PU-H71 was administered as an intravenous infusion over 1 hour two times a week on a 2 week on and 1 week off (Q21 day) schedule with a starting dose of 10 mg/m2.

Hsp90 inhibition was measured by fluorescence polarization assay. The assay buffer contained 20 mM HEPES (K) pH 7.3, 50 mM KCl, 5 mM MgCl2, 20 mM Na2MoO4, 0.01% NP40. Before each use, 0.1 mg/mL bovine gamma globulin (BGG) and 2 mM DTT were freshly added. GM-BODIPY was synthesized as previously reported22a and was dissolved in DMSO to form 10 uM solutions. Cell lysates were prepared rupturing cellular membranes by freezing at -70 °C and dissolving the cellular extract in HFB with added protease and phosphotase inhibitors. Saturation curves were recorded in which GM-BODIPY (5 nM) was treated with increasing amounts of cellular lysates. The amount of lysate that resulted in polarization (mP) readings corresponding to 20 nM recombinant Hsp90R was chosen for the competition study. For the competition studies, each 96-well contained 5 nM fluorescent GM, cellular lysate (amounts as determined above and normalized to total Hsp90 as determined by Western blot analysis using as standard Hsp90 purified from HeLa cells and tested inhibitor (initial stock in DMSO) in a final volume of 100 uL. The plate was left on a shaker at 4 °C for 24 h, and the FP values in mP were recorded. EC50 values were determined as the competitor concentrations at which 50% of the fluorescent GM was displaced.