A Tandem-Affinity Purification Method for Identification of Primary Intracellular Drug-Binding Proteins

  • ACS Chem Biol. 2024 Feb 16;19(2):233-242. doi: 10.1021/acschembio.3c00570.
Sehbanul Islam  1 ,  Jitendra Gour  2 ,  Thomas Beer  2 ,  Hsin-Yao Tang  2 ,  Joel Cassel  3 ,  Joseph M Salvino  2 ,  Luca Busino  1
Affiliations
  • 1. University of Pennsylvania, Perelman School of Medicine, Department of Cancer Biology, Philadelphia, Pennsylvania 19104, United States.
  • 2. Medicinal Chemistry and Molecular and Cellular Oncogenesis (MCO) Program, The Wistar Institute, Philadelphia, Pennsylvania 19104, United States.
  • 3. Molecular Screening and Protein Expression Shared Resource, The Wistar Institute, Philadelphia, Pennsylvania 19104, United States.
PMID: 38271588 DOI: 10.1021/acschembio.3c00570
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 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.

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