HSP90 binding assay

H1975 cells, cultured in RPMI-1640 and 10% FBS, were seeded at a density of 3 × 10⁵ cells per well in 6 well plates. Twenty-four hours later, cells were treated with ganetespib as indicated and incubated at 37°C. Cells were washed twice in cold PBS then lysed in cold HSP90 binding buffer (20mM HEPES pH 7.3, 1mM EDTA, 100mM KCl, 5mM MgCl, 0.01% v/v NP-40, 0.5mg/mL bovine gamma globulin, 1mM TCEP) by incubation on ice for 10 minutes followed by three freeze/thaw cycles. Lysates were clarified by centrifugation at 14,000 x g. To remove unbound ganetespib, lysates were passed over 40K MWCO size exclusion columns (Thermo Fisher Scientific). To titrate unoccupied HSP90 binding sites, 10 μM of a deuterated form of ganetespib (D3-ganetespib) was added to eluates and incubated at 4°C for 2 hours then passed over a size exclusion column to remove unbound D3-ganetespib. Total protein from flow through was quantified by BCA protein assay and all samples diluted to 1 mg/mL. The concentrations of ganetespib and D3-ganetespib were measured by LC/MS-MS. A Phenomenex Kinetex column (C18, 30 × 2.1 mm, 2.6 µm) was used with a run time of 3.5 minutes per sample. The following equation was used to calculate the percent of ganetespib bound to HSP90 (HSP90 occupancy): [ganetespib]/([ganetespib] + [D3-ganetespib]) x 100.