Hsc70, Hsp70 and Grp78 fluorescence polarisation (FP) assay

The FP assay for Hsp70 was conducted in aqueous buffer consisting of 100 mM Tris pH 7.4, 150 mM KCl and 5 mM CaCl2, in a final assay volume of 100 uL, using 96-well black polystyrene high bind plates with a Fusion plate reader. N6-(6-amino)hexyl-ATP-5-FAM and the in-house protein preparation of GST-HSP70 3-382 had final concentrations in the assay of 20 nM and 400 nM, respectively. Compounds were tested as 10-point IC50, with a final DMSO concentration of 5%. Assay mixtures were incubated for 3 hrs prior to reading on the Fusion (ex 485 nm; em 535 nm). The data was fitted using a 4 parameter logistical data model by XLFit 4. The FP assay for Hsc70 and Grp78 was carried out as described for Hsp70 using the same N6-(6-amino)hexyl-ATP-5-FAM as the FP probe with the following modifications. For Hsc70, the protein and probe concentrations were 0.3 μM and 20 nM, respectively, with a 30 min incubation at 22 ℃ while for Grp78, the protein and probe concentrations were 2 μM and 10 nM, respectively, with a 2 hr incubation at 22 ℃. The KD for the FAM-ATP probe was 0.24 μM for Hsc70 and 2 μM for Grp78.

Cell lines

BT474, MB-468, HCT116 and HT29 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

50 μM; 72 hrs

Applications

In human breast and colon cancer cell lines BT474, MB-468, HCT116 and HT29, VER 155008 inhibited cell proliferation, with the GI50 values ranging from 5.3 μM to 14.4 μM. In addition, VER 155008 induced Hsp90 client protein degradation in both HCT116 and BT474 cells.

Animal models

Mice bearing HCT116 tumors

Dosage form

25 or 40 mg/kg; i.v.

Applications

In mice bearing HCT116 tumors, VER 155008 showed rapid metabolism and clearance, along with tumor levels below the predicted pharmacologically active level.

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.

VER 155008 is a potent inhibitor of heat shock protein 70 (Hsp70) family of chaperones, which have been shown to contribute to cancer cell survival via anti-apoptotic functions, that inhibits Hsp70 (with the value of 50% inhibition concentration IC₅₀of 0.5 μM) as well as heat shock cognate 71 kDa protein (Hsc70) and 78 kDa glucose-regulated protein (Grp78) but to a lesser extent. X-ray crystallography analysis reveals that VER 155008 binds to the ATPase pocket of Hsp70 leading to the inhibition of the intrinsic ATPase activity of Hsp70. Study results also have demonstrated that VER 155008 induces cell proliferation and apoptosis in human cancer cell lines.

Reference

[1].Massey AJ, Williamson DS, Browne H, Murray JB, Dokurno P, Shaw T, Macias AT, Daniels Z, Geoffroy S, Dopson M, Lavan P, Matassova N, Francis GL, Graham CJ, Parsons R, Wang Y, Padfield A, Comer M, Drysdale MJ, Wood M. A novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells. Cancer Chemother Pharmacol. 2010; 66(3): 535-545