RESUMEN
Background and purpose: J147, a novel neurotrophic compound, was originally developed to treat aging-associated neurological diseases. Based on the broad spectrum of cytoprotective effects exhibited by this compound, we investigated whether J147 has cerebroprotection for acute ischemic stroke and whether it can enhance the effectiveness of thrombolytic therapy with tissue plasminogen activator (tPA). Methods: Rats were subjected to transient occlusion of the middle cerebral artery (tMCAO) by insertion of an intraluminal suture or embolic middle cerebral artery occlusion (eMCAO), and treated intravenously with J147 alone or in combination with tPA. Results: We found that J147 treatment significantly reduced infarct volume when administered at 2 h after stroke onset in the tMCAO model, but had no effect in eMCAO without tPA. However, combination treatment with J147 plus tPA at 4 h after stroke onset significantly reduced infarct volume and neurological deficits at 72 h after stroke compared with saline or tPA alone groups in the eMCAO model. Importantly, the combination treatment significantly reduced delayed tPA-associated brain hemorrhage and secondary microvascular thrombosis. These protective effects were associated with J147-mediated inhibition of matrix metalloproteinase-9 (MMP9), 15-lipoxygenase-1, and plasminogen activator inhibitor (PAI) expression in the ischemic hemispheres (predominantly in ischemic cerebral endothelium). Moreover, the combination treatment significantly reduced circulating platelet activation and platelet-leukocyte aggregation compared with saline or tPA alone groups at 24 h after stroke, which might also contribute to reduced microvascular thrombosis and neuroinflammation (as demonstrated by reduced neutrophil brain infiltration and microglial activation). Conclusion: Our results demonstrate that J147 treatment alone exerts cerebral cytoprotective effects in a suture model of acute ischemic stroke, while in an embolic stroke model co-administration of J147 with tPA reduces delayed tPA-induced intracerebral hemorrhage and confers cerebroprotection. These findings suggest that J147-tPA combination therapy could be a promising approach to improving the treatment of ischemic stroke.
RESUMEN
The discovery of 5,5'- and 6,6'-dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC50 <0.10 microM). In vitro DMPK data for selected compounds as well as crystal structures of representative inhibitors complexed with the NS5B protein are also disclosed.
Asunto(s)
Antivirales/química , Inhibidores Enzimáticos/química , Hepacivirus/enzimología , Piridonas/química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Animales , Antivirales/síntesis química , Antivirales/farmacología , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Humanos , Macaca fascicularis , Microsomas Hepáticos/metabolismo , Piridonas/síntesis química , Piridonas/farmacología , ARN Polimerasa Dependiente del ARN/metabolismo , Relación Estructura-ActividadRESUMEN
A novel series of non-nucleoside small molecules containing a tricyclic dihydropyridinone structural motif was identified as potent HCV NS5B polymerase inhibitors. Driven by structure-based design and building on our previous efforts in related series of molecules, we undertook extensive SAR studies, in which we identified a number of metabolically stable and very potent compounds in genotype 1a and 1b replicon assays. This work culminated in the discovery of several inhibitors, which combined potent in vitro antiviral activity against both 1a and 1b genotypes, metabolic stability, good oral bioavailability, and high C(12) (PO)/EC(50) ratios.
Asunto(s)
Disponibilidad Biológica , Diseño de Fármacos , Relación Estructura-Actividad , Antivirales/farmacocinética , Química Farmacéutica , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Genotipo , Hepacivirus/efectos de los fármacos , Hepatitis C , Estructura Molecular , ARN Polimerasa Dependiente del ARN , Proteínas no Estructurales Virales/antagonistas & inhibidoresRESUMEN
5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. The structure-activity relationship (SAR) associated with variation of the pyridazinone 2- and 6-substituents is discussed. The synthesis and metabolic stability of this new class of compounds are also described.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Piridazinas/química , Piridazinas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Cristalografía por Rayos X , Diseño de Fármacos , Estabilidad de Medicamentos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Humanos , Concentración 50 Inhibidora , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Piridazinas/síntesis química , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , Tiadiazinas/síntesis química , Tiadiazinas/química , Tiadiazinas/farmacología , Proteínas no Estructurales Virales/metabolismoRESUMEN
5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. The synthesis, structure-activity relationships (SAR), metabolic stability, and structure-based design approach for this new class of compounds are discussed.
Asunto(s)
Benzotiadiazinas/química , Benzotiadiazinas/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Piridazinas/química , Piridazinas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Benzotiadiazinas/metabolismo , Cristalografía por Rayos X , Diseño de Fármacos , Estabilidad de Medicamentos , Inhibidores Enzimáticos/metabolismo , Humanos , Enlace de Hidrógeno , Concentración 50 Inhibidora , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Piridazinas/metabolismo , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismoRESUMEN
The synthesis of 4-(1',1'-dioxo-1',4'-dihydro-1'lambda(6)-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones bearing 6-amino substituents as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC(50)<0.10 microM). In vitro DMPK data (microsome t(1/2), Caco-2 P(app)) for many of the compounds are also disclosed, and a crystal structure of a representative inhibitor complexed with the NS5B protein is discussed.
Asunto(s)
Antivirales/síntesis química , Química Farmacéutica/métodos , Óxidos S-Cíclicos/síntesis química , Piridazinas/química , Piridazinas/síntesis química , Tiadiazinas/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Antivirales/farmacología , Células CACO-2 , Cristalografía por Rayos X/métodos , Óxidos S-Cíclicos/farmacología , ARN Polimerasas Dirigidas por ADN/química , Diseño de Fármacos , Genotipo , Humanos , Concentración 50 Inhibidora , Microsomas/metabolismo , Modelos Químicos , Conformación Molecular , Piridazinas/farmacología , Relación Estructura-Actividad , Tiadiazinas/farmacologíaRESUMEN
Hexahydro-pyrrolo- and hexahydro-1H-pyrido[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Among these, compound 4c displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b) <10 nM; EC(50) (1b)=34 nM) as well as good stability towards human liver microsomes (HLM t(1/2) =59 min).
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , Piridazinas/síntesis química , Piridazinas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/química , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Estructura Molecular , Piridazinas/química , Relación Estructura-ActividadRESUMEN
5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. Lead optimization led to the discovery of compound 3a, which displayed potent inhibitory activities in biochemical and replicon assays [IC(50) (1b)<10nM; IC(50) (1a)=22 nM; EC(50) (1b)=5nM], good stability toward human liver microsomes (HLM t(1/2)>60 min), and high ratios of liver to plasma concentrations 12h after a single oral administration to rats.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacocinética , Hepacivirus/efectos de los fármacos , Piridazinas/síntesis química , Piridazinas/farmacocinética , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Proteínas no Estructurales Virales/antagonistas & inhibidores , Administración Oral , Animales , Antivirales/sangre , Antivirales/química , Técnicas Químicas Combinatorias , Diseño de Fármacos , Humanos , Microsomas Hepáticos/efectos de los fármacos , Estructura Molecular , Piridazinas/sangre , Piridazinas/química , Ratas , Relación Estructura-ActividadRESUMEN
A novel series of HCV NS5B polymerase inhibitors comprising 1,1-dioxoisothiazoles and benzo[b]thiophene-1,1-dioxides were designed, synthesized, and evaluated. SAR studies guided by structure-based design led to the identification of a number of potent NS5B inhibitors with nanomolar IC(50) values. The most potent compound exhibited IC(50) less than 10nM against the genotype 1b HCV polymerase and EC(50) of 70 nM against a genotype 1b replicon in cell culture. The DMPK properties of selected compounds were also evaluated.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacocinética , Inhibidores Enzimáticos/farmacocinética , Tiazoles/síntesis química , Tiofenos/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Química Farmacéutica/métodos , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Genotipo , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Conformación Molecular , ARN Viral/metabolismo , Relación Estructura-Actividad , Tiazoles/farmacocinética , Tiofenos/farmacocinéticaRESUMEN
Pyrrolo[1,2-b]pyridazin-2-one analogs were discovered as a novel class of inhibitors of genotype 1 HCV NS5B polymerase. Structure-based design led to the discovery of compound 3 k, which displayed potent inhibitory activities in biochemical and replicon assays (IC(50) (1b)<10nM; EC(50) (1b)=12 nM) as well as good stability towards human liver microsomes (HLM t(1/2)>60 min).
Asunto(s)
Antivirales/farmacología , Piridazinas/farmacología , Pirroles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/síntesis química , Antivirales/química , Sitios de Unión/efectos de los fármacos , Línea Celular , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Piridazinas/síntesis química , Piridazinas/química , Pirroles/síntesis química , Pirroles/química , Relación Estructura-Actividad , Proteínas no Estructurales Virales/químicaRESUMEN
4-(1,1-Dioxo-1,4-dihydro-1lambda(6)-benzo[1,4]thiazin-3-yl)-5-hydroxy-2H-pyridazin-3-one analogs were discovered as a novel class of inhibitors of HCV NS5B polymerase. Structure-based design led to the identification of compound 3a that displayed potent inhibitory activities in biochemical and replicon assays (1b IC(50)<10 nM; 1b EC(50)=1.1 nM) as well as good stability toward human liver microsomes (HLM t(1/2)>60 min).
Asunto(s)
Química Farmacéutica/métodos , Hepacivirus/enzimología , Microsomas Hepáticos/enzimología , Piridazinas/síntesis química , Piridazinas/farmacología , Tiazinas/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Células CACO-2 , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Hepacivirus/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Conformación Molecular , Piridazinas/química , Relación Estructura-Actividad , Tiazinas/química , Tiazinas/farmacología , Factores de TiempoRESUMEN
The enzyme 17beta-hydroxysteroid dehydrogenase type 10 (HSD10), also known as amyloid beta-peptide-binding alcohol dehydrogenase (ABAD), has been implicated in the development of Alzheimer's disease. This protein, a member of the short-chain dehydrogenase/reductase family of enzymes, has been shown to bind beta-amyloid and to participate in beta-amyloid neurotoxicity. We have determined the crystal structure of human ABAD/HSD10 complexed with NAD(+) and an inhibitory small molecule. The inhibitor occupies the substrate-binding site and forms a covalent adduct with the NAD(+) cofactor. The crystal structure provides a basis for the design of potent, highly specific ABAD/HSD10 inhibitors with potential application in the treatment of Alzheimer's disease.
Asunto(s)
3-Hidroxiacil-CoA Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiacil-CoA Deshidrogenasas/química , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , 3-Hidroxiacil-CoA Deshidrogenasas/genética , 3-Hidroxiacil-CoA Deshidrogenasas/metabolismo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/metabolismo , Secuencia de Bases , Dominio Catalítico , Cristalografía por Rayos X , ADN Complementario/genética , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NAD/química , Conformación Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The hepatitis C (HCV) internal ribosome entry site (IRES) element plays a central role in cap-independent translation of the viral genomic RNA. The unique conformation of IRES domain II is critical for 80S ribosomal assembly and initiation of viral translation. Here, the crystal structure of subdomain IIa of the HCV IRES has been determined at 2.3 A resolution, revealing the positions of divalent metal ions and complex inter-strand interactions that stabilize the L-shaped conformation of the RNA. The presence of divalent metal ions was necessary for crystal formation. Magnesium ions occupy specific sites that appear to be critical for the formation of the folded conformation. Subdomain IIa also was crystallized in the presence of strontium, which improved the diffraction quality of the crystals and the ability to identify interactions of the RNA with metal ions and tightly bound water molecules. The hinge region and noncanonical G-U base-pair motifs are stabilized by divalent metal ions and provide unique structural features that are potential interaction sites for small-molecule ligands. The information obtained from the crystal structure provides a basis for structure-guided design of HCV translation inhibitors targeting disruption of ribosomal assembly.