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By Vera Rakhmanova, Ph.D., Cecilia Po and Rich Meyer, Ph.D. AnaSpec, Inc. San Jose, CA 95131

Introduction
secretase (BACE1) is an aspartic protease enzyme involved in the pathogenesis of Alzheimer disease (AD). This enzyme is responsible for the cleavage of Amyloid Precursor Protein (APP) resulting in the production of amyloid neurotoxic peptides that aggregate in the brain of Alzheimer patients. BACE1 is an attractive target for anti-amyloid treatment of AD. To detect secretase activity and screen secretase inhibitors, we developed a SensoLyte^{/sup> 520 secretase assay kit using a novel FRET peptide, HiLyte Fluor/sup> 488-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys (QXL/sup> 520)-OH. The sequence of this FRET peptide is derived from the secretase cleavage site of APP with Swedish double mutation (K670N/M671L).1 This double mutation enhances the susceptibility of APP to secretase and results in an early onset of AD. In the FRET peptide, the fluorescence of HiLyte Fluor/sup> 488 is quenched by QXL/sup> 520 (Fig.1) Upon cleavage of peptide into two separate fragments by secretase at the Leu-Asp bond, the fluorescence of HiLyte Fluor/sup> 488 can be monitored at excitation/emission = 488 nm/520 nm (Fig. 2). This novel substrate offers several advantages compared to a previously described EDANS/DABCYL FRET substrate. The long wavelength fluorescence of HiLyte Fluor/sup> 488/QXL/sup> 520-based substrate is less interfered by the short wavelength autofluorescence of drug candidates and cellular components. HiLyte Fluor/sup> 488 is pH insensitive and well suited for use in buffers with low pH. Hydrophilicity of QXL/sup> 520 results in better solubility of the peptide substrate.}

Fig. 1. The absorption spectrum of QXL^{/sup>520 overlaps with the emission spectrum of HiLyte Fluor/sup> 488.}

Fig. 2. Proteolytic cleavage of HiLyte Fluor^{/sup>488/QXL/sup>520 FRET peptide by secretase. In the FRET peptide, the fluorescence of HiLyte Fluor/sup> 488 is quenched by QXL/sup> 520. Upon cleavage, the fluorescence of HiLyte Fluor/sup> 488 is recovered, and can be continuously monitored at Ex/Em = 488 nm/520 nm.}

Protocols
Assays are performed in a convenient 96-well microplate format. 384-well or 1536-well format can also be used as well with minor modifications.

A choice of two protocols based on the goal of one experiment can be used. Protocol A describes the detection of secretase activity in biological samples. The secretase substrate stock solution (4 mM) is diluted 1: 100 in 2X assay buffer. Controls recommended for Protocol A include: a). Substrate control (contains de-ionized water only); b). Negative control (samples without secretase). Volume of control and test samples is adjusted to 50 l. The enzymatic reaction starts upon addition of the diluted substrate (50 l/well).

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