A Tandem-Affinity Purification Method for Identification of Primary Intracellular Drug-Binding Proteins.
ACS Chem Biol
; 19(2): 233-242, 2024 02 16.
Article
en En
| MEDLINE
| ID: mdl-38271588
ABSTRACT
In the field of drug discovery, understanding how small molecule drugs interact with cellular components is crucial. Our study introduces a novel methodology to uncover primary drug targets using Tandem Affinity Purification for identification of Drug-Binding Proteins (TAP-DBP). Central to our approach is the generation of a FLAG-hemagglutinin (HA)-tagged chimeric protein featuring the FKBP12(F36V) adaptor protein and the TurboID enzyme. Conjugation of drug molecules with the FKBP12(F36V) ligand allows for the coordinated recruitment of drug-binding partners effectively enabling in-cell TurboID-mediated biotinylation. By employing a tandem affinity purification protocol based on FLAG-immunoprecipitation and streptavidin pulldown, alongside mass spectrometry analysis, TAP-DBP allows for the precise identification of drug-primary binding partners. Overall, this study introduces a systematic, unbiased method for identification of drug-protein interactions, contributing a clear understanding of target engagement and drug selectivity to advance the mode of action of a drug in cells.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Proteínas Portadoras
/
Purificación por Afinidad en Tándem
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
ACS Chem Biol
Año:
2024
Tipo del documento:
Article
País de afiliación:
Estados Unidos