BIIB021

Kinase experiment:

For fluorescence polarization competition measurements, the FITC-geldanamycin probe (20 nM) is reduced with 2 mM TCEP at room temperature for 3 hours, after which the solution is aliquoted and stored at -80°C until used. Recombinant human Hsp90α (0.8 nM) and reduced FITC-geldanamycin (2 nM) are incubated in a 96-well microplate at room temperature for 3 hours in the presence of assay buffer containing 20 mM HEPES (pH 7.4), 50 mM KCl, 5 mM MgCl2, 20 mM Na2MoO4, 2 mM DTT, 0.1 mg/mL BGG, and 0.1% (v/v) CHAPS. Following this preincubation, BIIB021 in 100% DMSO is then added to final concentrations of 0.2 nM to 10 μM (final volume 100 μL, 2% DMSO). The reaction is incubated for 16 hours at room temperature and fluorescence is then measured in an Analyst plate reader, excitation=485 nm, emission=535 nm. High and low controls contain no BIIB021 or no Hsp90, respectively. The data are fit to a four-parameter curve and IC50 is generated.

Cell experiment:

A modified tetrazolium salt assay is used to measure the IC50. Tumor cells are added to 96-well plates and propagated for 24 hours before BIIB021 addition. BIIB021 is added to the plated cells. DMSO (0.03-0.003%) is included as a vehicle control. After incubation phenazine methosulfate (stock concentration 1 mg/mL) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (stock concentration 2 mg/mL) are mixed at a ratio of 1:20 and added to each well of a 96-well plate. Reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt gives rise to a soluble formazan product that is secreted into the culture medium. After 4 hours incubation, the formazan product is quantitated spectrophotometrically at a wavelength of 490 nm. Data are acquired using SOFTmaxPRO software, and 100% viability is defined as the A490 of DMSO-treated cells stained with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (the mean A490 of cells treated with DMSO at a range of 0.03-0.003%). Percent viability of each sample is calculated from the A490 values as follows: % viability=(A490 nm sample/A490 nm DMSO-treated cells × 100). The IC50 is defined as the concentration that gives rise to 50% inhibition of cell viability.

Animal experiment:

BALB/c and athymic mice are obtained from Harlan Sprague-Dawley at age 6 to 8 weeks. The mice are maintained in sterilized cages in a ventilated caging system with a 12 h light/12 h dark photoperiod at temperature of 21°C to 23°C and a relative humidity of 50±5%. Irradiated pelleted food and autoclaved deionized water are provided ad libitum. Animals are identified by the use of individually numbered ear tags. N87 tumor fragments (appr 2 mm3) are implanted s.c. in the right flank of the animal. BIIB021 is administered to animals bearing N87 stomach carcinoma tumors at doses of 31, 62.5, and 125 mg/kg, once daily, from Monday to Friday, for 5 weeks. Tumor dimensions are measured using calipers and tumor volumes are calculated using the equation for an ellipsoid sphere (l×w2)/2=mm3, where l and w refer to the larger and smaller dimensions collected at each measurement, respectively. Tumor volumes are measured and animals are weighed and monitored for toxicity at least twice weekly. P values are calculated using the two-tailed Student's t test to assess the difference in tumor volumes between control and treated groups. P<0.05 is considered significant.

References:

[1]. Lundgren, Karen., et al. BIIB021, an orally available, fully synthetic small-molecule inhibitor of the heat shock protein Hsp90. Molecular Cancer Therapeutics (2009), 8(4), 921-929.
[2]. B ll B, et al. Heat shock protein 90 inhibitor BIIB021 (CNF2024) depletes NF-kappaB and sensitizes Hodgkin's lymphoma cells for natural killer cell-mediated cytotoxicity. Clin Cancer Res. 2009 Aug 15;15(16):5108-16.
[3]. Yin X, et al. BIIB021, a novel Hsp90 inhibitor, sensitizes head and neck squamous cell carcinoma to radiotherapy. Int J Cancer. 2010 Mar 1;126(5):1216-25
[4]. Zhang H, et al. BIIB021, a synthetic Hsp90 inhibitor, has broad application against tumors with acquired multidrug resistance. Int J Cancer. 2010 Mar 1;126(5):1226-34