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Assay Method Information

Assay Name:  HTRF FRET Assay
Description:  The compounds of the invention were determined to be potent inhibitors of BACE-1 using the following assay.The following reagents were used in this assay. Na+-Acetate pH 5.0; 1% Brij-35; Glycerol; Dimethyl Sulfoxide (DMSO); Recombinant human soluble BACE-1 catalytic domain (>95% pure); APP Swedish mutant peptide substrate (QSY7-APPswe-Eu): QSY7-EISEVNLDAEFC-Europium-amide.A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE-1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE-1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE-1 enzyme. Inhibition of BACE-1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.
Affinity data for this assay
 

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Last update November 1, 2007
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