A Tandem-Affinity Purification Method for Identification of Primary Intracellular Drug-Binding Proteins.
Sehbanul IslamJitendra GourThomas BeerHsin-Yao TangJoel CasselJoseph M SalvinoLuca BusinoPublished in: ACS chemical biology (2024)
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 T andem A ffinity P urification for identification of D rug- B inding P roteins (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.
Keyphrases
- small molecule
- mass spectrometry
- adverse drug
- drug induced
- protein protein
- cell proliferation
- induced apoptosis
- cell therapy
- mesenchymal stem cells
- oxidative stress
- social media
- hiv infected
- liquid chromatography
- amino acid
- transcription factor
- electronic health record
- endoplasmic reticulum stress
- data analysis
- hepatitis c virus