| Assay Method Information | |
| | RET Enzyme Assay |
| Description: | Compounds of formulas I, la to lx, Ila to IIx, and/or Illa to IIIx are screened for their ability to inhibit wildtype, V804M, and G810S mutant RET kinase using CisBio s HTRF KinEASE -TK assay technology. N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (1.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (1.25 nM enzyme; Catalog No. 14-760) or N-Terminal GST-tagged recombinant human G810S (aa-658-end) (1.25 nM Enzyme; produced in insect cells) is incubated with 62.5 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound (0.4% final DMSO in the assay) in a IX Cisbuio enzymatic buffer consisting of 1 nM DTT, 5 mM MgCh, 0.04% BSA and 0.05% Tween20 in a volume of 10 pL. Compounds are typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 40-60 min (40 min for V804M, 60 min for WT and G810S) incubation at 22 °C, the reaction is quenched by adding quench solution (10 pL) containing 7.8 nM Streptavidine-XL665 and 0.5X TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 60-80 min incubation (60 minutes for WT, 80 minutes for V804M and G810S) at 22 °C, the extent of reaction is determined using a PHERastar plate reader via HTRF detection at excitation/emission 337/665 nm. Percent of inhibition is calculated with 0% inhibition referring to control conditions containing no compound (0.4% DMSO only) and 100% inhibition represented by conditions containing no enzyme. The % inhibition values are fit to a 4 parameter logistic curve, and the determined IC5o value is defined as the estimated concentration of inhibitor at the inflexion point of the fitted curve. |
| Affinity data for this assay | |
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