RESUMO
The Duffy antigen receptor is a seven-transmembrane (7TM) protein expressed primarily at the surface of red blood cells and displays strikingly promiscuous binding to multiple inflammatory and homeostatic chemokines. It serves as the basis of the Duffy blood group system in humans and also acts as the primary attachment site for malarial parasite Plasmodium vivax and pore-forming toxins secreted by Staphylococcus aureus. Here, we comprehensively profile transducer coupling of this receptor, discover potential non-canonical signaling pathways, and determine the cryoelectron microscopy (cryo-EM) structure in complex with the chemokine CCL7. The structure reveals a distinct binding mode of chemokines, as reflected by relatively superficial binding and a partially formed orthosteric binding pocket. We also observe a dramatic shortening of TM5 and 6 on the intracellular side, which precludes the formation of the docking site for canonical signal transducers, thereby providing a possible explanation for the distinct pharmacological and functional phenotype of this receptor.
RESUMO
Chemokine receptors constitute an important subfamily of G protein-coupled receptors (GPCRs), and they are critically involved in a broad range of immune response mechanisms. Ligand promiscuity among these receptors makes them an interesting target to explore multiple aspects of biased agonism. Here, we comprehensively characterize two chemokine receptors namely, CXCR4 and CXCR7, in terms of their transducer-coupling and downstream signaling upon their stimulation by a common chemokine agonist, CXCL12, and a small molecule agonist, VUF11207. We observe that CXCR7 lacks G-protein-coupling while maintaining robust ßarr recruitment with a major contribution of GRK5/6. On the other hand, CXCR4 displays robust G-protein activation as expected but exhibits significantly reduced ßarr-coupling compared to CXCR7. These two receptors induce distinct ßarr conformations even when activated by the same agonist, and CXCR7, unlike CXCR4, fails to activate ERK1/2 MAP kinase. We also identify a key contribution of a single phosphorylation site in CXCR7 for ßarr recruitment and endosomal localization. Our study provides molecular insights into intrinsic-bias encoded in the CXCR4-CXCR7 system with broad implications for drug discovery.
Assuntos
Receptores CXCR , Receptores CXCR/genética , Receptores CXCR/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , Proteínas de Ligação ao GTP , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Quimiocina CXCL12/metabolismoRESUMO
Despite ongoing advancements in research, the inability of therapeutics to cross the blood-brain barrier (BBB) makes the treatment of neurological disorders (NDs) a challenging task, offering only partial symptomatic relief. Various adverse effects associated with existing approaches are another significant barrier that prompts the usage of structurally diverse phytochemicals as preventive/therapeutic lead against NDs in preclinical and clinical settings. Despite numerous beneficial properties, phytochemicals suffer from poor pharmacokinetic profile which limits their pharmacological activity and necessitates the utility of nanotechnology for efficient drug delivery. Nanocarriers have been shown to be proficient carriers that can enhance drug delivery, bioavailability, biocompatibility, and stability of phytochemicals. We, thus, conducted a meticulous literature survey using several electronic databases to gather relevant studies in order to provide a comprehensive summary about the use of nanocarriers in delivering phytochemicals as a treatment approach for NDs. Additionally, the review highlights the mechanisms of drug transport of nanocarriers across the BBB and explores their potential future applications in this emerging field.
Assuntos
Portadores de Fármacos , Nanopartículas , Portadores de Fármacos/química , Nanopartículas/química , Encéfalo , Barreira Hematoencefálica , Sistemas de Liberação de Medicamentos , Compostos Fitoquímicos/uso terapêutico , Compostos Fitoquímicos/farmacologiaRESUMO
The emergence and rapid evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused a global crisis that required a detailed characterization of the dynamics of mutational pattern of the viral genome for comprehending its epidemiology, pathogenesis and containment. We investigated the molecular evolution of the SASR-CoV-2 genome during the first, second and third waves of COVID-19 in Uttar Pradesh, India. Nanopore sequencing of the SARS-CoV-2 genome was undertaken in 544 confirmed cases of COVID-19, which included vaccinated and unvaccinated individuals. In the first wave (unvaccinated population), the 20A clade (56.32%) was superior that was replaced by 21A Delta in the second wave, which was more often seen in vaccinated individuals in comparison to unvaccinated (75.84% versus 16.17%, respectively). Subsequently, 21A delta got outcompeted by Omicron (71.8%), especially the 21L variant, in the third wave. We noticed that Q677H appeared in 20A Alpha and stayed up to Delta, D614G appeared in 20A Alpha and stayed in Delta and Omicron variants (got fixed), and several other mutations appeared in Delta and stayed in Omicron. A cross-sectional analysis of the vaccinated and unvaccinated individuals during the second wave revealed signature combinations of E156G, F157Del, L452R, T478K, D614G mutations in the Spike protein that might have facilitated vaccination breach in India. Interestingly, some of these mutation combinations were carried forward from Delta to Omicron. In silico protein docking showed that Omicron had a higher binding affinity with the host ACE2 receptor, resulting in enhanced infectivity of Omicron over the Delta variant. This work has identified the combinations of key mutations causing vaccination breach in India and provided insights into the change of [virus's] binding affinity with evolution, resulting in more virulence in Delta and more infectivity in Omicron variants of SARS-CoV-2. Our findings will help in understanding the COVID-19 disease biology and guide further surveillance of the SARS-CoV-2 genome to facilitate the development of vaccines with better efficacies.
RESUMO
Pain sensation is a normal physiological response to alert and prevent further tissue damage. It involves the perception of external stimuli by somatosensory neurons, then transmission of the message to various other types of neurons present in the spinal cord and brain to generate an appropriate response. Currently available analgesics exhibit very modest efficacy, and that too in only a subset of patients with chronic pain conditions, particularly neuropathic pain. The G protein-coupled receptors (GPCRs) are expressed on presynaptic, postsynaptic terminals, and soma of somatosensory neurons, which binds to various types of ligands to modulate neuronal activity and thus pain sensation in both directions. Fundamentally, neuropathic pain arises due to aberrant neuronal plasticity, which includes the sensitization of peripheral primary afferents (dorsal root ganglia and trigeminal ganglia) and the sensitization of central nociceptive neurons in the spinal cord or trigeminal nucleus or brain stem and cortex. Owing to the expression profiles of GPCRs in somatosensory neurons and other neuroanatomical regions involved in pain processing and transmission, this article shall focus only on four families of GPCRs: 1- Opioid receptors, 2-Cannabinoid receptors, 3-Adenosine receptors, and 4-Chemokine receptors.
Assuntos
Neuralgia , Humanos , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Gânglios Espinais/metabolismo , Medula Espinal/metabolismo , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Efforts have been devoted for the discovery and development of positive allosteric modulators (PAMs) of 5-HT2CR because of their potential advantages over the orthosteric agonist like Lorcaserin that was withdrawn from the market. On the other hand, pursuing a positive ago-allosteric modulator (PAAM) is considered as beneficial particularly when an agonist is not capable of affecting the potency of the endogenous agonist sufficiently. In search of a suitable PAAM of 5-HT2CR we adopted an in silico based approach that indicated the potential of the 3-(1-hydroxycycloalkyl) substituted isoquinolin-1-one derivatives against the 5-HT2CR as majority of these molecules interacted with the site other than that of Lorcaserin with superior docking scores. These compounds along with the regioisomeric 3-methyleneisoindolin-1-one derivatives were prepared via the Cu(OAc)2 catalyzed coupling of 2-iodobenzamide with 1-ethynylcycloalkanol under ultrasound irradiation. According to the in vitro studies, most of these compounds were not only found to be potent and selective agonists but also emerged as PAAM of 5-HT2CR whereas Lorcaserin did not show PAAM activities. According to the SAR study the isoquinolin-1(2H)-ones appeared as better PAAM than isoindolin-1-ones whereas the presence of hydroxyl group appeared to be crucial for the activity. With the potent PAAM activity for 5-HT2CR (EC50 = 1 nM) and 107 and 86-fold selectivity towards 5-HT2C over 5-HT2A and 5-HT2B the compound 4i was identified as a hit molecule. The compound showed good stability in male BALB/c mice brain homogenate (â¼85 % remaining after 2 h), moderate stability in the presence of rat liver microsomes (42 % remaining after 1 h) and acceptable PK properties with fast reaching in the brain maintaining â¼ 1:1 brain/plasma concentration ratio. The compound at a dose of 50 mg/kg exhibited decreased trend in the food intake starting from day 3 in S.D. rats, which reached significant by 5th day, and the effect was comparable to Lorcaserin (10 mg/kg) on day 5. Thus, being the first example of PAAM of 5-HT2CR the compound 4i is of further medicinal interest.
Assuntos
Indóis , Isoquinolinas , Agonistas do Receptor 5-HT2 de Serotonina , Animais , Masculino , Camundongos , Ratos , Encéfalo , Agonistas do Receptor 5-HT2 de Serotonina/síntese química , Agonistas do Receptor 5-HT2 de Serotonina/química , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Camundongos Endogâmicos BALB C , Isoquinolinas/síntese química , Isoquinolinas/química , Isoquinolinas/farmacologia , Indóis/síntese química , Indóis/química , Indóis/farmacologiaRESUMO
The synthesis of 5-formyl-6-aryl-6H-indolo[3,2,1-de][1,5] naphthyridine-2-carboxylates by reaction between 1-formyl-9H-ß-carbolines and cinnamaldehydes in the presence of pyrrolidine in water with microwave irradiation is described. Pharmacophoric modification of the formyl group offered several new fused ß-carboline derivatives, which were investigated for their κ-opioid receptor (KOR) agonistic activity. Two compounds 4 a and 4 c produced appreciable agonist activity on KOR with EC50 values of 46±19 and 134±9â nM, respectively. Moreover, compound-induced KOR signaling studies suggested both compounds to be extremely G-protein-biased agonists. The analgesic effect of 4 a was validated by the increase in tail flick latency in mice in a time-dependent manner, which was completely blocked by the KOR-selective antagonist norBNI. Moreover, unlike U50488, an unbiased full KOR agonist, 4 a did not induce sedation. The docking of 4 a with the human KOR was studied to rationalize the result.
Assuntos
Analgésicos/farmacologia , Carbolinas/farmacologia , Antagonistas de Entorpecentes/farmacologia , Dor/tratamento farmacológico , Receptores Opioides kappa/agonistas , Analgésicos/síntese química , Analgésicos/química , Animais , Carbolinas/síntese química , Carbolinas/química , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Antagonistas de Entorpecentes/síntese química , Antagonistas de Entorpecentes/químicaRESUMO
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
In this study, we presented rational designing and synthesis of coumarin-dihydroquinazolinone conjugates to evaluate their agonist activity at GPR109a receptor. Among the synthesized small molecule library, compound 10c displayed robust agonist action at GPR109a with EC50â¯<â¯11â¯nM. Homology model of human GPR109a protein was generated to realize the binding interaction of the active molecule with the active site of GPR109a. Further, the efficacy of active compound 10c was supported by in-vivo experiments which showed reduced body weight in diet induced obese mice model. Interestingly, compound 10c reduced leptin in blood plasma and total serum cholesterol. These results suggest that the coumarin-dihydroquinazolinone conjugate is a suitable scaffold to further expand the chemical diversity and make them potential niacin receptor 1 agonist.
Assuntos
Fármacos Antiobesidade/farmacologia , Cumarínicos/farmacologia , Descoberta de Drogas , Obesidade/tratamento farmacológico , Quinazolinonas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Animais , Fármacos Antiobesidade/síntese química , Fármacos Antiobesidade/química , Cumarínicos/síntese química , Cumarínicos/química , Dieta Hiperlipídica/efeitos adversos , Relação Dose-Resposta a Droga , Teste de Tolerância a Glucose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Obesidade/induzido quimicamente , Quinazolinonas/síntese química , Quinazolinonas/química , Receptores Nicotínicos , Relação Estrutura-AtividadeRESUMO
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
Beta-arrestins (ßarrs) critically mediate desensitization, endocytosis and signalling of G protein-coupled receptors (GPCRs), and they scaffold a large number of interaction partners. However, allosteric modulation of their scaffolding abilities and direct targeting of their interaction interfaces to modulate GPCR functions selectively have not been fully explored yet. Here we identified a series of synthetic antibody fragments (Fabs) against different conformations of ßarrs from phage display libraries. Several of these Fabs allosterically and selectively modulated the interaction of ßarrs with clathrin and ERK MAP kinase. Interestingly, one of these Fabs selectively disrupted ßarr-clathrin interaction, and when expressed as an intrabody, it robustly inhibited agonist-induced endocytosis of a broad set of GPCRs without affecting ERK MAP kinase activation. Our data therefore demonstrate the feasibility of selectively targeting ßarr interactions using intrabodies and provide a novel framework for fine-tuning GPCR functions with potential therapeutic implications.
Assuntos
Endocitose/efeitos dos fármacos , Fragmentos Fab das Imunoglobulinas , Biblioteca de Peptídeos , Receptores Acoplados a Proteínas G/metabolismo , Anticorpos de Cadeia Única , Clatrina/metabolismo , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/farmacologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/farmacologiaRESUMO
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
To obtain selective and potent opioid receptor ligands, we synthesized dehydro derivatives of alvimopan and found compound (28f), a selective but modest affinity MOR antagonist weaker than alvimopan (1). We replaced the arylpiperidine unit by an arylpiperazine to obtain the 1-(α-carboxycinnamyl)-4-arylpiperazines like 13h, which to our surprise had no MOR or DOR activity but was a KOR agonist with moderate affinity. In contrast, literature examples of arylpiperazines 4 and 5 were reported to be pan opioid receptor antagonists, while 6 was a MOR agonist. Two compounds (13l and 11b) showed analgesic response in tail flick test which was blocked by pretreatment with norbinaltorphimine (norBNI). Among 10 1-(α-carboxycinnamyl)-4-arylpiperidines, compound 28g and five others were specific MOR antagonists. Interestingly, compound 26b of this series was found to be more potent than naloxone but weaker than 1. Docking studies have explained differential activities of the above piperazines and piperidines.
Assuntos
Cinamatos/farmacologia , Piperazinas/farmacologia , Piperidinas/farmacologia , Receptores Opioides kappa/agonistas , Receptores Opioides mu/antagonistas & inibidores , Animais , Barreira Hematoencefálica/metabolismo , Cinamatos/síntese química , Células HEK293 , Humanos , Ligantes , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Naloxona/farmacologia , Antagonistas de Entorpecentes/síntese química , Antagonistas de Entorpecentes/farmacologia , Piperazinas/síntese química , Piperidinas/síntese químicaRESUMO
G protein-coupled receptors (GPCRs) exhibit highly conserved activation and signaling mechanisms by which agonist stimulation leads to coupling of heterotrimeric G proteins and generation of second messenger response. This is followed by receptor phosphorylation, primarily in the carboxyl terminus but also in the cytoplasmic loops, and subsequent binding of arrestins. GPCRs typically recruit arrestins through two different sets of interactions, one involving phosphorylated receptor tail and the other mediated by the receptor core. The engagement of both set of interactions (tail and core) is generally believed to be necessary for arrestin-dependent functional outcomes such as receptor desensitization, endocytosis, and G protein-independent signaling. Here we demonstrate that a vasopressin receptor (V2R) mutant with truncated third intracellular loop (V2RΔICL3) can interact with ß-arrestin 1 (ßarr1) only through the phosphorylated tail without engaging the core interaction. Of interest, such a partially engaged V2RΔICL3-ßarr1 complex can efficiently interact with clathrin terminal domain and ERK2 MAPK in vitro. Furthermore, this core interaction-deficient V2R mutant exhibits efficient endocytosis and ERK activation upon agonist stimulation. Our data suggest that core interaction with ßarr is dispensable for V2R endocytosis and ERK activation and therefore provide novel insights into refining the current understanding of functional requirements in biphasic GPCR-ßarr interaction.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Receptores de Vasopressinas/metabolismo , beta-Arrestina 1/metabolismo , Sequência de Aminoácidos , Arrestinas/metabolismo , Clatrina/metabolismo , Endocitose , Ativação Enzimática , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Fosforilação , Ligação Proteica , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Postnatal and adult neurogenesis are region- and modality-specific, but the significance of developmentally distinct neuronal populations remains unclear. We demonstrate that chemogenetic inactivation of a subset of forebrain and olfactory neurons generated at birth disrupts responses to an aversive odor. In contrast, novel appetitive odor learning is sensitive to inactivation of adult-born neurons, revealing that developmentally defined sets of neurons may differentially participate in hedonic aspects of sensory learning.
Assuntos
Comportamento Apetitivo/fisiologia , Aprendizagem da Esquiva/fisiologia , Comportamento Animal/fisiologia , Condicionamento Clássico/fisiologia , Olfato/fisiologia , Animais , Masculino , Camundongos , Neurogênese/fisiologia , Neurônios/fisiologia , Córtex Olfatório/fisiologiaRESUMO
Psychotomimetic and prodepressive effect by kappa opioid receptor (KOR) activation in rodents and human is widely known. Significantly, recent clinical investigations demonstrated the salutary effects of KOR antagonists in patients with treatment resistant depression, indicating essential role of KOR signaling in refractory depression. This study was undertaken to reveal the molecular determinant of KOR mediated depression and antidepressant response of KOR antagonist. We observed that chronic KOR activation by U50488, a selective KOR agonist, significantly increased depression like symptoms (behavioral despair, anhedonia and sociability) in C57BL/6J mice, which were blocked by KOR antagonist norBNI and antidepressant imipramine, but not by fluoxetine or citalopram. Further, chronic KOR activation increased phosphorylation of NR2B subunit of NMDA at tyrosine 1472 (pNR2B NMDA) in the hippocampus, but not in the cortex. Similar to behavioral effects norBNI and imipramine, but not SSRIs, blocked NR2B phosphorylation. Moreover, KOR induced depression like behaviors were reversed by NR2B selective inhibitor Ro 25-6981. Mechanistic studies in primary cultured neurons and brain tissues using genetic and pharmacological approaches revealed that stimulation of KOR modulates several molecular correlates of depression. Thus, these findings elucidate molecular mechanism of KOR signaling in treatment resistant depression like behaviors in mice.