| Assay Method Information | |
| | TR-FRET Assay |
| Description: | Activity of the compounds was investigated using TACE (ADAM17) isolated enzyme. The screening of ADAM17 inhibitors was performed with a profluorescent peptidic substrate (Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Ser-Arg-NH2) which contains a highly fluorescent 7-methoxycoumarin (Mca) group that is efficiently quenched by resonance energy transfer to the 2,4-dinitrophenyl group (Dpa). Mca fluorescence was quenched by the Dpa group until cleavage by ADAM17 at the Ala-Val bond separates the two moieties.The enzymatic reaction was performed in 384-well microplates with a final volume of 10 uL containing 1% DMSO. Recombinant Human ADAM17 catalytic domain (500 nM) was assayed with 40 uM of substrate and serial dilutions of tested inhibitors (from 10000 nM to 0.04 nM). After two hours of incubation at room temperature, the fluorescence was measured using a microplate reader (320/425 nm). |
| Affinity data for this assay | |
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