Fluorescence polarization (FP)-based competition binding assay

This assay utilized a boron difluoride dipyrromethene (BODIPY) labeled geldanamycin analogue (BODIPY-AG) as a probe and measured fluorescence polarization upon binding of the probe to a protein. Native human Hsp90 protein (α + β isoforms) is isolated from HeLa cells. BODIPY-AG solution was freshly prepared in FP assay buffer (20 mM HEPES-KOH, pH 7.3, 1.0 mM EDTA, 100 mM KCl, 5.0 mM MgCl2, 0.01% NP-40, 0.1 mg/mL fresh bovine γ-globulin (BGG), 1.0 mM fresh DTT, and protease inhibitor from stock solution in DMSO). Competition curves were obtained by mixing 10 μL each of a solution containing BODIPY-AG and Hsp90, and a serial dilution of 17-DMAG freshly prepared in FP assay buffer from stock solution in DMSO. Final concentrations were 10 nM BODIPY-AG, 40 or 60 nM Hsp90, varying concentration of 17-DMAG (0.10 nM ~ 10 μM), and ≤ 0.25% DMSO in a 384-well microplate. After 3-hr incubation at 30°C, fluorescence anisotropy (γEx = 485 nm, γEm = 535 nm) was measured on an EnVision 2100 multilabel plate reader. The IC50 value of 17-DMAG was obtained from the competition curves.

Cell lines

Chronic lymphocytic leukemia (CLL)

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

~ 1 μM; 24 or 48 hrs

Applications

In CLL cells, 17-DMAG effectively led to depletion of the Hsp90 client protein, decreasing NF-κB p50/p65 DNA binding, NF-κB target gene transcription and caspase-dependent apoptosis. By targeting the NF-κB family, 17-DMAG selectively mediated cytotoxicity against CLL cells (in dose- and time-dependent manner), but not normal T cells or NK cells important for immune surveillance.

Animal models

SCID mice engrafted with TCL1 leukemia cells

Dosage form

10 mg/kg; i.p.; 5 times per week for 16 days

Applications

In a TCL1-SCID transplant mouse model, the 17-DMAG treatment (10 mg/kg) significantly decreased the white blood cell count and prolonged the survival.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Jie Ge, Emmanuel Normant, James R. Porter, Janid A. Ali, Marlene S. Dembski, Yun Gao, Asimina T. Georges, Louis Grenier, Roger H. Pak, Jon Patterson, Jens R. Sydor, Thomas T. Tibbitts, Jeffrey K. Tong, Julian Adams, and Vito J. Palombella. Design, synthesis and biological evaluation of Hydroquinone derivatives of 17-Amino-17-demethoxygeldanamycin as potent, water-soluble inhibitors of Hsp90. J. Med. Chem. 2006, 49, 4606-4615.

[2]. Hertlein E, Wagner AJ, Jones J, Lin TS, Maddocks KJ, Towns WH 3rd, Goettl VM, Zhang X, Jarjoura D, Raymond CA, West DA, Croce CM, Byrd JC, Johnson AJ. 17-DMAG targets the nuclear factor-kappaB family of proteins to induce apoptosis in chronic lymphocytic leukemia: clinical implications of HSP90 inhibition. Blood. 2010 Jul 8;116(1):45-53.