Development of a Cell-Based Ligand-Screening System for Identifying Hsp90 Inhibitors

Because
of its critical roles in regulating cellular signal transduction,
the molecular chaperone heat-shock protein 90 (Hsp90) has become a
novel therapeutic target for various diseases, including cancer, inflammation,
and neurological diseases. However, the lack of methods that allow
us to directly evaluate the binding of small molecule ligands to intracellular
Hsp90 makes the inhibitor development more difficult. Here, we report
a simple cell-based assay system for the Hsp90 inhibitor in live-cell
environments. In this strategy, the binding activity of ligands of
interest is evaluated by competitive inhibition of ligand-directed N-acyl-N-alkyl sulfonamide (LDNASA) chemistry-mediated
Hsp90 labeling. Using several known Hsp90 inhibitors, we demonstrated
that our method could easily detect the ligand-binding event of Hsp90
in live cells. Our system is applicable to high-throughput ligand
screening, and we discovered a new small molecule candidate that binds
to the N-terminal ATP binding domain of Hsp90. These results demonstrate
the use of the competitive LDNASA-based approach to directly evaluate
ligand activity in live cells and identify potent drug candidates
from chemical libraries.