RESUMEN
Familial dysautonomia (FD) is an autonomic and sensory neuropathy caused by a mutation in the splice donor site of intron 20 of the ELP1 gene. Variable skipping of exon 20 leads to a tissue-specific reduction in the level of ELP1 protein. We have shown that the plant cytokinin kinetin is able to increase cellular ELP1 protein levels in vivo and in vitro through correction of ELP1 splicing. Studies in FD patients determined that kinetin is not a practical therapy due to low potency and rapid elimination. To identify molecules with improved potency and efficacy, we developed a cell-based luciferase splicing assay by inserting renilla (Rluc) and firefly (Fluc) luciferase reporters into our previously well-characterized ELP1 minigene construct. Evaluation of the Fluc/Rluc signal ratio enables a fast and accurate way to measure exon 20 inclusion. Further, we developed a secondary assay that measures ELP1 splicing in FD patient-derived fibroblasts. Here we demonstrate the quality and reproducibility of our screening method. Development and implementation of this screening platform has allowed us to efficiently screen for new compounds that robustly and specifically enhance ELP1 pre-mRNA splicing.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Disautonomía Familiar/genética , Precursores del ARN/genética , Empalme del ARN/efectos de los fármacos , ARN Mensajero/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Elongación Transcripcional/genética , Línea Celular , Citocininas/farmacología , Exones/efectos de los fármacos , Exones/genética , Células HEK293 , Humanos , Cinetina/farmacología , Empalme del ARN/genéticaRESUMEN
We have continued to explore spirobenzazepines as vasopressin receptor antagonists to follow up on RWJ-339489 (2), which had advanced into preclinical development. Further structural modifications were pursued to find a suitable backup compound for human clinical studies. Thus, we identified carboxylic acid derivative 3 (RWJ-676070; JNJ-17158063) as a potent, balanced vasopressin V(1a)/V(2) receptor antagonist with favorable properties for clinical development. Compound 3 is currently undergoing human clinical investigation.
Asunto(s)
Antagonistas de los Receptores de Hormonas Antidiuréticas , Benzazepinas/química , Compuestos de Espiro/química , Animales , Antihipertensivos/administración & dosificación , Antihipertensivos/química , Antihipertensivos/metabolismo , Antihipertensivos/farmacocinética , Benzazepinas/administración & dosificación , Benzazepinas/farmacocinética , Benzazepinas/farmacología , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Masculino , Ratas , Ratas Long-Evans , Receptores de Vasopresinas/metabolismo , Receptores de Vasopresinas/fisiología , Compuestos de Espiro/administración & dosificación , Compuestos de Espiro/metabolismo , Compuestos de Espiro/farmacocinética , Compuestos de Espiro/farmacología , Vasopresinas/metabolismoRESUMEN
The no-wash calcium assay kits developed by Molecular Devices Corporation have greatly enhanced the throughput of cell-based calcium mobilization high-throughput screening (HTS) assays and enabled screening using nonadherent cells. The fluorescent imaging plate reader (FLIPR) Calcium 3 Assay Kit, optimal for targets that have proteins or peptides as agonists, has 2 potential drawbacks: 1) a significant downward spike in fluorescence signal upon liquid transfer that can be the same magnitude as the agonist response, making data analysis difficult; and 2) medium removal is required for some targets, which essentially reintroduces a wash step. Several no-wash products were introduced in 2005. The authors compare the Fluo-4 NW Calcium Assay Kit and the BD Calcium Assay Kit with the FLIPR Calcium 3 Assay Kit using human native rhabdomyosarcoma cells expressing the urotensin-II receptor (UT). The BDtrade mark Calcium Assay Kit gives the best performance in the true no-wash mode, in which both agonist and antagonist activity are easily quantified. Although these new products provide additional options for measuring calcium mobilization, the different results observed with each kit, using the UT receptor as an example, suggest that one should characterize all dyes against each target in a systematic way prior to choosing one for HTS.