Kinase experiment:

His-tagged Hsp90α N-terminal domain protein (N-Hsp90α-His) is diluted to 2× the final concentration in the assay buffer consisting of 50 mM Hepes, pH 7, 6 mM MgCl2, 20 mM KCl and 0.1% BSA. The Hsp90 is dispensed into 96 well polypropylene plates at 50 μL per well. In a separate polypropylene plate, test compounds (NVP-HSP990) are diluted to 40× their final concentration in 100% DMSO. Serial dilutions in DMSO are made in 3-fold increments. 2.5 μL of diluted compounds are transferred to the 50 μL of Hsp90 and mixed. Background wells receive 25 μM (final concentration) radicicol. Biotin-radicicol is diluted into assay buffer at 2× the final concentration and 50 μL are added to the Hsp90/compound plate. DMSO is at a final concentration of 2.5%. Samples are incubated at room temperature for 2 hours before 50 μL are transferred to NeutrAvidin-coated plates. Plates are incubated 1 hour, washed 3× with DELFIA wash buffer (5 mM Tris, pH 7.5, 0.1% Tween 20, 0.1% sodium azide, 0.9% NaCl), and then 50 μL per well of 3 nM Eu-anti-His diluted into DELFIA assay buffer are added. The plates are next incubated 2 hours at room temperature, washed 4×, and then 50 μL enhancement solution are added. Plates are gently shaken for 7-10 minutes before reading in a VICTOR2 instrument[2].

Cell experiment:

GTL-16 Cells (1 × 103) are plated into 96 well tissue culture plates and cultured at 37℃, 5% CO2. Serially diluted compounds (NVP-HSP990) are added to the cells and are incubated for 72 hrs. at 37℃, 5% CO2. Cell proliferation is determined using Cell Viability assay[2].

Animal experiment:

Human tumor xenograft models GTL-16, NCI-H1975, BT474, and MV4;11 are implanted subcutaneously with 50% Matrigel in nude or severe combined immunodeficient mice. Mice are randomized into cohorts (10 mice/group for efficacy) when tumors reach 200 to 500 mm3. NVP-HSP990 is administered orally in a vehicle of 100% polyethylene glycol (PEG400). Tumor caliper measurements are converted into tumor volumes using the formula: [length × (width)2]/2. Relative tumor inhibition is calculated as %T/C = 100 × dT/dC, where, dT or dC = difference of mean tumor volume of drug treatment (T) or vehicle (C) on the final day of the study and the randomization volume. Statistical comparisons are conducted using a one-way ANOVA, followed by the Dunn or Tukey post hoc test. Differences are statistically significant at P< 0.05[1].

HSP990 (NVP-HSP990) is a potent and selective inhibitor of HSP90 with IC50 values of 0.6, 0.8 and 8.5 nM for Hsp90α, Hsp90β and Grp94, respectively [1].

Heat shock protein 90 (HSP90) is a chaperone protein that stabilizes proteins against heat stress, assists proteins to fold properly and aids in protein degradation. Also, HSP90 stabilizes proteins required for tumor growth.

HSP990 (NVP-HSP990) is a potent and selective HSP90 inhibitor. NVP-HSP990 bound to the N-terminal ATP-binding domain of Hsp90. NVP-HSP990 inhibited TRAP1 ATPase activity by 90% with IC50 value of 320 nM. In GTL-16 cells, NVP-HSP990 destabilized the Hsp90-p23 complex in a concentration- and time-dependent way. Also, NVP-HSP990 decreased c-Met with EC50 value of 37 nM and induced Hsp70 with EC50 value of 20 nM. NVP-HSP990 inhibited ERK and AKT phosphorylation with EC50 values of 11 and 6 nM respectively and thus inhibited ERK and AKT activation. In five human tumor cell lines, NVP-HSP990 inhibited cell growth with GI50 values of 4-40 nM [1]. In glioma tumor-initiating cells, NVP-HSP990 inhibited cell growth with IC50 value of 10-500 nM in a dose-dependent way and reduced CDK2 and CDK4, thus disrupting cell-cycle control [2].

In the GTL-16 gastric cancer mice model, NVP-HSP990 exhibited antitumor efficacy [1].

References:
[1].  Menezes DL, Taverna P, Jensen MR, et al. The novel oral Hsp90 inhibitor NVP-HSP990 exhibits potent and broad-spectrum antitumor activities in vitro and in vivo. Mol Cancer Ther, 2012, 11(3): 730-739.
[2].  Fu J, Koul D, Yao J, et al. Novel HSP90 inhibitor NVP-HSP990 targets cell-cycle regulators to ablate Olig2-positive glioma tumor-initiating cells. Cancer Res, 2013, 73(10): 3062-3074.