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℃, 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.

17-DMAG is an inhibitor of Hsp90 with IC50 value of 62±29nM [1].

17-DMAG can bind to the ATP-binding motif of Hsp90 and inhibit the protein chaperoning activity of Hsp90. It will cause misfolding and subsequent degradation of Hsp90’s client proteins, such as EGFR, AKT, mutant p53, and IKK. Since there is more specific conformation Hsp90 required for 17-DMAG binding in tumor cells and many client proteins of Hsp90 contribute to tumor cell growth, 17-DMAG is usually more toxic to tumor cells than to normal cells [2].

17-DMAG is reported as an antitumor agent with more broadly exploitable activity and more pharmaceutically tractable characteristics in the in vitro and initial in vivo assay. 17-DMAG can effect cell growth when treating the NCI 60 cell lines with it, the mean GI50 is 0.053mM. The in vivo activity of 17-DMAG is tested in four melanoma models using the Freiburg human tumor xenograft panel and two lung xenografts. It shows that 17-DMAG has high activity in the two lung xenografts and two of the four melanoma models, but not in another two, MEXF 462 and MEXF 514 [3].

Reference:
[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] Xiaoping Sun, Jillian A.  Bristol, Satoko Iwahori, Stacy R. Hagemeier, Qiao Meng, Elizabeth A. Barlow, Joyce D. Fingeroth, Vera L. Tarakanova, Robert F. Kalejta, Shannon C. Kenney. Hsp90 Inhibitor 17-DMAG Decreases Expression of Conserved Herpesvirus Protein Kinases and Reduces Virus Production in
Epstein-Barr Virus-Infected Cells.  Journal of Virology. 2013, 87 (18): 10126–10138.
[3] Melinda Hollingshead, Michael Alley, Angelika M.  Burger, Suzanne Borgel,
Christine Pacula-Cox, Heinz-Herbert Fiebig, Edward A.  Sausville. In vivo antitumor efficacy of 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride), a water-soluble geldanamycin derivative. Cancer Chemother Pharmacol. 2005, 56: 115–125.