Isothermal titration calorimetry (ITC) of nucelotide binding

The titration experiments were performed using the MSC system. In each experiment, 16 aliquots of 15 μL of Geldanamycin (300 μM in 1% DMSO) were injected into 1.3 mL of protein (31 μM in 20 mM Tris-HCl, pH 7.5, 1 mM EDTA) at 25°C, and the resulting data were fit after subtracting the heats of dilution. Heats of dilution were determined in separate experiments from addition of Geldanamycin into buffer and buffer into protein. No evidence for binding of DMSO in the nucleotide binding site was observed. Titration data were fit using a nonlinear least-squares curve-fitting algorithm with three floating variables: stoichiometry, binding constant (Kb = 1/Kd), and change of enthalpy of interaction (ΔH°). The dissociation constant estimated for Geldanamycin binding to intact yeast Hsp90 was 1.22 μM, and for binding to Hsp90 N-terminal domain was 0.78 μM. No meaningful heat was observed with binding to the C-terminal fragment.

Cell lines

A2780 cells

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

0.001 ~ 10 μM; 3 hrs

Applications

In human ovarian cell line A2780, Geldanamycin caused a dose-dependent G2 arrest and reversible inhibiton of entry into the S phase.

Animal models

Mice bearing FRE/erbB-2 tumors

Dosage form

50, 100, 200 and 400 mg/kg; i.p.; b.i.d., for 5 days

Applications

In mice bearing FRE/erbB-2 tumors, Geldanamycin (50 mg/kg) shows 30% inhibition on p185-associated phosphotyrosine levels.

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]. Roe SM, Prodromou C, O'Brien R, Ladbury JE, Piper PW, Pearl LH. Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin. J Med Chem. 1999 Jan 28;42(2):260-6.

[2]. McIlwrath AJ, Brunton VG, Brown R. Cell-cycle arrest and p53 accumulation induced by geldanamycin in human ovarian tumour cells. Cancer Chemother Pharmacol. 1996;37(5):423-8.

[3]. Schnur RC, Corman ML, Gallaschun RJ, Cooper BA, Dee MF, Doty JL, Muzzi ML, Moyer JD, DiOrio CI, Barbacci EG, et al. Inhibition of the oncogene product p185erbB-2 in vitro and in vivo by geldanamycin and dihydrogeldanamycin derivatives. J Med Chem. 1995 Sep 15;38(19):3806-12.