RESUMEN
Synthesis and structure-activity relationship (SAR) of a series of alkyl and cycloalkyl containing non-steroidal dissociated glucocorticoid receptor (GR) agonists is reported. This series of compounds was identified as part of an effort to replace the CF3 group in a scaffold represented by 1a. The study culminated in the identification of compound 14, a t-butyl containing derivative, which has shown potent activity for GR, selectivity against the progesterone receptor (PR) and the mineralocorticoid receptor (MR), in vitro anti-inflammatory activity in an IL-6 transrepression assay, and dissociation in a MMTV transactivation counter-screen. In a collagen-induced arthritis mouse model, 14 displayed prednisolone-like efficacy, and lower impact on body fat and free fatty acids than prednisolone at an equivalent anti-inflammatory dose.
Asunto(s)
Descubrimiento de Drogas , Glucocorticoides/síntesis química , Metanol/química , Receptores de Glucocorticoides/agonistas , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/química , Antiinflamatorios/farmacología , Artritis/tratamiento farmacológico , Sitios de Unión , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Glucocorticoides/química , Glucocorticoides/farmacología , Humanos , Concentración 50 Inhibidora , Metanol/síntesis química , Metanol/farmacología , Ratones , Modelos Moleculares , Estructura Molecular , Prednisolona/química , Prednisolona/farmacología , Unión Proteica/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
We report a SAR of non-steroidal glucocorticoid mimetics that utilize indoles as A-ring mimetics. Detailed SAR is discussed with a focus on improving PR and MR selectivity, GR agonism, and in vitro dissociation profile. SAR analysis led to compound (R)-33 which showed high PR and MR selectivity, potent agonist activity, and reduced transactivation activity in the MMTV and aromatase assays. The compound is equipotent to prednisolone in the LPS-TNF model of inflammation. In mouse CIA, at 30 mg/kg compound (R)-33 inhibited disease progression with an efficacy similar to the 3 mg/kg dose of prednisolone.
Asunto(s)
Glucocorticoides/química , Glucocorticoides/farmacología , Indoles/química , Indoles/farmacología , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/metabolismo , Animales , Células HeLa , Humanos , Ratones , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
Numerous assay methods have been developed to identify small-molecule effectors of protein kinases, but no single method can be applied to all isolated kinases. The authors developed a set of 3 high-throughput screening (HTS)-compatible biochemical assays that can measure 3 mechanistically distinct properties of a kinase active site, with the goal that at least 1 of the 3 would be applicable to any kinase selected as a target for drug discovery efforts. Two assays measure catalytically active enzyme: A dissociation-enhanced lanthanide fluoroimmuno assay (DELFIA) uses an antibody to quantitate the generation of phosphorylated substrate; a second assay uses luciferase to measure the consumption of adenosine triphosphate (ATP) during either phosphoryl-transfer to a peptide substrate or to water (intrinsic ATPase activity). A third assay, which is not dependent on a catalytically active enzyme, measures the competition for binding to kinase between an inhibitor and a fluorescent ATP binding site probe. To evaluate the suitability of these assays for drug discovery, the authors compared their ability to identify inhibitors of a nonreceptor protein tyrosine kinase from the Tec family, interleukin-2-inducible T cell kinase (ITK). The 3 assays agreed on 57% of the combined confirmed hit set identified from screening a 10,208-compound library enriched with known kinase inhibitors and molecules that were structurally similar. Among the 3 assays, the one measuring intrinsic ATPase activity produced the largest number of unique hits, the fewest unique misses, and the most comprehensive hit set, missing only 2.7% of the confirmed inhibitors identified by the other 2 assays combined. Based on these data, all 3 assay formats are viable for screening and together provide greater options for assay design depending on the targeted kinase.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Bioensayo/métodos , Inhibidores de Proteínas Quinasas/análisis , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Sitios de Unión/efectos de los fármacos , Colorantes Fluorescentes/química , Humanos , CinéticaRESUMEN
With the transition from manual to robotic HTS in the last several years, assay optimization has become a significant bottleneck. Recent advances in robotic liquid handling have made it feasible to reduce assay optimization timelines with the application of statistically designed experiments. When implemented, they can efficiently optimize assays by rapidly identifying significant factors, complex interactions, and nonlinear responses. This article focuses on the use of statistically designed experiments in assay optimization.
Asunto(s)
Bioensayo/métodos , Proyectos de Investigación , Automatización , Interpretación Estadística de Datos , Procesamiento de Imagen Asistido por Computador , Reproducibilidad de los ResultadosRESUMEN
We report on the nuclear receptor binding affinities, cellular activities of transrepression and transactivation, and anti-inflammatory properties of a quinol-4-one and other A-ring mimetic containing nonsteroidal class of glucocorticoid agonists.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Glucocorticoides/agonistas , Imitación Molecular , Quinolonas/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Transactivadores/farmacología , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Aromatasa/metabolismo , Dexametasona/farmacología , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HeLa , Humanos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos BALB C , Quinolonas/síntesis química , Quinolonas/química , Transactivadores/síntesis química , Transactivadores/química , Activación TranscripcionalRESUMEN
Sphingosine kinase 1 (SphK1) is a lipid kinase that phosphorylates sphingosine to produce the bioactive sphingolipid, sphingosine-1-phosphate (S1P), and therefore represents a potential drug target for a variety of pathological processes such as fibrosis, inflammation, and cancer. We developed two assays compatible with high-throughput screening to identify small-molecule inhibitors of SphK1: a purified component enzyme assay and a genetic complementation assay in yeast cells. The biochemical enzyme assay measures the phosphorylation of sphingosine-fluorescein to S1P-fluorescein by recombinant human full-length SphK1 using an immobilized metal affinity for phosphochemicals (IMAP) time-resolved fluorescence resonance energy transfer format. The yeast assay employs an engineered strain of Saccharomyces cerevisiae, in which the human gene encoding SphK1 replaced the yeast ortholog and quantitates cell viability by measuring intracellular adenosine 5'-triphosphate (ATP) using a luciferase-based luminescent readout. In this assay, expression of human SphK1 was toxic, and the resulting yeast cell death was prevented by SphK1 inhibitors. We optimized both assays in a 384-well format and screened â¼10(6) compounds selected from the Boehringer Ingelheim library. The biochemical IMAP high-throughput screen identified 5,561 concentration-responsive hits, most of which were ATP competitive and not selective over sphingosine kinase 2 (SphK2). The yeast screen identified 205 concentration-responsive hits, including several distinct compound series that were selective against SphK2 and were not ATP competitive.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Pruebas de Enzimas/métodos , Humanos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
The emergence of sphingosine-1-phosphate lyase (SPL) as a promising therapeutic target for inflammatory diseases has heightened interest in the identification of small molecules that modulate its activity. The enzymatic activity of SPL is typically measured using radiometric or fluorescence-based assays that require a lipid extraction step, or by direct quantitation of reaction products using mass spectrometry (MS). To facilitate testing large numbers of compounds to identify SPL modulators, we developed a robust scintillation proximity assay (SPA) that is compatible with high-throughput screening (HTS). This assay employs recombinant human full-length SPL in insect cell membrane preparations to catalyze the conversion of biotinylated aminosphingosine-1-[(33)P]phosphate (S1(33)P-biotin) to trans-2-hexadecenal-biotin and ethanolamine [(33)P]phosphate. To validate the SPA and confirm the fidelity of its measurement of SPL enzyme activity, we developed a Rapid-Fire MS method that quantitates nonradiolabeled S1P-biotin. In addition, we developed a simple, scalable method to produce S1(33)P-biotin in quantities sufficient for HTS. The optimized SPA screen in 384-well microplates produced a mean plate-wise Z'-statistic of 0.58 across approximately 3,000 plates and identified several distinct structural classes of SPL inhibitor. Among the inhibitors that the screen identified was one compound with an IC50 of 1.6 µM in the SPA that induced dose-dependent lymphopenia in mice.
Asunto(s)
Aldehído-Liasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Conteo por Cintilación , Aldehído-Liasas/metabolismo , Animales , Biocatálisis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Humanos , Linfopenia/tratamiento farmacológico , Linfopenia/enzimología , Linfopenia/metabolismo , Espectrometría de Masas , Ratones , Estructura Molecular , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Synthesis and structure-activity relationship (SAR) of a series of nonsteroidal glucocorticoid receptor (GR) agonists are described. These compounds contain "diazaindole" moieties and display different transcriptional regulatory profiles in vitro and are considered "dissociated" between gene transrepression and transactivation. The lead optimization effort described in this article focused in particular on limiting the transactivation of genes which result in bone side effects and these were assessed in vitro in MG-63 osteosarcoma cells, leading to the identification of (R)-18 and (R)-21. These compounds maintained anti-inflammatory activity in vivo in collagen induced arthritis studies in mouse but had reduced effects on bone relevant parameters compared to the widely used synthetic glucocorticoid prednisolone 2 in vivo. To our knowledge, we are the first to report on selective glucocorticoid ligands with reduced bone loss in a preclinical in vivo model.
Asunto(s)
Huesos/efectos de los fármacos , Receptores de Glucocorticoides/agonistas , Animales , Línea Celular Tumoral , Femenino , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Relación Estructura-ActividadRESUMEN
A series of nonsteroidal "dissociated" glucocorticoid receptor agonists was optimized for drug-like properties such as cytochrome P450 inhibition, metabolic stability, aqueous solubility, and hERG ion channel inhibition. This effort culminated in the identification of the clinical candidate compound ( R )-39.
RESUMEN
Syntheses and structure-activity relationships (SAR) of nonsteroidal glucocorticoid receptor (GR) agonists are described. These compounds contain azaindole moieties as A-ring mimetics and display various degrees of in vitro dissociation between gene transrepression and transactivation. Collagen induced arthritis studies in mouse have demonstrated that in vitro dissociated compounds (R)-16 and (R)-37 have steroid-like anti-inflammatory properties with improved metabolic side effect profiles, such as a reduced increase in body fat and serum insulin levels, compared to steroids.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Piridinas/síntesis química , Pirroles/síntesis química , Receptores de Glucocorticoides/agonistas , Esteroides/química , Tejido Adiposo/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/efectos adversos , Antiinflamatorios no Esteroideos/farmacología , Aromatasa/biosíntesis , Aromatasa/genética , Inhibidores de la Aromatasa/farmacología , Artritis Experimental/tratamiento farmacológico , Disponibilidad Biológica , Células Cultivadas , Inducción Enzimática , Femenino , Humanos , Enlace de Hidrógeno , Insulina/sangre , Interleucina-1/farmacología , Interleucina-6/antagonistas & inhibidores , Interleucina-6/biosíntesis , Interleucina-6/genética , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Piridinas/efectos adversos , Piridinas/farmacología , Pirroles/efectos adversos , Pirroles/farmacología , Ratas , Ratas Sprague-Dawley , Estereoisomerismo , Relación Estructura-Actividad , Activación TranscripcionalRESUMEN
We have recently reported the discovery of a novel class of glucocorticoid receptor (GR) antagonists, exemplified by 3, containing a 1,2-dihydroquinoline molecular scaffold. Further SAR studies of these antagonists uncovered chemical modifications conveying agonist functional activity to this series. These agonists exhibit good GR binding affinity and are selective against other nuclear hormone receptors.
Asunto(s)
Quinolinas/química , Quinolinas/farmacología , Receptores de Glucocorticoides/agonistas , Quinolinas/metabolismo , Receptores de Glucocorticoides/metabolismo , Relación Estructura-ActividadRESUMEN
Discovery of the pyrazole-naphthyl urea class of p38 MAP kinase inhibitors typified by the clinical candidate BIRB 796 has encouraged further exploration of this particular scaffold. Modification to the part of the inhibitor that occupies the adenine/ATP binding site has resulted in a new way to obtain potent inhibitors that possess favorable in vitro and in vivo properties.
Asunto(s)
Adenina/metabolismo , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Sitios de Unión , Humanos , Modelos Moleculares , Inhibidores de Proteínas Quinasas/metabolismo , Relación Estructura-Actividad , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
We report the discovery of a novel class of glucocorticoid receptor (GR) ligands based on 1,2-dihydroquinoline molecular scaffold. The compounds exhibit good GR binding affinity and selectivity profile against other nuclear hormone receptors.
Asunto(s)
Quinolinas/síntesis química , Quinolinas/farmacología , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/metabolismo , Antiinflamatorios/farmacología , Dexametasona/farmacología , Células HeLa , Humanos , Interleucina-1/farmacología , Interleucina-6/metabolismo , Ligandos , Virus del Tumor Mamario del Ratón/genética , Modelos Moleculares , Estructura Molecular , Regiones Promotoras Genéticas/efectos de los fármacos , Quinolinas/química , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Receptores de Mineralocorticoides/efectos de los fármacos , Receptores de Mineralocorticoides/metabolismo , Receptores de Progesterona/efectos de los fármacos , Receptores de Progesterona/metabolismo , Relación Estructura-Actividad , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genéticaRESUMEN
Compound 1, a potent glucocorticoid receptor ligand, contains a quaternary carbon bearing trifluoromethyl and hydroxyl groups. This paper describes the effect of replacing the trifluoromethyl group on binding and agonist activity of the GR ligand 1. The results illustrate that replacing the CF3 group with a cyclohexylmethyl or benzyl group maintains the GR binding potency. These substitutions alter the functional behavior of the GR ligands from agonists to antagonists. Docking studies suggest that the benzyl analog 19 binds in a similar fashion as the GR antagonist, RU486. The central benzyl group of 19 and the C-11 dimethylaniline moiety of RU486 overlay. Binding of compound 19 is believed to force helix 12 to adopt an open conformation and this leads to the antagonist properties of the non-CF3 ligands carrying a large group at the center of the molecule.