Chaperone mediated detection of small molecule target binding in cells.
Nat Commun
; 11(1): 465, 2020 01 23.
Article
em En
| MEDLINE
| ID: mdl-31974362
ABSTRACT
The ability to quantitatively measure a small molecule's interactions with its protein target(s) is crucial for both mechanistic studies of signaling pathways and in drug discovery. However, current methods to achieve this have specific requirements that can limit their application or interpretation. Here we describe a complementary target-engagement method, HIPStA (Heat Shock Protein Inhibition Protein Stability Assay), a high-throughput method to assess small molecule binding to endogenous, unmodified target protein(s) in cells. The methodology relies on the change in protein turnover when chaperones, such as HSP90, are inhibited and the stabilization effect that drug-target binding has on this change. We use HIPStA to measure drug binding to three different classes of drug targets (receptor tyrosine kinases, nuclear hormone receptors, and cytoplasmic protein kinases), via quantitative fluorescence imaging. We further demonstrate its utility by pairing the method with quantitative mass spectrometry to identify previously unknown targets of a receptor tyrosine kinase inhibitor.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Chaperonas Moleculares
/
Proteínas de Choque Térmico HSP90
/
Bibliotecas de Moléculas Pequenas
/
Ensaios de Triagem em Larga Escala
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Nat Commun
Assunto da revista:
BIOLOGIA
/
CIENCIA
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Estados Unidos