Protein-Small Molecule Interaction Analysis
Application Note NT-MO-001
Binding of the geldanamycin derivative 17-DMAG to Hsp90
measured with fluorescence label and label-free
Stephen H. McLaughlin
MRC Laboratory for Molecular Biology, Cambridge, UK
Abstract
For proper folding, many proteins involved in
signal-transduction pathways, cell-cycle
regulation and apoptosis depend upon the
ATP-dependent molecular chaperone Hsp90.
Consequently Hsp90 turned out to be an
attractive target for cancer therapeutics. In this
study we demonstrate the binding of the
geldanamycin derivative 17-DMAG to Hsp90
using MicroScale Thermophoresis (MST). The
study also highlights the high content
information of the MST measurements as one
important benefit of MicroScale
Thermophoresis.
Introduction
The cytosolic heat shock protein 90 (Hsp90) is the
focus of several drug discovery programs for anti-
cancer therapy. The action of Hsp90 underpins
the maintenance of the transformed state through
its function in the conformational maturation and
activation of many client proteins involved in many
of the pathways that hallmark cancer.
Consequently, cancer cells are vulnerable to
Hsp90 inactivation (Whitesell et al., 2005).
Geldanamycin, its derivates like 17-DMAG and all
the Hsp90 inhibitors in current clinical trials (Trepel
et al., 2010) target the ATP-binding site of Hsp90
(Fig. 1) preventing its ATPase dependent activity,
which is essential for function in vivo (Panaretou
et al. 1998). Inhibition of Hsp90 leads to a
reduction of cellular levels of oncogenic client
proteins, such as mutated p53, Akt, Bcr-Abl, and
ErbB2 (McDonald et al. 2006). Wild-type human
Hsp90 binds DMAG with a K
d
of 0.35 ± 0.04 μM in
vitro. The stoichiometry of binding was close to a
1:1 ratio of ligand to Hsp90 monomer (Onuoha et
al. 2007).
Fig. 1 Crystal structure showing 17-DMAG in complex with
Hsp90.
Results
In this study, we have investigated the binding of
the geldanamycin derivative 17-DMAG to wild-
type human Hsp90 using MicroScale
Thermophoresis.