RESUMO
Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.
Assuntos
Antivirais/farmacologia , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Orthomyxoviridae/fisiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Cristalografia por Raios X , Modelos Animais de Doenças , Ensaios de Triagem em Larga Escala , Hidrodinâmica , Camundongos , Modelos Moleculares , Nucleoproteínas/ultraestrutura , Orthomyxoviridae/efeitos dos fármacos , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Bibliotecas de Moléculas Pequenas/uso terapêutico , SoluçõesRESUMO
The purpose of this study was to develop a mechanistic pharmacokinetic-pharmacodynamic (PK-PD) model to describe the effects of rifampicin on hepatic Cyp3a11 RNA, enzymatic activity, and triazolam pharmacokinetics. Rifampicin was administered to steroid and xenobiotic X receptor (SXR) humanized mice at 10 mg/kg p.o. (every day for 3 days) followed by triazolam (4 mg/kg p.o.) 24 h after the last dose of rifampicin. Rifampicin and triazolam concentrations and Cyp3a11 RNA expression and activity in the liver were measured over the 4-day period. Elevations in Cyp3a11 RNA expression were observed 24 h after the first dose of rifampicin, reaching a maximum (â¼10 times baseline) after the third dose and were sustained until day 4 and began declining 48 h after the last rifampicin dose. Similar changes in enzymatic activity were also observed. The triazolam serum area under the curve (AUC) was 5-fold lower in mice pretreated with rifampicin, consistent with enzyme induction. The final PK-PD model incorporated rifampicin liver concentration as the driving force for the time-delayed Cyp3a11 induction governed by in vitro potency estimates, which in turn regulated the turnover of enzyme activity. The PK-PD model was able to recapitulate the delayed induction of Cyp3a11 mRNA and enzymatic activity by rifampicin. Furthermore, the model was able to accurately anticipate the reduction in the triazolam plasma AUC by integrating a ratio of the predicted induced enzyme activity and basal activity into the equations describing triazolam pharmacokinetics. In conjunction with the SXR humanized mouse model, this mathematical approach may serve as a tool for predicting clinically relevant drug-drug interactions via pregnane X receptor-mediated enzyme induction and possibly extended to other induction pathways (e.g., constitutive androstane receptor).
Assuntos
Citocromo P-450 CYP3A/biossíntese , Proteínas de Membrana/biossíntese , Receptores de Esteroides/metabolismo , Rifampina/farmacologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Humanos , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Receptor de Pregnano X , Rifampina/farmacocinéticaRESUMO
Monkeys have been proposed as an animal model to predict the magnitude of human clinical drug-drug interactions caused by CYP3A4 enzyme induction. To evaluate whether the cynomolgus monkey can be an effective in vivo model, human CYP3A4 inducers were evaluated both in vitro and in vivo. First, a full-length pregnane X receptor (PXR) was cloned from the cynomolgus monkey, and the sequence was compared with those of rhesus monkey and human PXR. Cynomolgus and rhesus monkey PXR differed by only one amino acid (A68V), and both were highly homologous to human PXR (approximately 96%). When the transactivation profiles of 30 compounds, including known inducers of CYP3A4, were compared between cynomolgus and human PXR, a high degree of correlation with EC(50) values was observed. These results suggest that cynomolgus and human PXR respond in a similar fashion to these ligands. Second, two known human CYP3A4 inducers, rifampicin and hyperforin, were tested in monkey and human primary hepatocytes for induction of CYP3A enzymes. Both monkey and human hepatocytes responded similarly to the inducers and resulted in increased RNA and enzyme activity changes of CYP3A8 and CYP3A4, respectively. Lastly, in vivo induction of CYP3A8 by rifampicin and hyperforin was shown by significant reductions of midazolam exposure that were comparable with those in humans. These results show that the cynomolgus monkey can be a predictive in vivo animal model of PXR-mediated induction of human CYP3A4 and can provide a useful assessment of the resulting pharmacokinetic changes of affected drugs.
Assuntos
Citocromo P-450 CYP3A/biossíntese , Hepatócitos/metabolismo , Macaca fascicularis , Receptores de Esteroides/metabolismo , Xenobióticos/farmacocinética , Adulto , Sequência de Aminoácidos , Animais , Compostos Bicíclicos com Pontes/sangue , Compostos Bicíclicos com Pontes/farmacocinética , Compostos Bicíclicos com Pontes/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas/genética , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Hypericum/química , Macaca mulatta , Masculino , Midazolam/sangue , Midazolam/metabolismo , Midazolam/farmacocinética , Pessoa de Meia-Idade , Modelos Animais , Dados de Sequência Molecular , Floroglucinol/análogos & derivados , Floroglucinol/sangue , Floroglucinol/farmacocinética , Floroglucinol/farmacologia , Extratos Vegetais/sangue , Extratos Vegetais/farmacocinética , Receptor de Pregnano X , Receptores de Esteroides/genética , Rifampina/sangue , Rifampina/farmacocinética , Rifampina/farmacologia , Homologia de Sequência de Aminoácidos , Terpenos/sangue , Terpenos/farmacocinética , Terpenos/farmacologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , TransfecçãoRESUMO
The SXR humanized mouse model was used to quantitatively assess an in vivo induction response of the human PXR agonist, rifampicin. Three days of rifampicin treatment increased RNA expression and microsomal enzyme activity of CYP3A11, as well as significantly reduced triazolam plasma exposure. These results indicate that the humanized SXR mouse can be used as a model to predict human CYP3A4 induction and the resulting pharmacokinetic changes of CYP3A4 substrates in humans.
Assuntos
Citocromo P-450 CYP3A/efeitos dos fármacos , Proteínas de Membrana/efeitos dos fármacos , Receptores de Esteroides/agonistas , Rifampina/farmacologia , Animais , Antibióticos Antituberculose/administração & dosagem , Antibióticos Antituberculose/farmacologia , Citocromo P-450 CYP3A/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Indução Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Modelos Animais , Receptor de Pregnano X , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Rifampina/administração & dosagem , Especificidade da Espécie , Triazolam/farmacocinéticaRESUMO
A series of 3-(1H-benzo[d]imidazol-2-yl)pyridin-2(1H)-one inhibitors of insulin-like growth factor receptor-1 (IGF-1R) were examined in which the pendant imidazole moiety was replaced to improve selectivity for IGF-1R inhibition over cytochrome P450 (CYP). Synthesis and SAR of these compounds is presented.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Imidazóis/química , Imidazóis/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Animais , Sistema Enzimático do Citocromo P-450/análise , Imidazóis/síntese química , Concentração Inibidora 50 , Relação Estrutura-AtividadeRESUMO
A novel class of 1H-(benzimidazol-2-yl)-1H-pyridin-2-one inhibitors of insulin-like growth factor I (IGF-1R) kinase is described. This report discusses the SAR of 4-(2-hydroxy-2-phenylethylamino)-substituted pyridones with improved IGF-1R potency.
Assuntos
Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Fosfotransferases/antagonistas & inibidores , Animais , Baculoviridae/enzimologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Indicadores e Reagentes , Camundongos , Modelos Moleculares , Piridonas/farmacologia , Relação Estrutura-Atividade , Timidina/metabolismoRESUMO
Compound 3 (BMS-536924), a novel small-molecule inhibitor of the insulin-like growth factor receptor kinase with equal potency against the insulin receptor is described. The in vitro and in vivo biological activity of this interesting compound is also reported.
Assuntos
Antineoplásicos/síntese química , Benzimidazóis/síntese química , Piridinas/síntese química , Piridonas/síntese química , Receptor IGF Tipo 1/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzimidazóis/química , Benzimidazóis/farmacologia , Disponibilidade Biológica , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Haplorrinos , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Estrutura Molecular , Transplante de Neoplasias , Piridinas/química , Piridinas/farmacologia , Piridonas/química , Piridonas/farmacologia , Ratos , Receptor de Insulina/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
The evolution of 2, a C-4-methylcarbonate analogue of paclitaxel with minimal oral bioavailability and oral efficacy, into its C-3'-t-butyl-3'-N-t-butyloxycarbonyl analogue (15i), a novel taxane with oral efficacy in preclinical models that is comparable to iv administered paclitaxel, is described.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/farmacocinética , Taxoides/farmacocinética , Administração Oral , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Disponibilidade Biológica , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Camundongos , Ratos , Relação Estrutura-Atividade , Taxoides/química , Taxoides/farmacologiaRESUMO
The formation of a reactive intermediate was found to be responsible for CYP3A4 metabolism-dependent inhibition (MDI) observed with (S)-N-[1-(3-morpholin-4-ylphenyl)ethyl]-3-phenyl-acrylamide (1). Structure-3A4 MDI relationship studies culminated in the discovery of a difluoro analogue, (S)-N-[1-(4-fluoro-3-morpholin-4-ylphenyl)ethyl]-3-(4-fluoro-phenyl)acrylamide (2), as an orally bioavailable KCNQ2 opener free of CYP3A4 MDI.
Assuntos
Cinamatos/síntese química , Inibidores das Enzimas do Citocromo P-450 , Flúor/química , Morfolinas/síntese química , Canais de Potássio/efeitos dos fármacos , Administração Oral , Animais , Disponibilidade Biológica , Linhagem Celular , Cinamatos/metabolismo , Cinamatos/farmacologia , Citocromo P-450 CYP3A , Modelos Animais de Doenças , Injeções Intravenosas , Ativação do Canal Iônico , Canal de Potássio KCNQ2 , Masculino , Potenciais da Membrana , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/fisiopatologia , Morfolinas/metabolismo , Morfolinas/farmacologia , Lobo Parietal/efeitos dos fármacos , Lobo Parietal/fisiopatologia , Técnicas de Patch-Clamp , Canais de Potássio/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The synthesis and antifungal activity of 5'- and 5'-6'-substituted azasordarin derivatives are described. Modification of the 5'-position led to the discovery of the spirocyclopentyl analogue 7g, which is the first azasordarin to register single-digit MIC values versus Aspergillus spp. Further investigation identified the 5'-i-Pr derivative 7b, which displays superior pharmacokinetic properties compared to other azasordarins.