RESUMO
Several liganded nuclear receptors have alternative ligands acting in a tissue-specific fashion and playing important biological roles. We present evidence that 3,5-diiodothyronine (T(2)), a naturally occurring iodothyronine that results from T(3) outer-ring deiodination, is an alternative ligand for thyroid hormone receptor ß1 (TRß1). In tilapia, 2 TRß isoforms differing by 9 amino acids in the ligand-binding domain were cloned. Binding and transactivation studies showed that T(2) activates the human and the long tilapia TRß1 isoform, but not the short one. A chimeric human TRß1 (hTRß1) that contained the 9-amino-acid insert showed no response to T(2), suggesting that the conformation of the hTRß1 naturally allows T(2) binding and that other regions of the receptor are implicated in TR activation by T(2). Indeed, further analysis showed that the N terminus is essential for T(2)-mediated transactivation but not for that by T(3) in the long and hTRß1, suggesting a functional interaction between the N-terminal domain and the insertion in the ligand-binding domain. To establish the functional relevance of T(2)-mediated TRß1 binding and activation, mRNA expression and its regulation by T(2) and T(3) was evaluated for both isoforms. Our data show that long TRß1expression is 10(6)-fold higher than that of the short isoform, and T(3) and T(2) differentially regulate the expression of these 2 TRß1 isoforms in vivo. Taken together, our results prompted a reevaluation of the role and mechanism of action of thyroid hormone metabolites previously believed to be inactive. More generally, we propose that classical liganded receptors are only partially locked to very specific ligands and that alternative ligands may play a role in the tissue-specific action of receptors.
Assuntos
Di-Iodotironinas/metabolismo , Proteínas de Peixes/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Receptores beta dos Hormônios Tireóideos/metabolismo , Animais , Sítios de Ligação/genética , Ligação Competitiva , Linhagem Celular , Linhagem Celular Tumoral , Di-Iodotironinas/farmacologia , Relação Dose-Resposta a Droga , Proteínas de Peixes/agonistas , Proteínas de Peixes/genética , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Cinética , Ligantes , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores beta dos Hormônios Tireóideos/agonistas , Receptores beta dos Hormônios Tireóideos/genética , Tilápia , Ativação Transcricional/efeitos dos fármacos , Transfecção , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologiaRESUMO
OBJECTIVES: Semagacestat, is a γ-secretase inhibitor, which belongs to a class of drugs that are being developed as therapeutic agents for Alzheimer's disease (AD). This study aims to evaluate another potential effect of semagacestat, namely its ability to stimulate the growth hormone secretagogue receptor (GHS-R1a), which may also contribute to its therapeutic efficacy. METHODS: The GHS-R1a-activating potential of semagacestat and its synthetic precursor was assessed in an in vitro calcium mobilization assay in cells expressing the GHS-R1a receptor and compared with that of the endogenous peptide GHS-R1a agonist, acyl-ghrelin, as well as the non-peptidyl synthetic GHS-R1a agonist, MK0677. In addition, semagacestat-mediated cellular trafficking of the GHS-R1a receptor, expressed as an enhanced green fluorescent protein tagged fusion protein, was analysed. KEY FINDINGS: Semagacestat and its precursor were shown to activate the GHS-R1a receptor, as demonstrated by an increased GHS-R1a-mediated intracellular calcium influx. Moreover, a synergistic GHS-R1a receptor activation was shown following a combined exposure to ghrelin and semagacestat. In addition, GHS-R1a receptor internalization was observed upon exposure to semagacestat and its precursor. CONCLUSION: These data suggest a novel molecular mechanism of action for semagacestat via modest GHS-R1a receptor activation. Studies focusing on the relative functional consequence of such effects in vivo are now warranted.
Assuntos
Alanina/análogos & derivados , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Azepinas/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas do Tecido Nervoso/agonistas , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Receptores de Grelina/agonistas , Acilação , Alanina/síntese química , Alanina/metabolismo , Alanina/farmacologia , Azepinas/síntese química , Azepinas/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Grelina/análogos & derivados , Grelina/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Indóis/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/metabolismo , Nootrópicos/síntese química , Nootrópicos/metabolismo , Nootrópicos/farmacologia , Pró-Fármacos/síntese química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Transporte Proteico/efeitos dos fármacos , Receptores de Grelina/genética , Receptores de Grelina/metabolismo , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/metabolismo , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) receptors facilitate synaptic plasticity and can improve various forms of learning and memory. These modulators show promise as therapeutic agents for the treatment of neurological disorders such as schizophrenia, ADHD, and mental depression. Three classes of positive modulator, the benzamides, the thiadiazides, and the biarylsulfonamides differentially occupy a solvent accessible binding pocket at the interface between the two subunits that form the AMPA receptor ligand-binding pocket. Here, we describe the electrophysiological properties of a new chemotype derived from a structure-based drug design strategy (SBDD), which makes similar receptor interactions compared to previously reported classes of modulator. This pyrazole amide derivative, JAMI1001A, with a promising developability profile, efficaciously modulates AMPA receptor deactivation and desensitization of both flip and flop receptor isoforms. This article is part of a Special Issue entitled 'Cognitive Enhancers'.
Assuntos
Acetamidas/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Nootrópicos/farmacologia , Pirazóis/farmacologia , Receptores de AMPA/agonistas , Tiofenos/farmacologia , Acetamidas/química , Acetamidas/metabolismo , Sítio Alostérico , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacologia , Química Farmacêutica/métodos , Bases de Dados de Proteínas , Desenho de Fármacos , Agonistas de Aminoácidos Excitatórios/química , Agonistas de Aminoácidos Excitatórios/metabolismo , Células HEK293 , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Cinética , Ligantes , Nootrópicos/química , Nootrópicos/metabolismo , Conformação Proteica , Subunidades Proteicas/agonistas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Pirazóis/química , Pirazóis/metabolismo , Receptores de AMPA/química , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacologia , Tiofenos/química , Tiofenos/metabolismoRESUMO
AIM: To establish a drug screening model of CB(2) agonist in vitro based on signal regulation pathway. METHODS: Plasmid pIRES(2)-EGFP-CB(2), pGL(4), 29[luc2P/CRE/Hygro] and PRL-TK were co-transfected into CHO cells in 96 wells plate, to screen agonists of CB(2) receptor by detecting the expressing levels of dual luciferase activity. The concentration and acting time of the agonist were optimized and the stability of the model were investigated. RESULTS: The largest relative induction activity was obtained after 8h drug administration. Establishment of a high throughput screening model for CB(2) receptor agonist. The Z' factor is 0.75 demonstrating its perfect stability. CONCLUSION: Successfully establish a drug screening model of CB(2) agonist, which provided a basis for searching valid material from traditional Chinese medicine.
Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Ensaios de Triagem em Larga Escala , Receptor CB2 de Canabinoide/agonistas , Animais , Células CHO , Cricetinae , Relação Dose-Resposta a Droga , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismo , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos TestesRESUMO
To avoid high systemic doses, strategies involving antigen-specific delivery of cytokine via linked antibodies or antibody fragments have been used. Targeting cancer-associated peptides presented by major histocompatibility complex (MHC) molecules (pepMHC) increases the number of potential target antigens and takes advantage of cross-presentation on tumor stroma and in draining lymph nodes. Here, we use a soluble, high-affinity single-chain T cell receptor Vα-Vß (scTv), to deliver cytokines to intracellular tumor-associated antigens presented as pepMHC. As typical wild-type T cell receptors (TCRs) exhibit low affinity (K(d) = 1-100 µM or more), we used an engineered TCR, m33, that binds its antigenic peptide SIYRYYGL (SIY) bound to the murine class I major histocompatability complex protein H2-K(b) (SIY/K(b) ) with nanomolar affinity (K(d) = 30 nM). We generated constructs consisting of m33 scTv fused to murine interleukin 2 (IL-2), interleukin 15 (IL-15), or IL-15/IL-15Rα (IL-15 linked to IL-15Rα sushi domain, called "superfusion"). The fusions were purified with good yields and bound specifically to SIY/K(b) with high affinity. Proper cytokine folding and binding were confirmed, and the fusions were capable of stimulating proliferation of cytokine-dependent cells, both when added directly and when presented in trans, bound to cells with the target pepMHC. The m33 superfusion was particularly potent and stable and represents a promising design for targeted antitumor immunomodulation.
Assuntos
Subunidade alfa de Receptor de Interleucina-15/química , Interleucina-15/química , Engenharia de Proteínas/métodos , Receptores de Antígenos de Linfócitos T/química , Proteínas Recombinantes de Fusão/química , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sistemas de Liberação de Medicamentos , Humanos , Interleucina-15/agonistas , Interleucina-15/genética , Interleucina-15/metabolismo , Subunidade alfa de Receptor de Interleucina-15/agonistas , Subunidade alfa de Receptor de Interleucina-15/genética , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Camundongos , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Ligação Proteica , Ratos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Drug development for nicotinic acetylcholine receptors (nAChR) is challenged by subtype diversity arising from variations in subunit composition. On-target activity for neuronal heteromeric receptors is typically associated with CNS receptors that contain α4 and other subunits, while off-target activity could be associated with ganglionic-type receptors containing α3ß4 binding sites and other subunits, including ß4, ß2, α5, or α3 as a structural subunit in the pentamer. Additional interest in α3 ß4 α5-containing receptors arises from genome-wide association studies linking these genes, and a single nucleotide polymorphism (SNP) in α5 in particular, to lung cancer and heavy smoking. While α3 and ß4 readily form receptors in expression system such as the Xenopus oocyte, since α5 is not required for function, simple co-expression approaches may under-represent α5-containing receptors. We used a concatamer of human α3 and ß4 subunits to form ligand-binding domains, and show that we can force the insertions of alternative structural subunits into the functional pentamers. These α3ß4 variants differ in sensitivity to ACh, nicotine, varenicline, and cytisine. Our data indicated lower efficacy for varenicline and cytisine than expected for ß4-containing receptors, based on previous studies of rodent receptors. We confirm that these therapeutically important α4 receptor partial agonists may present different autonomic-based side-effect profiles in humans than will be seen in rodent models, with varenicline being more potent for human than rat receptors and cytisine less potent. Our initial characterizations failed to find functional effects of the α5 SNP. However, our data validate this approach for further investigations.
Assuntos
Acetilcolina/metabolismo , Gânglios/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Agonistas Nicotínicos/metabolismo , Antagonistas Nicotínicos/metabolismo , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/metabolismo , Acetilcolina/agonistas , Acetilcolina/antagonistas & inibidores , Alcaloides/metabolismo , Alcaloides/farmacologia , Animais , Azocinas/metabolismo , Azocinas/farmacologia , Benzazepinas/metabolismo , Benzazepinas/farmacologia , Agonismo Parcial de Drogas , Potenciais Evocados/efeitos dos fármacos , Humanos , Ligantes , Mutagênese Sítio-Dirigida , Proteínas Mutantes/agonistas , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Nicotina/agonistas , Nicotina/antagonistas & inibidores , Nicotina/metabolismo , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Subunidades Proteicas/agonistas , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Quinolizinas/metabolismo , Quinolizinas/farmacologia , Quinoxalinas/metabolismo , Quinoxalinas/farmacologia , Ratos , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , Vareniclina , Xenopus laevisRESUMO
The optimization of a series of thieno[3,2-b]thiophene-2-carboxylic acid derivatives for agonist activity against the GPR35 is reported. Compounds were optimized to achieve ß-arrestin-biased agonism for developing probe molecules that may be useful for elucidating the biology and physiology of GPR35. Compound 13 was identified to the most potent GPR35 agonist, and compounds 30 and 36 exhibited the highest efficacy to cause ß-arrestin translocation.
Assuntos
Anti-Inflamatórios/síntese química , Arrestinas/agonistas , Receptores Acoplados a Proteínas G/agonistas , Tiofenos/síntese química , Anti-Inflamatórios/farmacologia , Arrestinas/química , Arrestinas/genética , Bioensaio , Relação Dose-Resposta a Droga , Células HT29 , Humanos , Ligantes , Transporte Proteico/efeitos dos fármacos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Tiofenos/farmacologia , beta-ArrestinasRESUMO
It is known that the ATP-binding cassette transporter A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. Several laboratories have demonstrated that ABCA1 binding to lipid-poor apolipoprotein A-I (apoA-I) will mediate the assembly of nascent HDL and cellular cholesterol efflux, which suggests a possible receptor-ligand interaction between ABCA1 and apoA-I. In this study, a cell-based-ELISA-like high-throughput screening (HTS) method was developed to identify the synthetic and natural compounds that can regulate binding activity of ABCA1 to apoA-I. The cell-based-ELISA-like high-throughput screen was conducted in a 96-well format using Chinese hamster ovary (CHO) cells stably transfected with ABCA1 pIRE2-EGFP (Enhanced Green Fluorecence Protein) expression vector and the known ABCA1 inhibitor glibenclamide as the antagonist control. From 2,600 compounds, a xanthone compound (IMB 2026791) was selected using this HTS assay, and it was proved as an apoA-I binding agonist to ABCA1 by a flow cytometry assay and western blot analysis. The [3H] cholesterol efflux assay of IMB2026791 treated ABCA1-CHO cells and PMA induced THP-1 macrophages (human acute monocytic leukemia cell) further confirmed the compound as an accelerator of cholesterol efflux in a dose-dependent manner with an EC(50) of 25.23 µM.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , Xantonas/farmacologia , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/agonistas , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Relação Dose-Resposta a Droga , Glibureto/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Ligação Proteica , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Two fluorescent derivatives of the M1 muscarinic selective agonist AC-42 were synthesized by coupling the lissamine rhodamine B fluorophore (in ortho and para positions) to AC42-NH(2). This precursor, prepared according to an original seven-step procedure, was included in the study together with the LRB fluorophore (alone or linked to an alkyl chain). All these compounds are antagonists, but examination of their ability to inhibit or modulate orthosteric [(3)H]NMS binding revealed that para-LRB-AC42 shared several properties with AC-42. Carefully designed experiments allowed para-LRB-AC42 to be used as a FRET tracer on EGFP-fused M1 receptors. Under equilibrium binding conditions, orthosteric ligands, AC-42, and the allosteric modulator gallamine behaved as competitors of para-LRB-AC42 binding whereas other allosteric compounds such as WIN 51,708 and N-desmethylclozapine were noncompetitive inhibitors. Finally, molecular modeling studies focused on putative orthosteric/allosteric bitopic poses for AC-42 and para-LRB-AC42 in a 3D model of the human M1 receptor.
Assuntos
Corantes Fluorescentes/síntese química , Sondas Moleculares/síntese química , Piperidinas/síntese química , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/metabolismo , Rodaminas/síntese química , Regulação Alostérica , Cálcio/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Modelos Moleculares , Sondas Moleculares/química , Sondas Moleculares/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Ensaio Radioligante , Receptor Muscarínico M1/antagonistas & inibidores , Receptor Muscarínico M1/genética , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rodaminas/química , Rodaminas/farmacologia , Solubilidade , Relação Estrutura-AtividadeRESUMO
Vasopressin (AVP) and CRH synergistically regulate adrenocorticotropin and insulin release at the level of the pituitary and pancreas, respectively. Here, we first extended these AVP and CRH coregulation processes to the adrenal medulla. We demonstrate that costimulation of chromaffin cells by AVP and CRH simultaneously induces a catecholamine secretion exceeding the one induced by each hormone alone, thus demonstrating a net potentiation. To further elucidate the molecular mechanisms underlying this synergism, we coexpressed human V1b and CRH receptor (CRHR)1 receptor in HEK293 cells. In this heterologous system, AVP also potentiated CRH-stimulated cAMP accumulation in a dose-dependent and saturable manner. This effect was only partially mimicked by phorbol ester or inhibited by a phospholipase C inhibitor respectively. This finding suggests the existence of an new molecular mechanism, independent from second messenger cross talk. Similarly, CRH potentiated the AVP-induced inositol phosphates production. Using bioluminescence resonance energy transfer, coimmunoprecipitation, and receptor rescue experiments, we demonstrate that V1b and CRHR1 receptors assemble as heterodimers. Moreover, new pharmacological properties emerged upon receptors cotransfection. Taken together, these data strongly suggest that direct molecular interactions between V1b and CRHR1 receptors play an important role in mediating the synergistic interactions between these two receptors.
Assuntos
Hormônio Liberador da Corticotropina/fisiologia , Multimerização Proteica , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de Vasopressinas/metabolismo , Vasopressinas/fisiologia , Glândulas Suprarrenais/citologia , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos , Catecolaminas/metabolismo , Bovinos , Células Cultivadas , Células Cromafins/metabolismo , Hormônio Liberador da Corticotropina/farmacologia , AMP Cíclico/metabolismo , Ativação Enzimática , Estrenos/farmacologia , Células HEK293 , Humanos , Hidrocarbonetos Halogenados/farmacologia , Indóis/farmacologia , Inositol Polifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/metabolismo , Cultura Primária de Células , Ligação Proteica , Pirrolidinas/farmacologia , Pirrolidinonas/farmacologia , Receptor Cross-Talk , Receptores de Hormônio Liberador da Corticotropina/agonistas , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Receptores de Vasopressinas/agonistas , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/metabolismo , Sistemas do Segundo Mensageiro , Tiazinas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo , Vasopressinas/farmacologiaRESUMO
Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5'-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5'-monophosphonate, ACP) directly activated the adenosine A(1) receptor (A(1)R). In contrast, AMP only activated the adenosine A(2B) receptor (A(2B)R) after hydrolysis to adenosine by ecto-5'-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A(1)R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A(1)R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A(1)R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A(1)R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine.
Assuntos
Agonistas do Receptor A1 de Adenosina/farmacologia , Monofosfato de Adenosina/farmacologia , Receptor A1 de Adenosina/metabolismo , 5'-Nucleotidase/metabolismo , Adenosina/metabolismo , Agonistas do Receptor A1 de Adenosina/química , Agonistas do Receptor A1 de Adenosina/metabolismo , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/metabolismo , Animais , Córtex Cerebral/citologia , Colforsina/farmacologia , Células HEK293 , Histidina , Humanos , Hidrólise/efeitos dos fármacos , Ligantes , Camundongos , Imagem Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Receptor A1 de Adenosina/química , Receptor A2B de Adenosina/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Análise de Célula ÚnicaRESUMO
Bivalent histamine H(2) receptor (H(2)R) agonists were synthesized by connecting pharmacophoric 3-(2-amino-4-methylthiazol-5-yl)-, 3-(2-aminothiazol-5-yl)-, 3-(imidazol-4-yl)-, or 3-(1,2,4-triazol-5-yl)propylguanidine moieties by N(G)-acylation with alkanedioic acids of various chain lengths. The compounds were investigated for H(2)R agonism in GTPase and [(35)S]GTPγS binding assays at guinea pig (gp) and human (h) H(2)R-Gsα(S) fusion proteins including various H(2)R mutants, at the isolated gp right atrium, and in GTPase assays for activity on recombinant H(1), H(3), and H(4) receptors. The bivalent ligands are H(2)R partial or full agonists, up to 2 orders of magnitude more potent than monovalent acylguanidines and, with octanedioyl or decanedioyl spacers, up to 4000 times more potent than histamine at the gpH(2)R. In contrast to their imidazole analogues, the aminothiazoles are highly selective for H(2)R vs other HR subtypes. Compounds with (theoretically) sufficient spacer length (20 CH(2) groups) to simultaneously occupy two orthosteric binding sites in H(2)R dimers are nearly inactive, whereas the highest potency resides in compounds with considerably shorter spacers. Thus, there is no evidence for interaction with H(2)R dimers. The high agonistic potency may result from interaction with an accessory binding site at the same receptor protomer.
Assuntos
Guanidinas/síntese química , Agonistas dos Receptores Histamínicos/síntese química , Receptores Histamínicos H2/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Guanidinas/química , Guanidinas/farmacologia , Cobaias , Coração/efeitos dos fármacos , Coração/fisiologia , Agonistas dos Receptores Histamínicos/química , Agonistas dos Receptores Histamínicos/farmacologia , Humanos , Técnicas In Vitro , Ligantes , Mutação , Regiões Promotoras Genéticas , Multimerização Proteica , Receptores Histamínicos H2/genética , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/genética , Relação Estrutura-AtividadeRESUMO
Preclinical in vivo assessment of the pharmacologic activity of nonpeptidyl thrombopoietin receptor (TPOR) agonists is very difficult because of the high species specificity of such agonists. In this study, we have developed a novel and simple in vivo hollow-fiber assay to preclinically evaluate TPOR agonists. The 32D-mpl cell line was generated by stable transfection of human TPOR into 32D lymphoblast cells and shown to be a specific model for nonpeptide TPOR agonists in vitro. Stably transfected 32D-mpl cells were then sealed in hollow fibers and implanted into nude mice. Cells in hollow fibers specifically responded to TPOR agonists, including thrombopoietin and eltrombopag, a nonpeptide small-molecule TPOR agonist, but not to granulocyte colony-stimulating factor or erythropoietin. Oral administration of eltrombopag stimulated 32D-mpl cell proliferation, prevented 32D-mpl cell apoptosis, and stimulated the phosphorylation of cellular signaling transducers and activators of transcription in a TPOR- and dose-dependent manner. These results indicate that the hollow-fiber assay is a specific and efficient model for rapidly evaluating the in vivo activity of small-molecule TPOR agonists.
Assuntos
Avaliação Pré-Clínica de Medicamentos/instrumentação , Receptores de Trombopoetina/agonistas , Animais , Benzoatos/farmacologia , Linhagem Celular/transplante , Ensaio de Unidades Formadoras de Colônias , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Eritropoetina/farmacologia , Feminino , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Hidrazinas/farmacologia , Implantes Experimentais , Interleucina-3/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Pirazóis/farmacologia , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/metabolismo , Trombopoetina/farmacologia , TransfecçãoRESUMO
We previously showed that phorbol-12-myristate-13-acetate (PMA) mediates a robust PKC-dependent sensitization and desensitization of the highly homologous human Gs protein and adenylyl cyclase (AC)-linked D1 (hD1R) and D5 (hD5R) dopaminergic receptors, respectively. Here, we demonstrate using forskolin-mediated AC stimulation that PMA-mediated hD1R sensitization and hD5R desensitization is not associated with changes in AC activity. We next employed a series of chimeric hD1R and hD5R to delineate the underlying structural determinants dictating the subtype-specific regulation of human D1-like receptors by PMA. We first used chimeric receptors in which the whole terminal region (TR) spanning from the extracellular face of transmembrane domain 6 to the end of cytoplasmic tail (CT) or CT alone were exchanged between hD1R and hD5R. CT and TR swaps lead to chimeric hD1R and hD5R retaining PMA-induced sensitization and desensitization of wild type parent receptors. In striking contrast, hD1R sensitization and hD5R desensitization mediated by PMA are correspondingly switched to PMA-induced receptor desensitization and sensitization following the IL3 swap between hD1R and hD5R. Cell treatment with the PKC blocker, Gö6983, inhibits PMA-induced regulation of these chimeric receptors in a similar fashion to wild type receptors. Further studies with chimeras constructed by exchanging IL3 and TR show that PMA-induced regulation of these chimeras remains fully switched relative to their respective wild type parent receptor. Interestingly, results obtained with the exchange of IL3 and TR also reveal that the D1-like subtype-specific regulation by PMA, while fully dictated by IL3, can be modulated in a receptor conformation-dependent manner. Overall, our results strongly suggest that IL3 is the critical determinant underlying the subtype-specific regulation of human D1-like receptor responsiveness by PKC.
Assuntos
Proteína Quinase C/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D5/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Adenilil Ciclases/metabolismo , Motivos de Aminoácidos , Colforsina/farmacologia , AMP Cíclico/metabolismo , Dopamina/farmacologia , Dopamina/fisiologia , Relação Dose-Resposta a Droga , Ativadores de Enzimas/farmacologia , Células HEK293 , Humanos , Indóis/farmacologia , Isoenzimas/metabolismo , Maleimidas/farmacologia , Fosforilação , Conformação Proteica , Proteína Quinase C/antagonistas & inibidores , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/química , Receptores de Dopamina D5/agonistas , Receptores de Dopamina D5/química , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/química , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
IκB-ζ, an essential inflammatory regulator, is specifically induced by Toll-like receptor ligands or interleukin (IL)-1ß by post-transcriptional activation mediated via a 165-nucleotide element in IκB-ζ mRNA. Here, we analyzed the Toll-like receptor-IL-1 receptor signaling components involved in the post-transcriptional regulation of IκB-ζ with mutated estrogen receptor [ER(T2)] fusion proteins. Upon 4-hydroxytamoxifen treatment, the ER(T2) fusion proteins with IL-1 receptor-associated kinase (IRAK)1 and IRAK4 elicited specific activation of a reporter gene for the post-transcriptional regulation of IκB-ζ. The tumor necrosis factor receptor-associated factor (TRAF)6-ER(T2) protein activated nuclear factor-κB, but not post-transcriptional regulation, indicating that activation of IRAK1/4, but not of TRAF6, is sufficient to activate the 165-nucleotide element-mediated post-transcriptional mechanism. Interestingly, the post-transcriptional mechanism was not activated in TRAF6-deficient cells, indicating an essential role for TRAF6. Thus, the signaling pathway leading to nuclear factor-κB activation and the post-transcriptional activation bifurcates at IRAK1, suggesting a new pathway activated by IRAK1.
Assuntos
Quinases Associadas a Receptores de Interleucina-1/metabolismo , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Moduladores de Receptor Estrogênico/farmacologia , Receptor beta de Estrogênio/agonistas , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Inativação Gênica , Genes Reporter/efeitos dos fármacos , Células HEK293 , Humanos , Proteínas I-kappa B , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Quinases Associadas a Receptores de Interleucina-1/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
Traditional risk assessment of potential endocrine-disruptive pollutants, including PCBs, focus mainly on the effects of parent compounds. Still, biotransformation results in systemic exposure to PCBs and their bioactive metabolites. In the present paper, the effects of twenty ultra-pure non-dioxin-like (NDL) PCBs and their environmentally relevant hydroxy- (OH-) and methylsulfonyl- (MeSO(2)-) metabolites on aromatase activity and their glucocorticoid properties were investigated. Although most NDL-PCBs were inactive, PCB28 inhibited aromatase activity in human placenta microsomes with an IC(50) of 2.2µM. Most of these NDL-PCBs were weak (ant-)agonist of the glucocorticoid receptor (GR). Interestingly, four OH-metabolites of the commonly found NDL-PCB180 were able to inhibit aromatase activity (LOECs in the low µM range) and showed anti-glucocorticoid properties (LOECs in the low nM range), in a concentration-dependent manner. Further, four MeSO(2)-PCBs slightly inhibited aromatase activity and showed anti-glucocorticoid properties. Although, these effects were also associated with cytotoxicity, they were dependent on the position of the MeSO(2)-group on the biphenyl ring. Our results are the first to show that OH-PCBs are both anti-glucocorticoids and aromatase inhibitors. Taken together, these results for PCBs again support the common idea that risk assessment of the endocrine disruptive potential of PCBs should also include their metabolites.
Assuntos
Inibidores da Aromatase/toxicidade , Disruptores Endócrinos/toxicidade , Mesilatos/toxicidade , Bifenilos Policlorados/toxicidade , Receptores de Glucocorticoides/antagonistas & inibidores , Aromatase/biossíntese , Aromatase/química , Inibidores da Aromatase/química , Inibidores da Aromatase/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Disruptores Endócrinos/química , Disruptores Endócrinos/metabolismo , Indução Enzimática/efeitos dos fármacos , Feminino , Genes Reporter/efeitos dos fármacos , Humanos , Hidroxilação , Mesilatos/química , Microssomos/enzimologia , Concentração Osmolar , Placenta/enzimologia , Bifenilos Policlorados/química , Bifenilos Policlorados/metabolismo , Gravidez , Proteínas da Gravidez/antagonistas & inibidores , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta/efeitos dos fármacos , Medição de Risco/métodosRESUMO
The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2, we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). We show that Neh2 domain is sufficient for recognition, ubiquitination, and proteasomal degradation of Neh2-luciferase fusion protein. The Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time course of reporter activation. The reporter was used to screen the Spectrum library of 2000 biologically active compounds to identify activators of Nrf2. The most robust and yet nontoxic Nrf2 activators found--nordihydroguaiaretic acid, fisetin, and gedunin--induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism.
Assuntos
Genes Reporter , Ensaios de Triagem em Larga Escala , Fator 2 Relacionado a NF-E2/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Simulação por Computador , Flavonoides/química , Flavonoides/farmacologia , Flavonóis , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch , Cinética , Limoninas/química , Limoninas/farmacologia , Luciferases/genética , Luciferases/metabolismo , Masoprocol/química , Masoprocol/farmacologia , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , UbiquitinaçãoRESUMO
Toll-like receptor 4 (TLR4) is involved in activation of the innate immune response in a large number of different diseases. Despite numerous studies, the role of separate domains of TLR4 in the regulation of receptor activation is poorly understood. Replacement of the TLR4 ectodomain with LPS-binding proteins MD-2 or CD14 resulted in a robust ligand-independent constitutive activation comparable with the maximal stimulation of the receptor with LPS. The same effect was achieved by the replacement of the ectodomain with a monomeric fluorescent protein or a 24-kDa gyrase B fragment. This demonstrates an intrinsic dimerization propensity of the transmembrane and cytoplasmic domains of TLR4 and reveals a previously unknown function of the ectodomain in inhibiting spontaneous receptor dimerization. Constitutive activation was abolished by the replacement of the ectodomain by a bulkier protein ovalbumin. N-terminal deletion variants of TLR4 revealed that the smallest segment of the ectodomain that already prevents constitutive activity comprises only 90 residues (542 to 631) of the total 608 residues. We conclude that TLR4 represents a receptor with a low threshold of activation that can be rapidly activated by the release of inhibition exerted by its ectodomain. This is important for the sensitivity of TLR4 to activation by different agonists. The TLR4 ectodomain has multiple roles in enabling ligand regulated activation, providing proper localization while serving as an inhibitor to prevent spontaneous, ligand-independent dimerization.
Assuntos
Multimerização Proteica/fisiologia , Receptor 4 Toll-Like/metabolismo , Células HEK293 , Humanos , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Antígeno 96 de Linfócito/agonistas , Antígeno 96 de Linfócito/genética , Antígeno 96 de Linfócito/imunologia , Antígeno 96 de Linfócito/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/agonistas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
We wanted to analyze the basis for the distinction between OX(1) and OX(2) orexin receptors by the known agonists, orexin-A, orexin-B and Ala(11), D-Leu(15)-orexin-B, of which the latter two show some selectivity for OX(2). For this, chimaeric OX(1)/OX(2) and OX(2)/OX(1) orexin receptors were generated. The receptors were transiently expressed in HEK-293 cells, and potencies of the agonists to elicit cytosolic Ca(2+) elevation were measured. The results show that the N-terminal regions of the receptor are most important, and the exchange of the area from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 is enough to lead to an almost total change of the receptor's ligand profile.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Neuropeptídeos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores de Neuropeptídeos/agonistas , Proteínas Recombinantes de Fusão/agonistas , Sequência de Aminoácidos , Sítios de Ligação/genética , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Neuropeptídeos/química , Receptores de Orexina , Orexinas , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estrutura Secundária de Proteína , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/química , Receptores de Neuropeptídeos/genética , Proteínas Recombinantes de Fusão/químicaRESUMO
The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Hydrogen/deuterium exchange (HDX) data indicate that the CAR activation function 2 (AF-2) is more stable in CAR(TCPOBOP):RXR and CAR(meclizine):RXR than in CAR:RXR. HDX kinetics also show significant differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Unlike CAR(meclizine):RXR, CAR(TCPOBOP):RXR shows a higher overall stabilization that extends into RXR. We identify residues 339-345 in CAR as an allosteric regulatory site with a greater magnitude reduction in exchange kinetics in CAR(TCPOBOP):RXR than CAR(meclizine):RXR. Accordingly, assays with mutations on CAR at leucine-340 and leucine-343 confirm this region as an important determinant of CAR activity.