A standard operating procedure for identification of drug targets in living cells based on cell thermal shift assay (CETSA)

Abstract:

[Objective] Cell thermal shift assay (CETSA) is a biophysical technique allowing the direct study of drug (ligand) binding to proteins (targets) in cells and tissues by measuring variation in the proteins’ thermal stability upon ligand binding. In this paper, taking panobinostat, a drug targeting multiple myeloma, as an example, we developed a standard operating procedure for the identification of drug targets in K562 cells with Western blotting and CETSA. [Methods] Experimental procedures included treating cells with the drug, heat treatment of cells, cell lysis, total soluble protein extraction and quantitation by Western blotting with specific antibodies. [Results] Through quantification by Western blotting and curve fitting, we obtained the CETSA melting curve and isothermal dose-response curve of histone deacetylase (HDAC1), syntaxin-4 (STX4), and tetratricopeptide repeat protein 38 (TTC38) in K562 cells, respectively.[Conclusion] HDAC1, STX4, and TTC38 were the targets of panobinostat in K562 cells. A standard operating procedure for identifying the target proteins in living cells was established by CESTA and Western blotting. The experiment can be completed in 2-3 d.

Key words:cell thermal shift assay (CETSA);living cell;Western blotting;identification of drug targets