PU-WS13

Kinase Assay [1]
The Hsp90 FP competition assays are carried out in black 96-well micro-plates in a total volume of 100 μL in each well. A stock of 10 μM cy3B-GM and PU-FITC3 is prepared in DMSO and diluted with Felts buffer (20 mM Hepes (K), pH 7.3, 50 mM KCl, 2 mM DTT, 5 mM MgCl2, 20 mM Na2MoO4 and 0.01% NP40 with 0.1 mg/mL BGG). To each well is added the fluorescent dye-labeled Hsp90 ligand (6 nM cy3B-GM for Hsp90α, Hsp90β and Grp94 and 3 nM PU-FITC3 for Trap-1), protein (10 nM Hsp90α, 10 nM Hsp90β, 10 nM Grp94, 30 nM Trap-1) and tested inhibitor (including PU-WS13, initial stock in DMSO) in a final volume of 100 μL Felts buffer. Compounds are added in duplicate or triplicate wells. For each assay, background wells (buffer only), tracer controls (free, fluorescent dye-labeled Hsp90 ligand only) and bound controls (fluorescent dye-labeled Hsp90 ligand in the presence of protein) are included on each assay plate. The assay plate is incubated on a shaker at 4°C for 24 h, and the FP values (in mP) are measured. The fraction of fluorescent dye-labeled Hsp90 ligand bound to Hsp90 is correlated to the mP value and plotted against values of competitor concentrations. The inhibitor concentration at which 50% of bound fluorescent dye-labeled Hsp90 ligand is displaced is obtained by fitting the data. For cy3B-GM, an excitation filter at 530 nm and an emission filter at 580 nm are used with a dichroic mirror of 561 nm. For PU-FITC3, an excitation filter at 485 nm and an emission filter at 530 nm are used with a dichroic mirror of 505 nm. All of the experimental data are analyzed, and binding affinity values are given as relative binding affinity values (EC50, concentration at which 50% of fluorescent ligand is competed off by compound)[1].

Cell Assay [1]
Cells are treated for 72 h with inhibitors (including PU-WS13) or transfected with Grp94 siRNA or control siRNA, and their viability is assessed using CellTiter-Glo luminescent Cell Viability Assay. The method determines the number of viable cells in culture based on quantification of the ATP present, which signals the presence of metabolically active cells[1].

References:
[1]. Patel PD, et al. Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2. Nat Chem Biol. 2013 Nov;9(11):677-684.