| Assay Method Information | |
| | MALT-1 Biochemical Assay |
| Description: | A Quenched Fluorescence Resonance Energy Transfer (Quench-FRET) enzyme assay was used to test the ability of exemplary MALT-1 inhibitors Example 1 to Example 39 to inhibit the cleavage of a MALT1 specific quenched fluorescent substrate of sequence [TAMRA-PEG2-Leu-Val-Ser-Arg-Gly-Ala-Ala-Ser-PEG2-K(QSY7) by human MALT-1 protein; where TAMRA is 6-carboxytetramethylrhodamine, PEG2 is 2-(2-(2-aminoethoxy)ethoxyamide linker, QSY7 is a non-fluorescent quenching dye (Thermo-Fisher catalog #Q10193). The assay was performed by pre-dispensing compounds into 384 well Proxiplates (PE 6008289). Human MALT-1 (6 nM) was prepared using the assay buffer (25 mM HEPES pH 7.5, 1 mM ethylenediaminetetraacetic acid, 0.8 M sodium citrate, 0.005% Bovine serum albumin (BSA), 2 mM dithiothreitol (DTT)) and added to the compound plates. The plates were incubated 40 minutes at room temperature. The reaction was carried out by adding the substrate (2 μM) to the plates and incubating for 60 minutes at room temperature. A fluorescence readout was then measured using a PE EnVision reader (Ex.535/Em.590). The results of the enzyme assay are provided subsequently in Table 2 and demonstrate the ability of the compounds of the present disclosure to inhibit MALT-1 protease activity. |
| Affinity data for this assay | |
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