USP7-IN-1

Kinase experiment:

USP7 is diluted in USP buffer (50 mM Tris HCl; 0.5 mM EDTA (Ethylenediaminetetraacetic acid); 5 mM DTT; 0.01 % Triton X-100; Bovine Serum Albumin 0.05 mg/mL pH7.6). Compounds stocks (10 mM) are stored at -20°C in DMSO. Compounds (including USP7-IN-1) are tested at different concentrations: from 200 μM to 91 nM. Reactions are performed as duplicates in Black 384 well plates (10 μL final reaction volume). The substrate concentration for USP7 is 300 nM Ub-AMC. The concentrations of the enzyme (USP7) in specificity assays is 100 pM. The concentrations are determined in order to perform specificity assays under initial velocities at fixed substrate concentration. Compounds are pre-incubated with enzymes for 30 minutes at 25°C. Reactions are initiated by addition of substrate to the plates containing the enzymes (+/- compounds) diluted in assay buffer. Reactions are incubated for 60 minutes at 37°C. Reactions are stopped by adding acetic acid (100 mM final). Readings are performed on a Pherastar Fluorescent Reader. λ Emission 380 nm; λ Excitation = 460 nm. Data (mean values +/- standard deviation) are analyzed as % of control (no compound) and plotted as percentage versus the Log of the compound concentration using GraphPad. Data are fitted to a sigmoidal model (variable slope)[1].

Cell experiment:

HCT116 colon cancer cells are maintained in Mc Coy's 5A medium containing 10% FBS, 3 mM glutamine and 1 % penicillin/streptomycin. Cells are incubated at 37°C in a humidified atmosphere containing 5% CO2. Cell viability is assayed using the MTS technique in 96-well culture plates. MTS (3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy- methoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-zolium) is a MTT-derived tetrazolium that is reduced in metabolically active cells into a soluble, cell-permeant formazan. The amount of formazan, detected by its absorbance at 492 nm is proportional to the number of living, metabolically active cells. 103 HCT116 cells are seeded per well. 24 hours later, the medium is changed and the cells treated in triplicate with the concentrations of each compound from 100 μM to 50 nM. The compounds (including USP7-IN-1) are diluted in 100% DMSO, whose final concentration on cells is kept at 0.5%. Cells are incubated with the compounds for 72 hours, and their viability then assayed by the addition of MTS for 2 hours. Absorbance at 492 nm is measured directly from the 96-well culture plates. GI50 (Growth Inhibition 50) concentrations for each compound are calculated using a sigmoidal variable slope fit. Values represent mean of three independent experiments[1].

References:

[1]. SELECTIVE AND REVERSIBLE INHIBITORS OF UBIQUITIN SPECIFIC PROTEASE 7. WO 2013030218 A1