| Assay Method Information | |
| | Enzymatic Assay |
| Description: | The IC50 is the concentration of an inhibitor where the measured enzyme activity is reduced by half. In the case of USP7 deubiquitinase, the IC50 of an USP7 inhibitor is the molar concentration of the compound that inhibits 50% of the activity observed in a USP7-mediated-ubiquitin-rhodamine cleavage assay. For the inhibitors disclosed herein, their potency was measured using the following method.A 45 μl reaction volume containing full-length USP7 (0.5 nM) in 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, 1 mg/ml BSA and 0.05% Tween20 was assembled in wells of 96 well half area black flat bottom plates. Test compounds were first dissolved to 25 mM stocks in DMSO and subsequently introduced to enzyme 15 min after USP7 incubation with DTT at room temperature. The enzymatic reaction was started by adding 0.5 μM Ubiquitin-Rhodamine 110 (final concentration) and allowed to proceed for 45 min at 37° C., 250 RPM shaking before Rhodamine fluorescence (485 nm excitation/520 nm emission) was measured using Tecan Spark M10 plate reader. The IC50 values were determined by data fitting to variable slope model four-parameter dose-response curve using GraphPad 7.05. |
| Affinity data for this assay | |
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