RESUMEN
2',5'-Oligoadenylate synthetase (OAS) enzymes and RNase-L constitute a major effector arm of interferon (IFN)-mediated antiviral defense. OAS produces a unique oligonucleotide second messenger, 2',5'-oligoadenylate (2-5A), that binds and activates RNase-L. This pathway is down-regulated by virus- and host-encoded enzymes that degrade 2-5A. Phosphodiesterase 12 (PDE12) was the first cellular 2-5A- degrading enzyme to be purified and described at a molecular level. Inhibition of PDE12 may up-regulate the OAS/RNase-L pathway in response to viral infection resulting in increased resistance to a variety of viral pathogens. We generated a PDE12-null cell line, HeLaΔPDE12, using transcription activator-like effector nuclease-mediated gene inactivation. This cell line has increased 2-5A levels in response to IFN and poly(I-C), a double-stranded RNA mimic compared with the parental cell line. Moreover, HeLaΔPDE12 cells were resistant to viral pathogens, including encephalomyocarditis virus, human rhinovirus, and respiratory syncytial virus. Based on these results, we used DNA-encoded chemical library screening to identify starting points for inhibitor lead optimization. Compounds derived from this effort raise 2-5A levels and exhibit antiviral activity comparable with the effects observed with PDE12 gene inactivation. The crystal structure of PDE12 complexed with an inhibitor was solved providing insights into the structure-activity relationships of inhibitor potency and selectivity.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/inmunología , Antivirales/farmacología , Endorribonucleasas/inmunología , Exorribonucleasas/química , Inmunidad Innata , Bibliotecas de Moléculas Pequeñas/farmacología , 2',5'-Oligoadenilato Sintetasa/genética , Nucleótidos de Adenina/inmunología , Nucleótidos de Adenina/metabolismo , Antivirales/síntesis química , Cristalografía por Rayos X , Virus de la Encefalomiocarditis/genética , Virus de la Encefalomiocarditis/metabolismo , Endorribonucleasas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Exorribonucleasas/antagonistas & inhibidores , Exorribonucleasas/genética , Exorribonucleasas/inmunología , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Interferón-alfa/farmacología , Modelos Moleculares , Oligorribonucleótidos/inmunología , Oligorribonucleótidos/metabolismo , Poli I-C/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Virus Sincitiales Respiratorios/genética , Virus Sincitiales Respiratorios/metabolismo , Rhinovirus/genética , Rhinovirus/metabolismo , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/síntesis química , Relación Estructura-ActividadRESUMEN
To identify novel antivirals to the hepatitis C virus (HCV) NS4B protein, we utilized encoded library technology (ELT), which enables purified proteins not amenable to standard biochemical screening methods to be tested against large combinatorial libraries in a short period of time. We tested NS4B against several DNA-encoded combinatorial libraries (DEL) and identified a single DEL feature that was subsequently progressed to off-DNA synthesis. The most active of the initial synthesized compounds had 50% inhibitory concentrations (IC50s) of 50 to 130 nM in a NS4B radioligand binding assay and 300 to 500 nM in an HCV replicon assay. Chemical optimization yielded compounds with potencies as low as 20 nM in an HCV genotype 1b replicon assay, 500 nM against genotype 1a, and 5 µM against genotype 2a. Through testing against other genotypes and genotype 2a-1b chimeric replicons and from resistance passage using the genotype 1b replicon, we confirmed that these compounds were acting on the proposed first transmembrane region of NS4B. A single sequence change (F98L) was identified as responsible for resistance, and it was thought to largely explain the relative lack of potency of this series against genotype 2a. Unlike other published series that appear to interact with this region, we did not observe sensitivity to amino acid substitutions at positions 94 and 105. The discovery of this novel compound series highlights ELT as a valuable approach for identifying direct-acting antivirals to nonenzymatic targets.
Asunto(s)
Hepacivirus/genética , Replicón/genética , Línea Celular , Genotipo , Humanos , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
Analysis of the x-ray crystal structure of mono-substituted acetylenic thienopyrimidine 6 complexed with the ErbB family enzyme ErbB-4 revealed a covalent bond between the terminal carbon of the acetylene moiety and the sulfhydryl group of Cys-803 at the solvent interface. The identification of this covalent adduct suggested that acetylenic thienopyrimidine 6 and related analogs might also be capable of forming an analogous covalent adduct with EGFR, which has a conserved cysteine (797) near the ATP binding pocket. To test this hypothesis, we treated a truncated, catalytically competent form of EGFR (678-1020) with a structurally related propargylic amine (8). An investigation of the resulting complex by mass spectrometry revealed the formation of a covalent complex of thienopyrimidine 8 with Cys-797 of EGFR. This finding enabled us to readily assess the irreversibility of various inhibitors and also facilitated a structure-activity relationship understanding of the covalent modifying potential and biological activity of a series of acetylenic thienopyrimidine compounds with potent antitumor activity. Several ErbB family enzyme and cell potent 6-ethynyl thienopyrimidine kinase inhibitors were found to form covalent adducts with EGFR.
Asunto(s)
Alquinos/metabolismo , Compuestos de Anilina/metabolismo , Receptores ErbB/metabolismo , Modelos Moleculares , Pirimidinas/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Femenino , Isatina/análogos & derivados , Isatina/metabolismo , Espectrometría de Masas , Ratones , Ratones SCID , Estructura Molecular , Pirimidinas/toxicidad , Proteínas Tirosina Quinasas Receptoras/metabolismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The discovery and development of a series of thiophenes as potent and selective inhibitors of PLK is described. Identification and characterization of 2, a useful in vitro PLK inhibitor tool compound, is also presented.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Química Farmacéutica/métodos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Tiofenos/antagonistas & inhibidores , Animales , Ciclo Celular , Proteínas de Ciclo Celular/química , Línea Celular Tumoral , Proliferación Celular , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Ratones , Mitosis , Modelos Químicos , Conformación Molecular , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/química , Tiofenos/química , Quinasa Tipo Polo 1RESUMEN
Aniline 'headgroups' were synthesized and incorporated into an alkynyl thienopyrimidine series of EGFR and ErbB-2 inhibitors. Potent inhibition of enzyme activity and cellular proliferation was observed. In certain instances, protein binding was reduced and oral exposure was found to be somewhat improved relative to compounds containing the reference aniline.
Asunto(s)
Compuestos de Anilina/síntesis química , Receptores ErbB/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Pirimidinas/síntesis química , Receptor ErbB-2/antagonistas & inhibidores , Administración Oral , Compuestos de Anilina/administración & dosificación , Compuestos de Anilina/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Receptores ErbB/metabolismo , Inhibidores de Crecimiento/administración & dosificación , Inhibidores de Crecimiento/síntesis química , Inhibidores de Crecimiento/farmacología , Humanos , Ratones , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/administración & dosificación , Pirimidinas/farmacología , Receptor ErbB-2/metabolismoRESUMEN
A novel class of pyrrolidinyl-acetyleneic thieno[3,2-d]pyrimidines has been identified which potently inhibit the EGFR and ErbB-2 receptor tyrosine kinases. Synthetic modifications of the pyrrolidine carbamate moiety result in a range of effects on enzyme and cellular potency. In addition, the impact of the absolute stereochemical configuration on cellular potency and oral mouse pharmacokinetics is described.
Asunto(s)
Antineoplásicos/química , Receptores ErbB/antagonistas & inhibidores , Pirimidinas/farmacología , Pirrolidinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Administración Oral , Animales , Ratones , Farmacocinética , Pirimidinas/síntesis química , Pirrolidinas/síntesis química , Relación Estructura-ActividadRESUMEN
A series of thiophene PLK1 inhibitors was optimized for increased solubility and reduced protein binding through the appendage of basic amine functionality. Interesting selectivity between PLK1 and PLK3 was also obtained through these modifications.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Química Farmacéutica/métodos , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Tiofenos/química , Ciclo Celular , Proteínas de Ciclo Celular/química , Línea Celular Tumoral , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Mitosis , Modelos Químicos , Conformación Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/química , Proteínas Proto-Oncogénicas/química , Solubilidad , Proteínas Supresoras de Tumor , Quinasa Tipo Polo 1RESUMEN
[structure: see text] The tetraacetylenic compound, (S)-minquartynoic acid (1), is synthesized in seven linear steps and 17% overall yield from commercially available azelaic acid monomethyl ester. The key step is a one-pot three-component Cadiot-Chodkiewicz reaction to construct the tetrayne unit without using either a diyne or a triyne intermediate.
Asunto(s)
Alquinos/síntesis química , Fármacos Anti-VIH/síntesis química , Antineoplásicos/síntesis química , Ácidos Grasos Insaturados/síntesis química , Alquinos/química , Antihelmínticos/síntesis química , Ácidos Dicarboxílicos/química , Humanos , Plantas Medicinales/química , Poliinos , EstereoisomerismoRESUMEN
A series of imidazo[1,2-a]pyridines which directly bind to HCV Non-Structural Protein 4B (NS4B) is described. This series demonstrates potent in vitro inhibition of HCV replication (EC50 < 10 nM), direct binding to purified NS4B protein (IC50 < 20 nM), and an HCV resistance pattern associated with NS4B (H94N/R, V105L/M, F98L) that are unique among reported HCV clinical assets, suggestive of the potential for additive or synergistic combination with other small molecule inhibitors of HCV replication.