A High-Throughput Screening Strategy for Development of RNF8-Ubc13 Protein-Protein Interaction Inhibitors.
SLAS Discov
; 22(3): 316-323, 2017 03.
Article
en En
| MEDLINE
| ID: mdl-27909234
ABSTRACT
The ubiquitin-proteasome system plays an essential role in a broad range of cellular signaling pathways. Ubiquitination is a posttranslational protein modification that involves the action of an enzymatic cascade (E1, E2, and E3 enzymes) for the covalent attachment of ubiquitin to target proteins. The emerging knowledge of the molecular mechanisms and correlation of deregulation of the ubiquitin system in human diseases is uncovering new opportunities for therapeutics development. The E3 ligase RNF8 acts in cooperation with the heterodimeric E2 enzyme Ubc13/Uev1a to generate ubiquitin conjugates at the sides of DNA double-strand breaks, and recent findings suggest RNF8 as a potential therapeutic target for the treatment of breast cancer. Here, we present a novel high-throughput screening (HTS)-compatible assay based on the AlphaScreen technology to identify inhibitors of the RNF8-Ubc13 protein-protein interaction, along with a follow-up strategy for subsequent validation. We have adapted the AlphaScreen assay to a 384-well format and demonstrate its reliability, reproducibility, and suitability for automated HTS campaigns. In addition, we have established a biochemical orthogonal homogeneous time-resolved fluorescence (HTRF) assay in HTS format and a cellular microscopy-based assay allowing verification of the primary hits. This strategy will be useful for drug screening programs aimed at RNF8-Ubc13 modulation.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Factores de Transcripción
/
Cisteína Endopeptidasas
/
Procesamiento Proteico-Postraduccional
/
Enzimas Ubiquitina-Conjugadoras
/
Ubiquitina-Proteína Ligasas
/
Proteínas de Unión al ADN
/
Roturas del ADN de Doble Cadena
/
Antineoplásicos
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
/
Screening_studies
Límite:
Humans
Idioma:
En
Revista:
SLAS Discov
Año:
2017
Tipo del documento:
Article
País de afiliación:
Alemania