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1.
Biotechniques ; 52(4): 273-5, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22482443

RESUMO

The use of subgenomic replicon systems has long been a valuable screening tool for the discovery of small molecule antivirals against Hepatitis C virus. While genotype 1a replicon systems have been widely used in stable systems, use in transient assays has been hampered by low signal. Here we describe the generation of a more robust genotype 1a (H77) replicon through the introduction of two fitness mutations, NS4A-K1691R and NS4B-E1726G, for use in transient transfections. While these mutations significantly improved the signal to noise ratio, leading to more robust data, they have no effect on the potency of tool compounds against various targets of HCV, thereby making this new system a powerful tool for screening of compounds against the genotype 1a replicon.


Assuntos
Aptidão Genética , Técnicas Genéticas , Hepacivirus/genética , Mutagênese/genética , Mutação/genética , Replicon/genética , Linhagem Celular Tumoral , Genótipo , Humanos , Transfecção
2.
Antimicrob Agents Chemother ; 56(1): 271-9, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22064535

RESUMO

Danoprevir is a hepatitis C virus (HCV) NS3/4A protease inhibitor that promotes multi-log(10) reductions in HCV RNA when administered as a 14-day monotherapy to patients with genotype 1 chronic HCV. Of these patients, 14/37 experienced a continuous decline in HCV RNA, 13/37 a plateau, and 10/37 a rebound. The rebound and continuous-decline groups experienced similar median declines in HCV RNA through day 7, but their results diverged notably at day 14. Plateau group patients experienced a lesser, but sustained, median HCV RNA decline. Baseline danoprevir susceptibility was similar across response groups but was reduced significantly at day 14 in the rebound group. Viral rebound in genotype 1b was uncommon (found in 2/23 patients). Population-based sequence analysis of NS3 and NS4A identified treatment-emergent substitutions at four amino acid positions in the protease domain of NS3 (positions 71, 155, 168, and 170), but only two (155 and 168) were in close proximity to the danoprevir binding site and carried substitutions that impacted danoprevir potency. R155K was the predominant route to reduced danoprevir susceptibility and was observed in virus isolated from all 10 rebound, 2/13 plateau, and 1/14 continuous-decline patients. Virus in one rebound patient additionally carried partial R155Q and D168E substitutions. Treatment-emergent substitutions in plateau patients were less frequently observed and more variable. Single-rebound patients carried virus with R155Q, D168V, or D168T. Clonal sequence analysis and drug susceptibility testing indicated that only a single patient displayed multiple resistance pathways. These data indicate the ascendant importance of R155K for viral escape during danoprevir treatment and may have implications for the clinical use of this agent.


Assuntos
Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepatite C Crônica/tratamento farmacológico , Lactamas/administração & dosagem , Inibidores de Proteases/administração & dosagem , Sulfonamidas/administração & dosagem , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Substituição de Aminoácidos , Antivirais/administração & dosagem , Antivirais/uso terapêutico , Ciclopropanos , Esquema de Medicação , Farmacorresistência Viral/efeitos dos fármacos , Genótipo , Hepacivirus/enzimologia , Hepatite C Crônica/virologia , Humanos , Isoindóis , Lactamas/uso terapêutico , Lactamas Macrocíclicas , Modelos Moleculares , Tipagem Molecular , Mutação , Prolina/análogos & derivados , Inibidores de Proteases/uso terapêutico , Estrutura Terciária de Proteína , RNA Viral/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recidiva , Especificidade da Espécie , Sulfonamidas/uso terapêutico , Carga Viral/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
3.
Chem Pharm Bull (Tokyo) ; 57(9): 1004-7, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19721266

RESUMO

Based upon the biphenyl 1-(2-naphthyl)-1H-pyrazole-5-carboxylamides reported in our previous communications, we designed and discovered 2-(6-chloro-3-methylsulfonyl)-naphthyl as an optimal factor Xa S1 binding element. Employing a key Diels-Alder reaction of 1,4-dihydro-2,3-benzoxathiin-3-oxide with maleic anhydride and a key Cu(I)-mediated methylsulfonylation, we prepared two biphenyl 1-(2-(6-chloro-3-methylsulfonyl)-naphthyl)-1H-pyrazole-5-carboxylamides as highly potent factor Xa inhibitors with K(i) values of 0.065 nM and 0.045 nM respectively, and demonstrated the synergistically enhanced binding interaction in the factor Xa S1 site.


Assuntos
Amidas/síntese química , Inibidores do Fator Xa , Inibidores de Serina Proteinase/síntese química , Amidas/química , Amidas/farmacologia , Sítios de Ligação , Desenho de Fármacos , Fator Xa/metabolismo , Ligação Proteica , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia
4.
Bioorg Med Chem Lett ; 19(8): 2179-85, 2009 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-19297154

RESUMO

Systematic SAR studies of in vitro factor Xa inhibitory activity around compound 1 were performed by modifying each of the three phenyl rings. A class of highly potent, selective, efficacious and orally bioavailable direct factor Xa inhibitors was discovered. These compounds were screened in hERG binding assays to examine the effects of substitution groups on the hERG channel affinity. From the leading compounds, betrixaban (compound 11, PRT054021) has been selected as the clinical candidate for development.


Assuntos
Anticoagulantes/síntese química , Anticoagulantes/farmacologia , Benzamidas/síntese química , Benzamidas/farmacologia , Descoberta de Drogas/métodos , Inibidores do Fator Xa , Piridinas/síntese química , Piridinas/farmacologia , Administração Oral , Animais , Anticoagulantes/administração & dosagem , Benzamidas/administração & dosagem , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/genética , Fator Xa/metabolismo , Humanos , Macaca fascicularis , Piridinas/administração & dosagem , Coelhos , Ratos
6.
Bioorg Med Chem Lett ; 14(9): 2073-8, 2004 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-15080981

RESUMO

A class of N,N-dialkylated 4-(4-arylsulfonylpiperazine-1-carbonyl)-benzamidines and 4-((4-arylsulfonyl)-2-oxo-piperazin-1-ylmethyl)-benzamidines has been discovered as potent factor Xa inhibitors with desirable in vitro and in vivo anticoagulant activity, but with low oral bioavailability. The 5-chloroindole and 6-chlorobenzo[b]thiophene groups are optimal as the factor Xa S1 binding elements. The strategy of incorporating a side chain on the piperazine nucleus to enhance binding affinity has been examined.


Assuntos
Benzamidinas/farmacologia , Inibidores do Fator Xa , Inibidores de Serina Proteinase/farmacologia , Benzamidinas/química , Benzamidinas/farmacocinética , Disponibilidade Biológica , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacocinética
7.
Bioorg Med Chem Lett ; 14(4): 983-7, 2004 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-15013006

RESUMO

Anthranilamides 4 and 5 were designed and synthesized as selective and orally bioavailable factor Xa inhibitors. Structural modifications aimed at lowering their lipophilicity were performed at the central phenyl ring and at the S4 binding biphenyl region by incorporating water solublizing substituents. The resulting compounds (e.g., 7, 8, 14, 30a, and 32b) are highly potent in vitro, and show improved activity in human plasma-based thrombin generation assay.


Assuntos
Amidas/síntese química , Amidas/farmacologia , Inibidores do Fator Xa , ortoaminobenzoatos/síntese química , ortoaminobenzoatos/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Trombose/tratamento farmacológico , Trombose/prevenção & controle
8.
Bioorg Med Chem Lett ; 14(4): 989-93, 2004 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-15013007

RESUMO

Compound 2 containing an aminomethylbenzoyl moiety as the S4 binding motif was synthesized in order to modulate hydrophlicity of anthranilamide-based factor Xa inhibitors with substituted biphenyl P4 groups. Structure-activity relationship studies around 2 have led to a series of potent factor Xa inhibitors which are highly active in the human plasma-based thrombin generation assay with 2XTG values less than 1 microM. Compound 55 shows strong antithrombotic activity in our rabbit deep vein thrombosis model, and also exhibits good oral bioavailability and a long half life in rats.


Assuntos
Amidas/síntese química , Amidas/farmacologia , Inibidores do Fator Xa , ortoaminobenzoatos/síntese química , ortoaminobenzoatos/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Desenho de Fármacos , Humanos , Estrutura Molecular , Coelhos , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Trombose/tratamento farmacológico
9.
Bioorg Med Chem Lett ; 14(5): 1221-7, 2004 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-14980670

RESUMO

A variety of P4 motifs have been examined to increase the binding affinity and in vitro anticoagulant potency of our biphenyl 1-(2-naphthyl)-1H-pyrazole-5-carboxylamide-based fXa inhibitors. Highly potent 2-naphthyl-P1 fXa inhibitors (K(i)< or =2 nM) with improved in vitro anticoagulant activity (2xTG< or =1 microM) and respectable pharmacokinetic properties have been discovered.


Assuntos
Amidas/química , Antitrombina III/química , Inibidores do Fator Xa , Pirazóis/química , Amidas/metabolismo , Amidas/farmacologia , Animais , Antitrombina III/metabolismo , Antitrombina III/farmacologia , Humanos , Ligação Proteica , Pirazóis/metabolismo , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley
11.
Arterioscler Thromb Vasc Biol ; 23(6): 1098-104, 2003 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-12750119

RESUMO

OBJECTIVE: In this study we test the hypothesis that blood/plasma-based prothrombinase assays, rather than inhibition of purified factor Xa (fXa), are predictive of in vivo antithrombotic activity. METHODS AND RESULTS: Six fXa inhibitors with equivalent nanomolar Ki were studied in thrombin generation assays using human plasma/blood and endogenous macromolecular substrate. In all assays, benzamidine inhibitors were more potent (100 to 800 nmol/L) than the aminoisoquinolines (5 to 58 micromol/L) or neutral inhibitors (3 to 10 micromol/L). A similar rank order of compound inhibition was also seen in purified prothrombinase assays as well as in a rabbit model of deep vein thrombosis. CONCLUSIONS: Assays using prothrombinase with protein substrates are better predictors of in vivo efficacy than fXa Ki using amidolytic substrates.


Assuntos
Benzamidinas/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores do Fator Xa , Fibrinolíticos/farmacologia , Isoquinolinas/farmacologia , Protrombina/metabolismo , Tromboplastina/antagonistas & inibidores , Animais , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/classificação , Fibrinolíticos/classificação , Humanos , Masculino , Estrutura Molecular , Sensibilidade e Especificidade , Especificidade por Substrato , Trombina/biossíntese , Trombose Venosa/prevenção & controle
12.
Bioorg Med Chem Lett ; 13(4): 723-8, 2003 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-12639567

RESUMO

A series of novel transition state factor Xa inhibitors containing a variety of lactam ring systems as central templates was synthesized in an expedient manner and allowed for a great deal of structural variability. Among them, the piperazinone-based inhibitors were found to be not only active against factor Xa but also selective over thrombin. Optimization of the P4 moiety yielded several potent compounds with IC(50) below 1 nM against factor Xa.


Assuntos
Inibidores do Fator Xa , Piperazinas/síntese química , Piperazinas/farmacocinética , Inibidores de Serina Proteinase/síntese química , Animais , Anticoagulantes/síntese química , Anticoagulantes/farmacocinética , Testes de Coagulação Sanguínea , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Piperazinas/farmacologia , Coelhos , Ratos , Ratos Sprague-Dawley , Inibidores de Serina Proteinase/farmacocinética , Inibidores de Serina Proteinase/farmacologia , Relação Estrutura-Atividade , Trombose/prevenção & controle
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