RESUMO
The farnesoid X receptor (FXR) may play a crucial role in a number of metabolic diseases and, as such, could potentially serve as a target for the development of therapeutics as a treatment for those diseases. Previous work has described GW4064 as an FXR agonist with an interesting activity profile. This manuscript will describe the synthesis of novel analogs of GW4064 and the activity profile of those analogs.
Assuntos
Isoxazóis/química , Isoxazóis/farmacologia , Oxazolidinonas/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Avaliação Pré-Clínica de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Humanos , Modelos Moleculares , Estrutura Molecular , Oxazolidinonas/química , Relação Estrutura-AtividadeRESUMO
A series of imidazo[1,2-a]pyridines which directly bind to HCV Non-Structural Protein 4B (NS4B) is described. This series demonstrates potent in vitro inhibition of HCV replication (EC50 < 10 nM), direct binding to purified NS4B protein (IC50 < 20 nM), and an HCV resistance pattern associated with NS4B (H94N/R, V105L/M, F98L) that are unique among reported HCV clinical assets, suggestive of the potential for additive or synergistic combination with other small molecule inhibitors of HCV replication.
RESUMO
To further explore the optimum placement of the acid moiety in conformationally constrained analogs of GW 4064 1a, a series of stilbene replacements were prepared. The benzothiophene 1f and the indole 1g display the optimal orientation of the carboxylate for enhanced FXR agonist potency.
Assuntos
Isoxazóis/química , Isoxazóis/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Estilbenos/química , Estilbenos/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Conformação Molecular , Dados de Sequência Molecular , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
To improve on the drug properties of GSK8062 1b, a series of heteroaryl bicyclic naphthalene replacements were prepared. The quinoline 1c was an equipotent FXR agonist with improved drug developability parameters relative to 1b. In addition, analog 1c lowered body weight gain and serum glucose in a DIO mouse model of diabetes.
Assuntos
Isoxazóis/química , Naftalenos/química , Quinolinas/química , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Sítios de Ligação , Glicemia/metabolismo , Cristalografia por Raios X , Diabetes Mellitus Experimental/metabolismo , Cães , Transferência Ressonante de Energia de Fluorescência , Humanos , Isoxazóis/síntese química , Isoxazóis/farmacocinética , Ligantes , Camundongos , Conformação Molecular , Estrutura Terciária de Proteína , Quinolinas/síntese química , Quinolinas/farmacocinética , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Aumento de Peso/efeitos dos fármacosRESUMO
The identification of nonporphyrin ligands for the orphan nuclear receptor Rev-erbα will enable studies of its role as a heme sensor and regulator of metabolic and circadian signaling. We describe the development of a biochemical assay measuring the interaction between Rev-erbα and a peptide from the nuclear receptor corepressor-1 (NCoR). The assay was utilized to identify a small molecule ligand for Rev-erbα, GSK4112 (1), that was competitive with heme. In cells, 1 profiled as a Rev-erbα agonist in cells to inhibit expression of the circadian target gene bmal1. In addition, 1 repressed the expression of gluconeogenic genes in liver cells and reduced glucose output in primary hepatocytes. Therefore, 1 is useful as a chemical tool to probe the function of Rev-erbα in transcriptional repression, regulation of circadian biology, and metabolic pathways. Additionally, 1 may serve as a starting point for design of Rev-erbα chemical probes with in vivo pharmacological activity.
Assuntos
Glicina/análogos & derivados , Correpressor 1 de Receptor Nuclear/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Peptídeos/metabolismo , Mapeamento de Interação de Proteínas/métodos , Bibliotecas de Moléculas Pequenas/metabolismo , Tiofenos/metabolismo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Linhagem Celular , Células Cultivadas , Ritmo Circadiano , Glicina/química , Glicina/metabolismo , Heme/metabolismo , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Correpressor 1 de Receptor Nuclear/química , Peptídeos/química , Bibliotecas de Moléculas Pequenas/química , Tiofenos/químicaRESUMO
Repression of gene transcription by the nuclear receptor Rev-erbalpha plays an integral role in the core molecular circadian clock. We report the crystal structure of a nuclear receptor-co-repressor (N-CoR) interaction domain 1 (ID1) peptide bound to truncated human Rev-erbalpha ligand-binding domain (LBD). The ID1 peptide forms an unprecedented antiparallel beta-sheet with Rev-erbalpha, as well as an alpha-helix similar to that seen in nuclear receptor ID2 crystal structures but out of register by four residues. Comparison with the structure of Rev-erbbeta bound to heme indicates that ID1 peptide and heme induce substantially different conformational changes in the LBD. Although heme is involved in Rev-erb repression, the structure suggests that Rev-erbalpha could also mediate repression via ID1 binding in the absence of heme. The previously uncharacterized secondary structure induced by ID1 peptide binding advances our understanding of nuclear receptor-co-repressor interactions.
Assuntos
Correpressor 1 de Receptor Nuclear/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/química , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Correpressor 1 de Receptor Nuclear/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
The liver X receptors (LXR) play a key role in cholesterol homeostasis and lipid metabolism. SAR studies around tertiary-amine lead molecule 2, an LXR full agonist, revealed that steric and conformational changes to the acetic acid and propanolamine groups produce dramatic effects on agonist efficacy and potency. The new analogs possess good functional activity, demonstrating the ability to upregulate LXR target genes, as well as promote cholesterol efflux in macrophages.
Assuntos
Aminas/química , Colesterol/metabolismo , Macrófagos/efeitos dos fármacos , Receptores Nucleares Órfãos/agonistas , Aminas/síntese química , Aminas/farmacocinética , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Humanos , Receptores X do Fígado , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Replacement of the metabolically labile stilbene with either benzothiophene or naphthalene rings led to the identification of potent full agonists 2a and 2g.
Assuntos
Isoxazóis/química , Naftalenos/química , Receptores Citoplasmáticos e Nucleares/agonistas , Tiofenos/química , Animais , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Humanos , Isoxazóis/farmacologia , Naftalenos/farmacocinética , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade , Tiofenos/farmacocinéticaRESUMO
Starting from the known FXR agonist GW 4064 1a, a series of alternately 3,5-substituted isoxazoles was prepared. Several of these analogs were potent full FXR agonists. A subset of this series, with a tether between the isoxazole ring and the 3-position aryl substituent, were equipotent FXR agonists to GW 4064 1a, with the 2,6-dimethyl phenol analog 1t having greater FRET FXR potency than GW 4064 1a.
Assuntos
Isoxazóis/síntese química , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Isoxazóis/química , Isoxazóis/farmacologia , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-AtividadeRESUMO
A novel series of 1H-indol-1-yl tertiary amine LXR agonists has been designed. Compounds from this series were potent agonists with good rat pharmacokinetic parameters. In addition, the crystal structure of an LXR agonist bound to LXRalpha will be disclosed.
Assuntos
Proteínas de Ligação a DNA/agonistas , Indóis/síntese química , Indóis/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Administração Oral , Animais , Colesterol/análise , Técnicas de Química Combinatória , Cristalografia por Raios X , Indóis/química , Receptores X do Fígado , Macrófagos/efeitos dos fármacos , Camundongos , Conformação Molecular , Estrutura Molecular , Receptores Nucleares Órfãos , Ratos , Relação Estrutura-AtividadeRESUMO
A cocrystal structure of T1317 (3) bound to hLXRbeta was utilized in the design of a series of substituted N-phenyl tertiary amines. Profiling in binding and functional assays led to the identification of LXR modulator GSK9772 ( 20) as a high-affinity LXRbeta ligand (IC 50 = 30 nM) that shows separation of anti-inflammatory and lipogenic activities in human macrophage and liver cell lines, respectively. A cocrystal structure of the structurally related analog 19 bound to LXRbeta reveals regions within the receptor that can affect receptor modulation through ligand modification. Mechanistic studies demonstrate that 20 is greater than 10-fold selective for LXR-mediated transrepression of proinflammatory gene expression versus transactivation of lipogenic signaling pathways, thus providing an opportunity for the identification of LXR modulators with improved therapeutic indexes.
Assuntos
Aminas/química , Aminas/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Proteínas de Ligação a DNA/efeitos dos fármacos , Desenho de Fármacos , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Cristalografia por Raios X , Receptores X do Fígado , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , Receptores Nucleares Órfãos , Relação Estrutura-AtividadeRESUMO
Starting from the known FXR agonist GW 4064 1a, a series of stilbene replacements were prepared. The 6-substituted 1-naphthoic acid 1b was an equipotent FXR agonist with improved developability parameters relative to 1a. Analog 1b also reduced the severity of cholestasis in the ANIT acute cholestatic rat model.
Assuntos
Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/farmacologia , Colestase/tratamento farmacológico , Proteínas de Ligação a DNA/agonistas , Isoxazóis/síntese química , Isoxazóis/farmacologia , Naftalenos/síntese química , Naftalenos/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/agonistas , Administração Oral , Animais , Ácidos Carboxílicos/química , Colestase/metabolismo , Colestase/patologia , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Modelos Animais de Doenças , Cães , Mucosa Gástrica/metabolismo , Haplorrinos , Isoxazóis/química , Camundongos , Conformação Molecular , Estrutura Molecular , Naftalenos/química , Ratos , Receptores Citoplasmáticos e Nucleares/química , Estômago/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Transcrição/químicaRESUMO
Antagonizing the action of the human nuclear xenobiotic receptor pregnane X receptor (PXR) may have important clinical implications in preventing drug-drug interactions and improving therapeutic efficacy. We provide evidence that a naturally occurring phytoestrogen, coumestrol, is an antagonist of the nuclear receptor PXR (NR1I2). In transient transfection assays, coumestrol was able to suppress the agonist effects of SR12813 on human PXR activity. PXR activity was assessed and correlated with effects on the metabolism of the anesthetic tribromoethanol and on gene expression in primary human hepatocytes. We found that coumestrol was able to suppress the effects of PXR agonists on the expression of the known PXR target genes, CYP3A4 and CYP2B6, in primary human hepatocytes as well as inhibit metabolism of tribromoethanol in humanized PXR mice. Coumestrol at concentrations above 1.0 microm competed in scintillation proximity assays with a labeled PXR agonist for binding to the ligand-binding cavity. However, mammalian two-hybrid assays and transient transcription data using ligand-binding-cavity mutant forms of PXR show that coumestrol also antagonizes coregulator recruitment. This effect is likely by binding to a surface outside the ligand-binding pocket. Taken together, these data imply that there are antagonist binding site(s) for coumestrol on the surface of PXR. These studies provide the basis for development of novel small molecule inhibitors of PXR with the ultimate goal of clinical applications toward preventing drug-drug interactions.
Assuntos
Cumestrol/farmacologia , Fitoestrógenos/farmacologia , Receptores de Esteroides/antagonistas & inibidores , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Linhagem Celular , Células Cultivadas , Receptor Constitutivo de Androstano , Cumestrol/química , Cumestrol/metabolismo , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Etanol/análogos & derivados , Etanol/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Imuno-Histoquímica , Espectrometria de Massas , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Coativador 1 de Receptor Nuclear , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Fitoestrógenos/química , Fitoestrógenos/metabolismo , Receptor de Pregnano X , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The circadian clock temporally coordinates metabolic homeostasis in mammals. Central to this is heme, an iron-containing porphyrin that serves as prosthetic group for enzymes involved in oxidative metabolism as well as transcription factors that regulate circadian rhythmicity. The circadian factor that integrates this dual function of heme is not known. We show that heme binds reversibly to the orphan nuclear receptor Rev-erbalpha, a critical negative component of the circadian core clock, and regulates its interaction with a nuclear receptor corepressor complex. Furthermore, heme suppresses hepatic gluconeogenic gene expression and glucose output through Rev-erbalpha-mediated gene repression. Thus, Rev-erbalpha serves as a heme sensor that coordinates the cellular clock, glucose homeostasis, and energy metabolism.
Assuntos
Ritmo Circadiano , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Heme/metabolismo , Redes e Vias Metabólicas , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Relógios Biológicos , Linhagem Celular , Linhagem Celular Tumoral , Ritmo Circadiano/genética , Metabolismo Energético , Gluconeogênese/genética , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/metabolismo , Hemina/farmacologia , Histona Desacetilases/metabolismo , Homeostase , Humanos , Masculino , Camundongos , Proteínas Nucleares/metabolismo , Correpressor 1 de Receptor Nuclear , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares , Proteínas Repressoras/metabolismoRESUMO
We report the identification of substituted cis-bicyclo[3.3.0]-oct-2-enes as small molecule agonists of subfamily V orphan nuclear receptors (NR5A), liver receptor homolog-1 (LRH-1) and steroidogenic factor-1 (SF-1). Using fluorescence resonance energy transfer (FRET)-based biochemical assays, compound 5a (GSK8470) was identified as a high-affinity ligand for LRH-1 and SF-1. In liver cells, 5a increased the expression of the LRH-1 target gene small heterodimer partner (SHP). Synthesis of analogues modified at three positions led to the development of compounds with functional selectivity between LRH-1 and SF-1.
Assuntos
Alcenos/síntese química , Compostos de Anilina/síntese química , Compostos Bicíclicos com Pontes/síntese química , Proteínas de Ligação a DNA/agonistas , Proteínas de Homeodomínio/agonistas , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/agonistas , Alcenos/química , Alcenos/farmacologia , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Sítios de Ligação , Compostos Bicíclicos com Pontes/química , Compostos Bicíclicos com Pontes/farmacologia , Células Cultivadas , Transferência Ressonante de Energia de Fluorescência , Genes Reporter , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Ligantes , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Estereoisomerismo , Fator Esteroidogênico 1 , Relação Estrutura-AtividadeRESUMO
Carbamate derivatives of bile acids were synthesized with the aim of systematically exploring the potential for farnesoid X receptor (FXR) modulation endowed with occupancy of the receptor's back door, localized between loops H1-H2 and H4-H5. Since it was previously shown that bile acids bind to FXR by projecting the carboxylic tail opposite the transactivation function 2 (AF-2, helix 12), functionalization of the side chain is not expected to interfere directly with the orientation of H12 but can result in a more indirect way of receptor modulation. The newly synthesized compounds were extensively characterized for their ability to modulate FXR function in a variety of assays, including the cell-free fluorescence resonance energy transfer (FRET) assay and the cell-based luciferase transactivation assay, and displayed a broad range of activity from full agonism to partial antagonism. Docking studies clearly indicate that the side chain of the new derivatives fits in a so far unexploited receptor cavity localized near the "back door" of FXR. We thus demonstrate the possibility of achieving a broad FXR modulation without directly affecting the H12 orientation.
Assuntos
Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/síntese química , Proteínas de Ligação a DNA/agonistas , Fatores de Transcrição/agonistas , Linhagem Celular Tumoral , Ácido Quenodesoxicólico/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desenho de Fármacos , Transferência Ressonante de Energia de Fluorescência , Genes Reporter , Histona Acetiltransferases , Humanos , Ligantes , Luciferases/genética , Modelos Moleculares , Coativador 1 de Receptor Nuclear , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides/metabolismo , Elementos de Resposta , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Substituted 3-(phenylamino)-1H-pyrrole-2,5-diones were identified from a high throughput screen as inducers of human ATP binding cassette transporter A1 expression. Mechanism of action studies led to the identification of GSK3987 as an LXR ligand. GSK3987 recruits the steroid receptor coactivator-1 to human LXRalpha and LXRbeta with EC(50)s of 40 nM, profiles as an LXR agonist in functional assays, and activates LXR though a mechanism that is similar to first generation LXR agonists.
Assuntos
Compostos de Anilina/síntese química , Proteínas de Ligação a DNA/agonistas , Maleimidas/síntese química , Receptores Citoplasmáticos e Nucleares/agonistas , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Genes Reporter , Histona Acetiltransferases , Humanos , Ligantes , Receptores X do Fígado , Luciferases/genética , Maleimidas/química , Maleimidas/farmacologia , Modelos Moleculares , Estrutura Molecular , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Coativador 1 de Receptor Nuclear , Receptores Nucleares Órfãos , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/química , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
There has been tremendous progress in the development of ligand binding assays for NRs during the past several years. A major development has been the advent of homogeneous assay formats, including those that do not require a radioligand. These high throughput, low volume assay formats will be powerful tools for the identification and characterization of novel NR ligands, including both natural ligands for orphan NRs and new drugs that mediate their therapeutic effects through this class of receptors.
Assuntos
Bioquímica/métodos , Receptores Citoplasmáticos e Nucleares/química , Animais , Sistema Livre de Células , Cromatografia em Gel , Proteínas de Ligação a DNA/química , Relação Dose-Resposta a Droga , Estradiol/química , Receptor beta de Estrogênio , Transferência Ressonante de Energia de Fluorescência/métodos , Concentração Inibidora 50 , Cinética , Ligantes , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/químicaRESUMO
The orphan nuclear constitutive androstane receptor (CAR) is proposed to play a central role in the response to xenochemical stress. Identification of CAR target genes in humans has been limited by the lack of a selective CAR agonist. We report the identification of 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) as a novel human CAR agonist with the following characteristics: (a) potent activity in an in vitro fluorescence-based CAR activation assay; (b) selectivity for CAR over other nuclear receptors, including the xenobiotic pregnane X receptor (PXR); (c) the ability to induce human CAR nuclear translocation; and (d) the ability to induce the prototypical CAR target gene CYP2B6 in primary human hepatocytes. Using primary cultures of human hepatocytes, the effects of CITCO on gene expression were compared with those of the PXR ligand rifampicin. The relative expression of a number of genes encoding proteins involved in various aspects of steroid and xenobiotic metabolism was analyzed. Notably, CAR and PXR activators differentially regulated the expression of several genes, demonstrating that these two nuclear receptors subserve overlapping but distinct biological functions in human hepatocytes.
Assuntos
Oximas/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Tiazóis/farmacologia , Fatores de Transcrição/agonistas , Células Cultivadas , Receptor Constitutivo de Androstano , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Receptores Citoplasmáticos e Nucleares/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/fisiologiaRESUMO
A series of 6alpha-alkyl-substituted analogues of chenodeoxycholic acid (CDCA) were synthesized and evaluated as potential farnesoid X receptor (FXR) ligands. Among them, 6alpha-ethyl-chenodeoxycholic acid (6-ECDCA) was shown to be a very potent and selective FXR agonist (EC(50) = 99 nM) and to be endowed with anticholeretic activity in an in vivo rat model of cholestasis.