Hsc70, Hsp70 and Grp78 fluorescence polarisation (FP) assay
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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.
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Applications
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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.
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References:
[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
[2]. Williamson DS, Borgognoni J, Clay A, Daniels Z, Dokurno P, Drysdale MJ, Foloppe N, Francis GL, Graham CJ, Howes R, Macias AT, Murray JB, Parsons R, Shaw T, Surgenor AE, Terry L, Wang Y, Wood M, Massey AJ. Novel adenosine-derived inhibitors of 70 kDa heat shock protein, discovered through structure-based design. J Med Chem. 2009 Mar 26;52(6):1510-3.
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