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1.
ACS Med Chem Lett ; 7(6): 590-4, 2016 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-27326332

RESUMO

BMS-711939 (3) is a potent and selective peroxisome proliferator-activated receptor (PPAR) α agonist, with an EC50 of 4 nM for human PPARα and >1000-fold selectivity vs human PPARγ (EC50 = 4.5 µM) and PPARδ (EC50 > 100 µM) in PPAR-GAL4 transactivation assays. Compound 3 also demonstrated excellent in vivo efficacy and safety profiles in preclinical studies and thus was chosen for further preclinical evaluation. The synthesis, structure-activity relationship (SAR) studies, and in vivo pharmacology of 3 in preclinical animal models as well as its ADME profile are described.

2.
J Pharmacol Exp Ther ; 350(2): 412-24, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24917546

RESUMO

Proprotein convertase subtilisin kexin-9 (PCSK9) is an important pharmacological target for decreasing low-density lipoprotein (LDL) in cardiovascular disease, although seemingly inaccessible to small molecule approaches. Compared with therapeutic IgG antibodies currently in development, targeting circulating PCSK9 with smaller molecular scaffolds could offer different profiles and reduced dose burdens. This inspired genesis of PCSK9-binding Adnectins, a protein family derived from human fibronectin-10th-type III-domain and engineered for high-affinity target binding. BMS-962476, an ∼11-kDa polypeptide conjugated to polyethylene glycol to enhance pharmacokinetics, binds with subnanomolar affinity to human. The X-ray cocrystal structure of PCSK9 with a progenitor Adnectin shows ∼910 Å(2) of PCSK9 surface covered next to the LDL receptor binding site, largely by residues of a single loop of the Adnectin. In hypercholesterolemic, overexpressing human PCSK9 transgenic mice, BMS-962476 rapidly lowered cholesterol and free PCSK9 levels. In genomic transgenic mice, BMS-962476 potently reduced free human PCSK9 (ED50 ∼0.01 mg/kg) followed by ∼2-fold increases in total PCSK9 before return to baseline. Treatment of cynomolgus monkeys with BMS-962476 rapidly suppressed free PCSK9 >99% and LDL-cholesterol ∼55% with subsequent 6-fold increase in total PCSK9, suggesting reduced clearance of circulating complex. Liver sterol response genes were consequently downregulated, following which LDL and total PCSK9 returned to baseline. These studies highlight the rapid dynamics of PCSK9 control over LDL and liver cholesterol metabolism and characterize BMS-962476 as a potent and efficacious PCSK9 inhibitor.


Assuntos
Anticolesterolemiantes/farmacologia , Lipoproteínas LDL/sangue , Polietilenoglicóis/farmacologia , Pró-Proteína Convertases/antagonistas & inibidores , Proteínas/farmacologia , Sequência de Aminoácidos , Animais , HDL-Colesterol/sangue , Cristalização , Feminino , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/química , Pró-Proteína Convertases/metabolismo , Ratos , Receptores de LDL/antagonistas & inibidores , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Especificidade da Espécie
3.
PLoS One ; 7(7): e41865, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22848640

RESUMO

Proprotein convertase subtilisin/kexin-9 (PCSK9) enhances the degradation of hepatic low-density lipoprotein receptor (LDLR). Deletion of PCSK9, and loss-of-function mutants in humans result in lower levels of circulating LDL-cholesterol and a strong protection against coronary heart disease. Accordingly, the quest for PCSK9 inhibitors has major clinical implications. We have previously identified annexin A2 (AnxA2) as an endogenous binding partner and functional inhibitor of PCSK9. Herein, we studied the relevance of AnxA2 in PCSK9 inhibition and lipid metabolism in vivo. Plasma analyses of AnxA2(-/-) mice revealed: i) a ∼1.4-fold increase in LDL-cholesterol without significant changes in VLDLs or HDLs, and ii) a ∼2-fold increase in circulating PCSK9 levels. Western blotting and immunohistochemistry of AnxA2(-/-) tissues revealed that the LDLR was decreased by ∼50% in extrahepatic tissues, such as adrenals and colon. We also show that AnxA2-derived synthetic peptides block the PCSK9≡LDLR interaction in vitro, and adenoviral overexpression of AnxA2 in mouse liver increases LDLR protein levels in vivo. These results suggest that AnxA2 acts as an endogenous regulator of LDLR degradation, mostly in extrahepatic tissues. Finally, we identified an AnxA2 coding polymorphism, V98L, that correlates with lower circulating levels of PCSK9 thereby extending our results on the physiological role of AnxA2 in humans.


Assuntos
Anexina A2/metabolismo , Fígado/metabolismo , Pró-Proteína Convertases/metabolismo , Proteólise , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Adenoviridae/genética , Sequência de Aminoácidos , Animais , Anexina A2/química , Anexina A2/deficiência , Anexina A2/genética , Linhagem Celular , LDL-Colesterol/sangue , Cricetinae , Éxons/genética , Espaço Extracelular/metabolismo , Humanos , Fígado/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/sangue , Estrutura Terciária de Proteína , Serina Endopeptidases/sangue
4.
J Biol Chem ; 285(52): 40965-78, 2010 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-20937814

RESUMO

PCSK9, a target for the treatment of dyslipidemia, enhances the degradation of the LDL receptor (LDLR) in endosomes/lysosomes, up-regulating LDL-cholesterol levels. Whereas the targeting and degradation of the PCSK9-LDLR complex are under scrutiny, the roles of the N- and C-terminal domains of PCSK9 are unknown. Although autocatalytic zymogen processing of PCSK9 occurs at Gln(152)↓, here we show that human PCSK9 can be further cleaved in its N-terminal prosegment at Arg(46)↓ by an endogenous enzyme of insect High Five cells and by a cellular mammalian protease, yielding an ∼4-fold enhanced activity. Removal of the prosegment acidic stretch resulted in ∼3-fold higher binding to LDLR in vitro, in ≥4-fold increased activity on cellular LDLR, and faster cellular internalization in endosome/lysosome-like compartments. Finally, swapping the acidic stretch of PCSK9 with a similar one found in the glycosylphosphatidylinositol-anchored heparin-binding protein 1 does not impair PCSK9 autoprocessing, secretion, or activity and confirmed that the acidic stretch acts as an inhibitor of PCSK9 function. We also show that upon short exposure to pH values 6.5 to 5.5, an ∼2.5-fold increase in PCSK9 activity on total and cell surface LDLR occurs, and PCSK9 undergoes a second cleavage at Arg(248), generating a two-chain PCSK9-ΔN(248). At pH values below 5.5, PCSK9 dissociates from its prosegment and loses its activity. This pH-dependent activation of PCSK9 represents a novel pathway to further activate PCSK9 in acidic endosomes. These data enhance our understanding of the functional role of the acidic prosegment and on the effect of pH in the regulation of PCSK9 activity.


Assuntos
Endossomos/enzimologia , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Animais , Endossomos/genética , Ativação Enzimática/fisiologia , Células HEK293 , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/enzimologia , Lisossomos/genética , Mariposas , Peptídeos/genética , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Ligação Proteica/fisiologia , Receptores de LDL/genética , Serina Endopeptidases/genética
5.
Bioorg Med Chem Lett ; 20(9): 2933-7, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20356736

RESUMO

The synthesis and follow-up SAR studies of our development candidate 1 by incorporating 2-aryl-4-oxazolylmethoxy and 2-aryl-4-thiazolylmethoxy moieties into the oxybenzylglycine framework of the PPARalpha/gamma dual agonist muraglitazar is described. SAR studies indicate that different substituents on the aryloxazole/thiazole moieties as well as the choice of carbamate substituent on the glycine moiety can significantly modulate the selectivity of PPARalpha versus PPARgamma. Potent, highly selective PPARalpha activators 2a and 2l, as well as PPARalpha activators with significant PPARgamma activity, such as 2s, were identified. The in vivo pharmacology of these compounds in preclinical animal models as well as their ADME profiles are discussed.


Assuntos
Anti-Inflamatórios/síntese química , Glicina/análogos & derivados , PPAR alfa/agonistas , PPAR gama/agonistas , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacocinética , Sítios de Ligação , Cricetinae , Cristalografia por Raios X , Glicina/síntese química , Glicina/farmacocinética , Humanos , Masculino , PPAR alfa/metabolismo , PPAR gama/metabolismo , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade
6.
J Med Chem ; 53(7): 2854-64, 2010 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-20218621

RESUMO

An 1,3-oxybenzylglycine based compound 2 (BMS-687453) was discovered to be a potent and selective peroxisome proliferator activated receptor (PPAR) alpha agonist, with an EC(50) of 10 nM for human PPARalpha and approximately 410-fold selectivity vs human PPARgamma in PPAR-GAL4 transactivation assays. Similar potencies and selectivity were also observed in the full length receptor co-transfection assays. Compound 2 has negligible cross-reactivity against a panel of human nuclear hormone receptors including PPARdelta. Compound 2 demonstrated an excellent pharmacological and safety profile in preclinical studies and thus was chosen as a development candidate for the treatment of atherosclerosis and dyslipidemia. The X-ray cocrystal structures of the early lead compound 12 and compound 2 in complex with PPARalpha ligand binding domain (LBD) were determined. The role of the crystal structure of compound 12 with PPARalpha in the development of the SAR that ultimately resulted in the discovery of compound 2 is discussed.


Assuntos
Descoberta de Drogas , Glicina/análogos & derivados , Oxazóis/química , Oxazóis/farmacologia , PPAR alfa/agonistas , Animais , Linhagem Celular , Cricetinae , Cristalografia por Raios X , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Glicina/síntese química , Glicina/química , Glicina/farmacologia , Glicina/toxicidade , Humanos , Masculino , Camundongos , Modelos Moleculares , Oxazóis/síntese química , Oxazóis/toxicidade , PPAR alfa/química , PPAR alfa/genética , Estrutura Terciária de Proteína , Especificidade por Substrato , Ativação Transcricional/efeitos dos fármacos
7.
J Pharmacol Exp Ther ; 327(3): 716-26, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18799592

RESUMO

The first generation peroxisome proliferator-activated receptor (PPAR) alpha agonist gemfibrozil reduces the risk of major cardiovascular events; therefore, more potent PPARalpha agonists for the treatment of cardiovascular diseases have been actively sought. We describe two novel, potent oxybenzylglycine PPARalpha-selective agonists, BMS-687453 [N-[[3-[[2-(4-chlorophenyl)-5-methyl-4-oxazolyl]methoxy]phenyl]methyl]-N-(methoxycarbonyl)-glycine] and BMS-711939 N-[[5-[[2-(4-chlorophenyl)-5-methyl-4-oxazolyl]methoxy]-2-fluorophenyl]methyl]-N-(methoxycarbonyl)-glycine], that robustly increase apolipoprotein (Apo) A1 and high-density lipoprotein cholesterol in human ApoA1 transgenic mice and lower low-density lipoprotein-cholesterol and triglycerides in fat-fed hamsters. These compounds have much lower potency against mouse PPARalpha than human PPARalpha; therefore, they were tested in PPARalpha-humanized mice that do not express murine PPARalpha but express human PPARalpha selectively in the liver. We developed hepatic gene induction as a novel biomarker for efficacy and demonstrate hepatic gene induction at very low doses of these compounds. BMS-711939 induces fecal cholesterol excretion, which is further increased upon cotreatment with a liver X receptor (LXR) agonist. It is surprising that this synergistic increase upon coadministration is also observed in mice that express PPARalpha in the liver only. BMS-711939 also prevented the LXR agonist-induced elevation of serum triglycerides. Such PPARalpha agonists could be attractive candidates to explore for the treatment of cardiovascular diseases, especially in combination with a suitable LXR agonist.


Assuntos
Colesterol/metabolismo , Proteínas de Ligação a DNA/agonistas , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , PPAR alfa/agonistas , Receptores Citoplasmáticos e Nucleares/agonistas , Triglicerídeos/sangue , Animais , Sinergismo Farmacológico , Humanos , Fígado/metabolismo , Receptores X do Fígado , Camundongos , Camundongos Transgênicos , Receptores Nucleares Órfãos , Ativação Transcricional/efeitos dos fármacos
8.
J Lipid Res ; 45(8): 1410-7, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15145986

RESUMO

Liver X receptors (LXRs) are ligand-activated transcription factors that belong to the nuclear receptor superfamily. LXRs activate transcription of a spectrum of genes that regulate reverse cholesterol transport, including the ATP binding cassette transporter A1 (ABCA1), and raise HDL cholesterol (HDL-C) levels. However, LXR agonists also induce genes that stimulate lipogenesis, including the sterol response element binding protein (SREBP1-c) and fatty acid synthetase (FAS). The induction of these genes in the liver cause increased hepatic triglyceride synthesis, hypertriglyceridemia, and hepatic steatosis. As LXR response elements have been identified in these promoters, it is not clear if these two processes can be separated. Herein, we demonstrate that plasma HDL-C elevation and intestinal ABCA1 induction can occur with relatively little induction of FAS and SREBP1-c in mouse liver via a selective LXR modulator GW3965. This is in contrast to the strong induction of hepatic lipogenic genes by the well-characterized LXR agonist T0901317 (T317). Consistent with the in vivo results, GW3965 is a very weak LXR activator compared with T317 in human hepatoma cells. GW3965-liganded LXR recruits selected coactivators less effectively than T317 and may explain in part the tissue selective gene induction. This demonstration that tissue and gene selective modulation is possible with selective LXR modulators has positive implications for the development of this class of antiatherosclerotic agents.


Assuntos
Benzoatos/farmacologia , Benzilaminas/farmacologia , HDL-Colesterol/sangue , Fígado Gorduroso/metabolismo , Hipertrigliceridemia/metabolismo , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Animais , Proteínas de Ligação a DNA , Ligantes , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Receptores Nucleares Órfãos
9.
J Biol Chem ; 278(12): 10028-32, 2003 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-12519787

RESUMO

The bile salt export pump (BSEP) plays an integral role in lipid homeostasis by regulating the canalicular excretion of bile acids. Induction of BSEP gene expression is mediated by the farnesoid X receptor (FXR), which binds as a heterodimer with the retinoid X receptor (RXR) to the FXR response element (FXRE) located upstream of the BSEP gene. RXR ligands mimic several partner ligands and show additive effects upon coadministration. Using real-time quantitative PCR and cotransfection reporter assays, we demonstrate that the RXR agonist LG100268 antagonizes induction of BSEP expression mediated by endogenous and synthetic FXR ligands, CDCA and GW4064, respectively. Moreover, this antagonism is a general feature of RXR agonists and is attributed to a decrease in binding of FXR/RXR heterodimers to the BSEP-FXRE coupled with the inability of RXR agonists to recruit coactivators to FXR/RXR. Our data suggest that FXR/RXR is a conditionally permissive heterodimer and is the first example of RXR ligand-mediated antagonism of FXR activity. Because FXR agonists lower triglyceride levels, our results suggest a novel role for RXR-mediated antagonism of FXR activity in the development of hypertriglyceridemia observed with RXR agonists in rodents and humans.


Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , DNA/metabolismo , Receptores do Ácido Retinoico/fisiologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/fisiologia , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Linhagem Celular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Dimerização , Humanos , Ácidos Nicotínicos/farmacologia , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares , Receptores do Ácido Retinoico/química , Elementos de Resposta , Receptores X Retinoide , Tetra-Hidronaftalenos/farmacologia , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
11.
J Steroid Biochem Mol Biol ; 81(3): 217-25, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12163133

RESUMO

The mechanism by which ligands of nuclear receptors show differential effects on gene transcription is not fully understood, but is believed to result in part from the preferential recruitment and/or displacement of coactivators and corepressors. We have explored the interaction of several known ligands and the nuclear receptor (peroxisome proliferator activated receptor alpha, PPARalpha) using scintillation proximity assay (SPA) and the interaction of LXXLL containing peptides derived from three coactivators (SRC-1, CBP and PGC-1) with PPARalpha in the presence of PPARalpha agonist ligands using fluorescence resonance energy transfer (FRET). The EC(50)s of the individual ligands for recruitment showed the same rank order regardless of the coactivator peptide used, with GW2331

Assuntos
Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , DNA Complementar/metabolismo , Transferência de Energia , Escherichia coli/metabolismo , Histona Acetiltransferases , Humanos , Cinética , Ligantes , Coativador 1 de Receptor Nuclear , Peptídeos/química , Peptídeos/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Espectrofotometria , Transfecção
12.
Gene ; 290(1-2): 35-43, 2002 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-12062799

RESUMO

Bile acid biosynthesis is regulated by both feed-forward and feedback mechanisms involving a cascade of nuclear hormone receptors. Feed-forward regulation of the rate limiting enzyme in bile acid biosynthesis is provided by oxysterols through liver-X-receptor alpha (NR1H3), while feedback regulation is provided by bile acids through farnesoid-X-receptor (FXR) (NR1H4). The Syrian golden hamster provides a useful model for studying lipid metabolism. The hamster metabolizes and transports dietary cholesterol in a similar manner to humans, with the resulting lipid profile being more similar to the human profile than that of other rodent models. Cloning of Fxr from Syrian golden hamster revealed four hamster Fxr splice variants that altered the N-terminal activation domain or the hinge region between the DNA and ligand binding domains. Human genomic sequence and data from hamster Fxr were used to identify and clone a novel human FXR isoform resulting from the use of an alternative promoter. RNA expression analysis indicates that the two human FXR isoforms are differentially expressed in developmental and tissue-specific patterns and are likely to provide a mechanism for cell-specific FXR-dependent transcriptional activity.


Assuntos
Processamento Alternativo , Proteínas de Ligação a DNA/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Ácido Quenodesoxicólico/farmacologia , Códon de Iniciação/genética , Cricetinae , DNA Complementar/química , DNA Complementar/genética , Éxons/genética , Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Genes/genética , Humanos , Mesocricetus , Dados de Sequência Molecular , Isoformas de Proteínas/genética , RNA/genética , RNA/metabolismo , Receptores Citoplasmáticos e Nucleares , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transcrição Genética , Células Tumorais Cultivadas
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