RESUMEN
Glucocorticoids (GCs)-ligands of the glucocorticoid receptor (GR)-are widely used to treat inflammatory diseases, but suffer from significant side effects and poor responsiveness in certain patient populations. Identification of chemical GR modulators may provide insights into the regulatory mechanisms of anti-inflammatory functions of GR and help improve GC-based therapy. Here we report the development and application of a high-throughput screening to identify compounds that either enhance or suppress the anti-inflammatory effect of GR function. Using a cell-based GR activity assay that measures Dexamethasone (Dex)-mediated NF-κB repression, we have screened ~8,000 compounds and identified several compounds that suppressed GR activity, including multiple GSK3ß inhibitors and anti-cancer agent camptothecin. Notably, we also identified two kinase IKK2 inhibitors, including TPCA-1, as GR enhancers that improve the anti-inflammatory effect of GR. In particular, TPCA-1 augmented the activity of Dex in NF-κB repression by attenuating GR down-regulation. Consistent with the observation, siRNA-mediated IKK2 knockdown decreased GR down-regulation and increased GR expression. Together, our results identified chemical compounds as novel modulators of GR and revealed an unexpected role for IKK2 in GR down-regulation. Furthermore, we have established a high-throughput screening platform for discovering GR-modulating compounds that may be repurposed to improve current GC-based therapies.
Asunto(s)
Amidas/farmacología , Antiinflamatorios/farmacología , Evaluación Preclínica de Medicamentos/métodos , Glucocorticoides/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Glucocorticoides/metabolismo , Tiofenos/farmacología , Células A549 , Amidas/análisis , Camptotecina/análisis , Camptotecina/farmacología , Línea Celular , Dexametasona/farmacología , Reposicionamiento de Medicamentos , Sinergismo Farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Humanos , FN-kappa B/metabolismo , Inhibidores de Proteínas Quinasas/análisis , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/farmacología , Tiofenos/análisisRESUMEN
Membrane transporters can be major determinants of the pharmacokinetic, safety and efficacy profiles of drugs. This presents several key questions for drug development, including which transporters are clinically important in drug absorption and disposition, and which in vitro methods are suitable for studying drug interactions with these transporters. In addition, what criteria should trigger follow-up clinical studies, and which clinical studies should be conducted if needed. In this article, we provide the recommendations of the International Transporter Consortium on these issues, and present decision trees that are intended to help guide clinical studies on the currently recognized most important drug transporter interactions. The recommendations are generally intended to support clinical development and filing of a new drug application. Overall, it is advised that the timing of transporter investigations should be driven by efficacy, safety and clinical trial enrolment questions (for example, exclusion and inclusion criteria), as well as a need for further understanding of the absorption, distribution, metabolism and excretion properties of the drug molecule, and information required for drug labelling.
Asunto(s)
Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Proteínas de Transporte de Membrana/efectos de los fármacos , Proteínas de Transporte de Membrana/metabolismo , Medicamentos bajo Prescripción/farmacocinética , Animales , Simulación por Computador , Árboles de Decisión , Aprobación de Drogas , Interacciones Farmacológicas , Humanos , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Noqueados , Medicamentos bajo Prescripción/efectos adversosRESUMEN
DNA vaccines are ideally suited for immunizing against tumor antigens because constructs can be formulated that not only encode the tumor antigen but also encode molecules chosen to improve the ability to elicit an antitumor response. Ligands expressed on antigen-presenting cells associated with stimulating a robust T-cell response are excellent candidates for inclusion in a DNA vaccine. Mice transgenic for the HER-2/neu homologue, rat neu, were immunized with full-length rat neu cDNA given alone or in combination with plasmids encoding costimulatory molecules CD80 or CD86 and the ligand for CD137 (CD137L). Intradermal injection of the plasmid constructs resulted in both plasmid transcript and antigen protein expression being detected in lymph nodes draining the injection site. Immunization with plasmids encoding the neu antigen along with plasmids encoding CD137L and either CD80 or CD86 resulted in the generation of neu-specific antibodies that induced phopshorylation of the neu tyrosine kinase and inhibited the growth of cultured tumor cells overexpressing neu. Survival of animals was significantly prolonged after immunization with vaccines encoding neu together with the costimulatory molecules. Although tumors eventually occurred in the vaccinated animals, they were markedly infiltrated with CD4+ T cells. DNA vaccines encoding neu, when given in combination with both CD137L and either CD80 or CD86, can induce cellular and humoral immunity and result in an antitumor effect.