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Binding of the geldanamycin derivative 17 DMAG to Hsp90 measured with fluorescence label and label free

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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.

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