Description

Geldanamycin is a natural existing HSP90 inhibitor with K_d of 1.2 μM.

Targets

IC₅₀

In Vitro

Geldanamycin binds in the ATP-binding site in the N-terminus domain of Hsp90s (residues 1-220). Geldanamycin inhibits the ATPase activity of Hsp90 in a dose-dependent manner. ^[1] Geldanamycin causes a dose-dependent G2 arrest and reversible inhibiton o f entry into the S phase in A2780 human ovarian cell line. This inhibition is accompanied by p53 increase and finally demonstrated to be p53 dependent. ^[2] Geldanamycin causes polyubiquitination and proteasomal degradation of the p185 receptor protein-tyrosin kinase and shows a IC50 with 70 nM. ^{[3, 4]} Geldanamycin is a typical anti-tumor reagent, shows a mean GI50 with 0.18 μM against the panel of 60 human tumor cell lines. ^[5]

In Vivo

Geldanamycin (50 mg//kg) shows 30% inhibition on pl85-associated phosphotyrosine levels in FRE/erbB-2 mice. ^[6]

Clinical Trials

Features

Isothermal Titration Calorimetry (ITC) of Nucelotide Binding

The titration experiments are performed using the MSC system. In each experiment, 16 aliquots of 15 μL of geldanamycin (300 μM in 1% DMSO) are injected into 1.3 mL of protein (31 μM in 20 mMTris-HCl, pH 7.5, 1 mMEDTA) at 25 °C, and the resulting data are fit after subtracting the heats of dilution. Heats of dilution are 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 is observed. Titration data are fit using a nonlinear least-squares curve-fitting algorithm with three floating variables: stoichiometry, binding constant (K_b) 1/K_d), and change of enthalpy of interaction (ΔH°). Dissociation constants estimated for geldanamycin binding to intact yeast Hsp90 is 1.22 μM, and for binding to Hsp90 N-terminal domain is 0.78 μM. No meaningful heat is observed with binding to the C-terminal fragment.

ATPase Assay

The ATPase assay is based on a regenerating coupled enzyme assay in which the phosphorylation of ADP by pyruvate kinase at the expense of phosphoenolpyruvate is coupled to the reduction of the resulting pyruvate by lactate dehydrogenase at the expense of NADH. Oxidation of NADH to NAD⁺ produces a loss of optical density at the NADH absorbance maximum of 340 nm, in direct stoichiometry to the amount of ADP phosphorylated. Each 1-mL assay contained 100 mM Tris-HCl (pH 7.4), 20 mM KCl, 6 mM MgCl₂, 0.8 mM ATP, 0.1 mM NADH, 2 mM phosphoenolpyruvate, 0.2 mg of pyruvate kinase, 0.05 mg of Llactate dehydrogenase, and between 2 and 3.5 nmol of Hsp90. Sufficient NADH is added to give an initial absorbance of 0.3 at 340 nm prior to addition of Hsp90s or fragments, and activity is detected as a decrease in absorbance. Inhibition of ATPase activity by geldanamycin is achieved by the addition of 1-10 μL of antibiotic dissolved in 100% DMSO to a final concentration of 1.5, 9, 15, and 30 μM. In control experiments, 1% DMSO present alone do not affect the measured ATPase activities, and stoichiometric rephosphorylation of ADP directly added to the assay system is unaffected by 1% DMSO or by Geldanamycin at all concentrations tested. All measurements are made on a Shimadzu UV-240 spectrophotometer.

Cell Lines

A2780 human ovarian cell line

Concentrations

0.001 – 10 μM

Incubation Time

3 hours

Methods

Animal Models

FRE/erbB-2 tumors in nu/nu mice

Formulation

Geldanamycin is dissolved in DMSO.

Doses

50 mg/kg

Administration

Administered via i.p.