| Assay Method Information | |
| | Fluorescence Polarisation (FP) Homogeneous Assay |
| Description: | Measurement of USP7 inhibitory activity. USP7 activity was monitored in a fluorescence polarisation (FP) homogeneous assay using the isopeptide ubiquitin-Lys-TAMRA substrate (U-558, Boston Biochem). Full-length USP7 was purchased from Boston Biochem (His6-USP7FL, E-519). Unless otherwise stated, all other reagents were purchased from Sigma-Aldrich. Enzymatic reactions were conducted in black flat-bottom low volume polystyrene 384-well plates (Grenier) and 15 μL total volume. USP7 (2.5 nM, 5 μL) was incubated in assay buffer (50 mM HEPES (pH 7.2), 150 mM NaCl, 5 mM DTT, 0.05% BSA (w/v), 0.05% CHAPS) in the presence or absence of inhibitor (5 μL). Inhibitors were stored as 10 mM DMSO stocks in an inert environment (low humidity, dark, low oxygen, room temperature) using a Storage Pod System and serial dilutions were prepared in buffer just prior to the assay (from 200 to 0.003 μM, 11 dp curve). Following incubation at room temperature for 30 min, the enzymatic reactions were initiated by dispensing the Ub substrate (50 nM, 5 μL). FP was measured every 5 min over a period of 1.5 h (within the linear range of the assay) using a Pherastar FSX (BMG Labtech) exciting at 530 nm and measuring the amount of parallel and perpendicular light at 575 nm. The FP signal was subsequently normalised to the control without compound present. Data were plotted and fitted, and the concentrations resulting in 50% inhibition (IC50) were calculated using the non-linear regression curve fitting model using GraphPad Prism. IC50 values for the inhibitors of the invention are compiled in Table 3 above and represent the average of at least duplicate experiments. |
| Affinity data for this assay | |
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