04 µM). Compound 4c did not exhibit any activity on the hERG ion channel and pan-assay interference compounds liability. Pharmacokinetic studies showed that 4c crosses the blood brain barrier, and in vivo studies demonstrated that 4c induces anorexia and weight loss in obese, but not in lean mice. These data reveal the therapeutic potential of 4c as an anti-obesity candidate drug via antagonizing the H3R.
Assuntos
Fármacos Antiobesidade/uso terapêutico , Antagonistas dos Receptores Histamínicos H3/uso terapêutico , Obesidade/tratamento farmacológico , Quinazolinas/uso terapêutico , Receptores Histamínicos H3/metabolismo , Animais , Fármacos Antiobesidade/síntese química , Fármacos Antiobesidade/farmacocinética , Glicemia/metabolismo , Dieta Hiperlipídica , Células HEK293 , Antagonistas dos Receptores Histamínicos H3/síntese química , Antagonistas dos Receptores Histamínicos H3/farmacocinética , Humanos , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Proteínas Proto-Oncogênicas c-fos/metabolismo , Quinazolinas/síntese química , Quinazolinas/farmacocinética , Estereoisomerismo , Relação Estrutura-Atividade , Redução de Peso/efeitos dos fármacosRESUMO
Allosteric modulators of G-protein-coupled receptors have lately gained significant traction in drug discovery. Recent studies have shown that allosteric modulation of serotonin 2C receptor (5-HT2C) as a viable strategy for the treatment of various central nervous system (CNS) disorders. Considering the critical role of 5-HT2C in the modulation of appetite, a selective positive allosteric modulator (PAM) of 5-HT2C offers a new opportunity for anti-obesity therapeutic development. In this study, phenyl cyclopropyl-linked N-heterocycles were synthesized and evaluated at 5-HT2C for agonist and PAM activity. Our study shows that imidazole linked phenyl cyclopropyl methanones has PAM activity on both 5-HT2C and serotonin 2B receptor (5-HT2B). Interestingly, piperazine linked phenyl cyclopropyl methanones (58) was active as PAM of 5-HT2C (increased the Emax of 5-HT to 139%), and as negative allosteric modulator (NAM) of 5-HT2B (decreases EC50 of 5-HT 10 times without affecting Emax). Similar effect of compound 58 was observed with synthetic orthosteric agonist lorcaserin on 5-HT2B. Molecular docking study revealed that all active compounds were binding to the predicted allosteric site on 5-HT2C and shared a common interacting residues. Finally, compound 58 suppressed food intake in Sprague Dawley (SD) rats similar to lorcaserin after i.c.v. administration. Therefore, these results suggest that piperazine moiety is essential for dual activity (PAM & NAM) of compounds 58, and supports the hypothesis of 5-HT2C PAM for the treatment of obesity similar to the full agonist.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Piperazina/farmacologia , Receptor 5-HT2B de Serotonina/efeitos dos fármacos , Receptor 5-HT2C de Serotonina/efeitos dos fármacos , Animais , Ingestão de Alimentos/efeitos dos fármacos , Compostos Heterocíclicos/síntese química , Simulação de Acoplamento Molecular , Obesidade/tratamento farmacológico , Piperazina/química , Ratos , Ratos Sprague-DawleyRESUMO
The last two decades of research has established histamine (HA) as a neurotransmitter. Since H3R antagonists are known to modulate several neurotransmitters besides HA, H3R antagonists have shown potential for the treatment of different central nervous system disorders, including depression. However, molecular mechanisms underlying the beneficial effects of H3R antagonism in depression are not clear, yet. In the present study, we investigated the antidepressant potential of ciproxifan, a selective H3R antagonist, in chronic unpredictable stress (CUS) model of depression in C57BL/6 J mice. We observed that chronic treatment of CUS mice with ciproxifan (3 mg/kg i.p.; for three weeks) alleviates depression-like symptoms such as helplessness measured by forced swim and tail suspension test (FST and TST), anhedonia measured by sucrose preference test (SPT) and social deficit measured in social behavior test. Chronic ciproxifan treatment restored CUS induced BDNF expression in the prefrontal cortex (PFC) and hippocampus. We also observed that ciproxifan modulates CUS induced NUCB2/nesfatin-1 and CRH expression in the hypothalamus and plasma corticosterone. We also determined the direct effect of HA on BDNF expression in neurons by western blotting and immunocytochemistry, and found that HA significantly induced BDNF expression, which was blocked by the H4R selective antagonist, but not by other HA receptor selective antagonists. Furthermore, ciproxifan significantly modulated NMDA glutamate receptor subunits NR2B and NR2A. Thus, these results suggest that increased HA signaling in the brain produces antidepressant-like effects in mice and modulates BDNF expression and HPA-axis.
Assuntos
Depressão/tratamento farmacológico , Antagonistas dos Receptores Histamínicos/farmacologia , Receptores Histamínicos H3/metabolismo , Animais , Antidepressivos/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Corticosterona/sangue , Depressão/metabolismo , Transtorno Depressivo/metabolismo , Modelos Animais de Doenças , Hipocampo/metabolismo , Histamina/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Hipotálamo/metabolismo , Imidazóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sistema Hipófise-Suprarrenal/metabolismo , Estresse Psicológico/metabolismoRESUMO
GPR40 (Free fatty acid receptor 1) has emerged as an important therapeutic target for diabetes. Several studies have demonstrated the association of comorbid psychiatric conditions with decreased n-3 polyunsaturated fatty acids, which may act as an agonist for GPR40. In this study, we for the first time provide evidence of reduced GPR40 signaling in the hippocampus and cortex which may be a critical underlying mechanism mediating cognitive deficits in diabesity (diabetes and obesity together). Specifically, we showed decreased GPR40 and brain-derived neurotrophic factor (BDNF) expression in the brain regions of high-fat-diet-induced obese and db/db mice. Next, we demonstrated that chronic treatment with docosahexaenoic acid (DHA) or the synthetic GPR40 agonist, GW9508, significantly alleviates cognitive functions in mice, which correlates with increased BDNF expression in the hippocampus. This supports the hypothesis that DHA improves cognitive function in diabesity via GPR40 agonism. We also showed that DHA specifically activates GPR40 and modulates BDNF expression in primary cortical neurons mediated by the extracellular receptor kinase (ERK) and P38-mitogen-activated protein kinase (MAPK) pathways. Finally, the central nervous system (CNS)-specific blockade of GPR40 signaling abrogated the memory potentiating effects of DHA, and induction of BDNF expression in the hippocampus. Thus, we provided evidence that DHA stimulation of GPR40 mediate some of DHA's beneficial effects in metabolic syndrome and identify GPR40 as a viable therapeutic target for the treatment of CNS-related comorbidities associated with diabesity.
Assuntos
Aprendizagem por Associação/fisiologia , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Ácidos Docosa-Hexaenoicos/uso terapêutico , Transtornos da Memória/metabolismo , Obesidade/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Animais , Aprendizagem por Associação/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/agonistas , Células Cultivadas , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/etiologia , Diabetes Mellitus/metabolismo , Dieta Hiperlipídica/efeitos adversos , Ácidos Docosa-Hexaenoicos/farmacologia , Masculino , Transtornos da Memória/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/tratamento farmacológico , Obesidade/etiologia , Receptores Acoplados a Proteínas G/agonistasRESUMO
Four series of structurally related ß-lactams, 2,5-pyrrolidinediones, azaspirodecatrienediones (ASDT) and dihydropyrroloquinoxalinetriones (DPQT) were synthesized by utilizing post-Ugi modifications in one-pot, and their activity towards human histamine-3 receptor (H3R) was evaluated. Out of 94 compounds, screened against histamine-3 receptor (H3R), 21 compounds showed high H3R selective agonist property with EC50 values ranging from 187â¯nM to 0.1â¯nM, whereas none of the compound was found to have the affinity towards other receptors of histamine family such as histamine H1, H2, and H4 receptor. All active compounds have no assay interference activity as determined by in-silico analysis and receptor independent luciferase assay and cell cytotoxicity assay. Given the important role of H3R in hypophagia, we also evaluated the in vivo effect of the representative compound 6k on the cumulative food intake in diet induce obese C57BL6/J mice. Interestingly, we observed that single dose administration (20â¯mg/kg, intraperitoneal injection) of 6k significantly suppressed cumulative food intake, while no significant effect was observed at 10â¯mg/kg. These results suggest that ß-lactams, 2,5-pyrrolidinediones, azaspirodecatrienediones (ASDT) and dihydropyrroloquinoxalinetriones (DPQT) could be useful for the development of anti-obesity candidate drugs.
Assuntos
Fármacos Antiobesidade/farmacologia , Obesidade/tratamento farmacológico , Pirrolidinonas/farmacologia , Receptores Histamínicos H3/metabolismo , beta-Lactamas/farmacologia , Animais , Fármacos Antiobesidade/administração & dosagem , Fármacos Antiobesidade/química , Sobrevivência Celular , Dieta Hiperlipídica/efeitos adversos , Relação Dose-Resposta a Droga , Ingestão de Alimentos/efeitos dos fármacos , Células HEK293 , Humanos , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Obesidade/induzido quimicamente , Obesidade/metabolismo , Pirrolidinonas/administração & dosagem , Pirrolidinonas/química , Relação Estrutura-Atividade , beta-Lactamas/administração & dosagem , beta-Lactamas/químicaRESUMO
G protein-coupled receptors (GPCRs) are modulators of almost every physiological process, and therefore, are most favorite therapeutic target for wide spectrum of diseases. Ideally, high-throughput functional assays should be implemented that allow the screening of large compound libraries in cost-effective manner to identify agonist, antagonist, and allosteric modulators in the same assay. Taking advantage of the increased understanding of the GPCR structure and signaling, several commercially available functional assays based on fluorescence or chemiluminescence detection are being used in both academia and industry. In this chapter, we provide step-by-step method and guidelines to perform cAMP measurement using GloSensor assay. Finally, we have also discussed the analysis and interpretation of results obtained using this assay by providing several examples of Gs- and Gi-coupled GPCRs.
Assuntos
Bioensaio/métodos , Técnicas Biossensoriais/métodos , AMP Cíclico/análise , Descoberta de Drogas/métodos , Receptores Acoplados a Proteínas G/metabolismo , Bioensaio/economia , Bioensaio/instrumentação , Técnicas Biossensoriais/economia , Técnicas Biossensoriais/instrumentação , Análise Custo-Benefício , Proteínas Quinases Dependentes de AMP Cíclico/química , Descoberta de Drogas/economia , Descoberta de Drogas/instrumentação , Imunofluorescência/economia , Imunofluorescência/instrumentação , Imunofluorescência/métodos , Células HEK293 , Humanos , Luciferases de Vaga-Lume/química , Medições Luminescentes/economia , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Transdução de Sinais/efeitos dos fármacosRESUMO
The efficiency of a fluorescence sensing device based on metal-enhanced fluorescence (MEF) is dependent on the optimization of interaction between the fluorophore and the metal nanoparticle (NP). Herewith, ultrasensitive and selective turn-on sensing of Au3+ is achieved by using a suitable combination of fluorophore and metal NP system through sequential MEF effect. Dansyl hydrazide-tagged Ag NPs in the polyacryloyl hydrazide cavity are utilized to sense the picomolar concentration of Au3+ in aqueous media. We demonstrated that the selective Au3+ sensing is due to the selective deposition of Au on the Ag NP surface over the 16 other metal ions studied. The sensitivity is assigned to the strong overlapping of the emission band of the fluorophore with the surface plasmon band of the Au and improvement of fluorescence signal through successive MEF by Ag and Au colloids. The sensing is associated with a fivefold increase in fluorescence intensity and appearance of violet color of the solution. These luminescent Ag-Au bimetallic NPs may be utilized to trace cancer cells in biological systems and for cell imaging applications.
RESUMO
Relocation is an important event in the lives of several social insects whereby all colony members have to be transferred to a new nest when conditions in the old nest become unfavorable. In the current study, network tools were used to examine the organization of this goal-oriented task in the Indian queenless ant Diacamma indicum which relocate their colonies by means of tandem running. Individual ants were used as nodes and tandem runs as directed edges to construct unweighted networks. Network parameters were characterized in control relocations (CRs) and in relocations where the node with the highest outdegree, that is, the Maximum tandem leader (Max TL) was experimentally removed. These were then compared to 1) randomized networks, 2) simulated networks in which Max TL was removed, and 3) simulated networks with removal of a random leader. Not only was there complete recovery of the task, but the manner in which it was organized when Max TL was removed was comparable to CRs. The results obtained from our empirical study were significantly different from the results predicted by simulations of leader removal. At an individual level, the Max TL had a significantly higher outdegree than expected by chance alone and in her absence the substitute Max TL did comparable work. In addition, the position of the Max TL in the pathway of information flow was conserved in control and experimentally manipulated conditions. Understanding the organization of this critical event as more than the sum of individual interactions using network parameters allows us to appreciate the dynamic response of groups to perturbations.
RESUMO
G protein-coupled receptors (GPCRs) act as a relay center through which extracellular signals, in the form of neurotransmitters or therapeutics, are converted into an intracellular response, which ultimately shapes the overall response at the tissue and behavioral level. Remarkably in similar ways, epigenetic mechanisms also modulate the expression pattern of a large number of genes in response to the dynamic environment inside and outside of the body, and consequently overall response. Emerging evidences from the pharmacogenomics and preclinical studies clearly suggest that these two distinct mechanisms criss-cross each other in several neurological disorders. At one hand such cross-talks between two distinct mechanisms make disease etiology more challenging to understand, while on the other hand if dealt appropriately, such situations might provide an opportunity to find novel druggable target and strategy for the treatment of complex diseases. In this review article, we have summarized and highlighted the main findings that tie epigenetic mechanisms to GPCR mediated signaling in the pathophysiology of central nervous system (CNS) disorders, including depression, addiction and pain.
Assuntos
Epigênese Genética , Transtornos Mentais/genética , Transtornos Mentais/patologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Animais , Humanos , Transtornos Mentais/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are widely known to modulate almost all physiological functions and have been demonstrated over the time as therapeutic targets for wide gamut of diseases. The design and implementation of high-throughput GPCR-based assays that permit the efficient screening of large compound libraries to discover novel drug candidates are essential for a successful drug discovery endeavor. Usually, GPCR-based functional assays depend primarily on the measurement of G protein-mediated second messenger generation. However, with advent of advanced molecular biology tools and increased understanding of GPCR signal transduction, many G protein-independent pathways such as ß-arrestin translocation are being utilized to detect the activity of GPCRs. These assays provide additional information on functional selectivity (also known as biased agonism) of compounds that could be harnessed to develop pathway-selective drug candidates to reduce the adverse effects associated with given GPCR target. In this chapter, we describe the basic principle, detailed methodologies and assay setup, result analysis and data interpretations of the ß-arrestin2 Tango assay, and its comparison with cell-based G protein-dependent GPCR assays, which could be employed in a simple academic setup to facilitate GPCR-based drug discovery.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestina 2/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293 , Humanos , Concentração Inibidora 50 , Ligantes , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
Division of labor is a central feature in social insects, wherein, simple individuals come together in groups to perform tasks that could be quite complex. It is generally believed that individuals who perform a specific task are themselves simple, interchangeable units. However, the variances in the performances of these individual insects need to be explored in greater detail. In this study, individual specialization in the context of colony relocation was examined in the Indian ponerine ant Diacamma indicum. One ant termed the maximum tandem leader (Max TL) was found to have a key role. Max TL performed 24% of the adult relocation in the colony and recruited more tandem leaders than other leaders thereby contributing to the organization of the relocation. The Max TL's role in the relocation process was further examined by comparing control relocations with experiments in which the Max TL was removed during the relocation process. Even though all the colonies relocated successfully, the relocation dynamics was significantly altered in the absence of the Max TL. We find that a single individual, the Max TL, takes up roles of a performer, organizer and catalyst during the colony relocation process, which challenges the norm that all workers are equal.