| Assay Method Information | |
| | PARG Enzymatic Assay |
| Description: | Inhibition of PARG enzymatic activity by compounds was determined as follows: Recombinant His-tagged human PARG protein expressed in Sf21 insect cells (Adipogen AG, #40T-0022) was aliquoted and stored at −80° C. until use. The artificial enzyme substrate 4-(trifluoromethyl)umbelliferone (TFMU)-ADPr was prepared essentially as described (Drown B S et al, Cell Chemical Biology 2018) and stored at −20° C. as 10 mM stock solutions in DMSO (dimethyl sulfoxide) until use. For reactions, typical final concentration of the enzyme was 1 nM and of the TFMU-ADPr 200 μM. Reactions were carried out in black 384-well low volume round bottom assay plates (Corning, #4514) in a final volume of 10 μL. To this end, PARG was diluted to 1.67 nM in reaction buffer (50 mM K2HPO4, 50 mM KCl, 10 mM β-mercaptoethanol, pH 7.4) and 6 μL were dispensed per assay well using a multichannel pipette. Then compound was dispensed into each well using an Echo dispenser (BeckmanCoulter) to yield the pre-specified final concentration of the range of concentrations to be tested (typically 8-point serial concentrations ranging from either from 0.01 to 10 μM, or from 0.001 to 1 μM, or from 0.0001 to 0.1 μM), and being volume-corrected with 100% DMSO (0.5% final DMSO concentration in reaction mixtures). Plates were mixed, and after 15 minutes 4 μL TFMU-ADPr was added into each well using a multichannel pipette followed by brief mixing. Fluorescence intensity signals were then measured after 30 minutes on an EnVision microplate reader (PerkinElmer) using 380/10 nm and 535/25 nm filters for the excitation and emission, respectively (gain: 150). The assay window was set using DMSO control (top) and a control containing no PARG enzyme (bottom). Delta values were plotted as concentration-response curves fitted to a sigmoidal 4-parameter logistic model to calculate IC50 values (Scigilian Analyze). |
| Affinity data for this assay | |
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