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| | Furin Enzyme Assay |
| Description: | Reactions were performed in black 384-well polystyrene low volume plates (Greiner). Furin (108-574-Tev-Flag-His) enzyme was expressed and purified from CHO cells. Compounds of the invention were dissolved in DMSO (1.0 mM) and serially diluted 1 to 3 with DMSO through eleven dilutions to provide a final compound concentration range from 0.00017 to 10 μM. 0.05 μL of each concentration was transferred to the corresponding well of an assay plate, and then 5 μL of 40 pM furin enzyme in assay buffer (100 mM HEPES pH7.5, 1 mM CaCl2 and 0.005% Triton X-100) was added using a Multidrop Combi (Thermo) to the compound plates, and mixed by inversion. Following a 30 min preincubation of enzyme with compound at room temperature (22° C.), the substrate FAM-QRVRRAVGIDK-TAMRA (SEQ ID NO:1) (5 μL of a 1 pM solution in assay buffer) was added using a Multidrop Combi to the entire assay plate. The plates were centrifuged at 500×g for 1 minute and incubated at room temperature for two hours. Enzyme inhibition is then quantified using an Envision instrument (PerkinElmer). Data were normalized to maximal inhibition determined by 1 μM Decanoyl-Arg-Val-Arg-Lys-Chloromethylketone (SEQ ID NO:2) (Calbiochem #344930 or AnaSpec #808143). |
| Affinity data for this assay | |
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