RESUMO
The peroxisome proliferator activated receptors PPARalpha, PPARgamma, and PPARdelta are ligand-activated transcription factors that play a key role in lipid homeostasis. The fibrates raise circulating levels of high-density lipoprotein cholesterol and lower levels of triglycerides in part through their activity as PPARalpha agonists; however, the low potency and restricted selectivity of the fibrates may limit their efficacy, and it would be desirable to develop more potent and selective PPARalpha agonists. Modification of the selective PPARdelta agonist 1 (GW501516) so as to incorporate the 2-aryl-2-methylpropionic acid group of the fibrates led to a marked shift in potency and selectivity toward PPARalpha agonism. Optimization of the series gave 25a, which shows EC50 = 4 nM on PPARalpha and at least 500-fold selectivity versus PPARdelta and PPARgamma. Compound 25a (GW590735) has been progressed to clinical trials for the treatment of diseases of lipid imbalance.
Assuntos
HDL-Colesterol/sangue , PPAR alfa/agonistas , Propionatos/síntese química , Tiazóis/síntese química , Animais , Apolipoproteína A-I/genética , VLDL-Colesterol/sangue , Cristalografia por Raios X , Cães , Dislipidemias/sangue , Dislipidemias/tratamento farmacológico , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Moleculares , PPAR alfa/química , Propionatos/farmacocinética , Propionatos/farmacologia , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Tiazóis/farmacocinética , Tiazóis/farmacologia , Triglicerídeos/sangueRESUMO
A series of tetrahydrobenzofuranyl and tetrahydrobenzothienyl propenoic acids that showed potent agonist activity against RXRalpha were synthesized via a structure-based design approach. Among the compounds studied, 46a,b showed not only very good potency against RXRalpha (K(i) = 6 nM) but was also found to be greater than 167-fold selective vs RARalpha (K(i) > 1000 nM). This compound profiled out as a full agonist in a cell-based transient transfection assay (EC(50) = 3 nM). The two antipodes were separated via chiral chromatography, and 46b was found to be 40-fold more potent than 46a. Interestingly, cocrystallization of 46a,b with the RXRalpha protein generated a liganded structure whereby the (S)-antipode was found in the binding pocket. Given orally in db/db mice or ZDF rats, 46a,b showed a significant glucose-lowering effect and an increase in liver mass. Triglycerides decreased significantly in db/db mice but increased in the ZDF rats. A dose-dependent decrease of nonesterified free fatty acids was seen in ZDF rats but not in db/db mice. These differences indicate a species specific effect of RXR agonists on lipid metabolism.
Assuntos
Acrilatos/síntese química , Benzofuranos/síntese química , Hipoglicemiantes/síntese química , Receptores do Ácido Retinoico/agonistas , Fatores de Transcrição/agonistas , Acrilatos/química , Acrilatos/farmacologia , Animais , Benzofuranos/química , Benzofuranos/farmacologia , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Haplorrinos , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Ligantes , Lipídeos/biossíntese , Masculino , Camundongos , Modelos Moleculares , Ensaio Radioligante , Ratos , Ratos Zucker , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/genética , Receptores X de Retinoides , Estereoisomerismo , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/genética , TransfecçãoRESUMO
Natural ligands for nuclear receptors are believed to activate gene transcription by causing dissociation of corepressors and promoting the association of coactivator proteins. Using multiple biophysical techniques, we find that peptides derived from one of the nuclear receptor interacting motifs of the corepressors nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid receptors (SMRT) are able to bind the ligand binding domains (LBD) of all three PPAR (peroxisome proliferator activated receptor) subtypes. Using these peptides as tools, we find that ligands designed as selective agonists for PPAR gamma promote the association of coactivator peptides and dissociation of corepressor peptides as expected on PPAR gamma but surprisingly have varied effects on the binding of corepressor peptides to the other PPAR subtypes. In particular, some members of a class of L-tyrosine-based compounds designed as selective agonists for PPAR gamma reduce the affinity for corepressor peptides on PPAR gamma but increase the affinity for the same peptides on PPAR delta and in one case on PPAR alpha. We provide structural data that suggests that the molecular basis for these observations are variations in the ligand binding pockets of the three PPAR subtypes that are perturbed differentially by individual ligands and result in altered presentations of the overlapping coactivator/corepressor binding surfaces.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Oxazóis/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Células Cultivadas , Cristalografia por Raios X , Fluorescência , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Correpressor 1 de Receptor Nuclear , Correpressor 2 de Receptor Nuclear , Oxazóis/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Isoformas de Proteínas , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/agonistas , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Transcriptional regulation by the glucocorticoid receptor (GR) is mediated by hormone binding, receptor dimerization, and coactivator recruitment. Here, we report the crystal structure of the human GR ligand binding domain (LBD) bound to dexamethasone and a coactivator motif derived from the transcriptional intermediary factor 2. Despite structural similarity to other steroid receptors, the GR LBD adopts a surprising dimer configuration involving formation of an intermolecular beta sheet. Functional studies demonstrate that the novel dimer interface is important for GR-mediated activation. The structure also reveals an additional charge clamp that determines the binding selectivity of a coactivator and a distinct ligand binding pocket that explains its selectivity for endogenous steroid hormones. These results establish a framework for understanding the roles of protein-hormone and protein-protein interactions in GR signaling pathways.
Assuntos
Dexametasona/química , Receptores de Glucocorticoides/química , Fatores de Transcrição/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Cristalização , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Coativador 2 de Receptor Nuclear , Conformação Proteica , Estrutura Terciária de Proteína , Receptores de Glucocorticoides/isolamento & purificação , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes de Fusão/química , SolubilidadeRESUMO
We report the synthesis and biological activity of a new series of small molecule agonists of the human Peroxisome Proliferator-Activated Receptor delta (PPARdelta). Several hits were identified from our original libraries containing lipophilic carboxylic acids. Optimization of these hits by structure-guided design led to 7k (GW501516) and 7l (GW0742), which shows an EC(50) of 1.1 nM against PPARdelta with 1000-fold selectivity over the other human subtypes.
Assuntos
Receptores Citoplasmáticos e Nucleares/agonistas , Tiazóis/síntese química , Fatores de Transcrição/agonistas , Animais , Células Cultivadas , Técnicas de Química Combinatória , Humanos , Camundongos , Ensaio Radioligante , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/farmacologia , Ativação TranscricionalRESUMO
Repression of gene transcription by nuclear receptors is mediated by interactions with co-repressor proteins such as SMRT and N-CoR, which in turn recruit histone deacetylases to the chromatin. Aberrant interactions between nuclear receptors and co-repressors contribute towards acute promyelocytic leukaemia and thyroid hormone resistance syndrome. The binding of co-repressors to nuclear receptors occurs in the unliganded state, and can be stabilized by antagonists. Here we report the crystal structure of a ternary complex containing the peroxisome proliferator-activated receptor-alpha ligand-binding domain bound to the antagonist GW6471 and a SMRT co-repressor motif. In this structure, the co-repressor motif adopts a three-turn alpha-helix that prevents the carboxy-terminal activation helix (AF-2) of the receptor from assuming the active conformation. Binding of the co-repressor motif is further reinforced by the antagonist, which blocks the AF-2 helix from adopting the active position. Biochemical analyses and structure-based mutagenesis indicate that this mode of co-repressor binding is highly conserved across nuclear receptors.