A Sensitive Fluorimetric Assay for Detection of ß-Secretase Activity

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

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

Fig. 1. The absorption spectrum of QXL^™520 overlaps with the emission spectrum of HiLyte Fluor^™ 488.

Fig. 2. Proteolytic cleavage of HiLyte Fluor^™488/QXL^™520 FRET peptide by ß-secretase. In the FRET peptide, the fluorescence of HiLyte Fluor^™ 488 is quenched by QXL^™ 520. Upon cleavage, the fluorescence of HiLyte Fluor^™ 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’s experiment can be used. Protocol A describes the detection of ß-secretase activity in biological samples. The ß-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 ß-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).

Protocol B is designed for screening ß-secretase inhibitors using purified enzyme. The amount of ß-secretase provided in this kit is sufficient to serve only as a positive control. ß-secretase is diluted in 20 mM Hepes, pH 7.4, 125 mM sodium chloride, 1 mg/mL BSA. The following controls for the protocol B are recommended: a). Positive control (enzyme only); b). Inhibitor control (enzyme and known inhibitor provided in the kit); c). Vehicle control (enzyme and vehicle used to deliver test compound); d). Test compound control (assay buffer and test compound); and e). Substrate control (contains assay buffer only). Volume of control and test samples is adjusted to 90 l. The 4 mM stock solution of ß-secretase substrate is diluted 1: 20 in 1X assay buffer and added to the 96-well plate at 10 l/well.

Fluorescence signal can be measured using kinetic reading or end-point reading at Ex/Em=488 nm/520 nm. In the latter case, using the stop solution provided in the kit is recommended. The enzymatic reaction can be performed at 37°C or 25°C.

The reading from the substrate control well must be subtracted from the readings of the other wells to get the fluorescence increase, measured in relative fluorescence units (RFU). For kinetic readings, data may be plotted as RFU versus time for each sample. A fluorescence reference standard is included in the kit for use in measuring RFU. RFU values can then be used to determine the concentration of the product of enzymatic reaction.

Results
For the newly designed HiLyte Fluor^™ 488/ QXL^™520 labeled FRET peptide, Km was determined after incubation of ß-secretase (1U/well) with a range of substrate concentrations. The Km, calculated using non-linear regression analysis, was 18.92 M (Fig.3). This homogeneous assay can be used to continuously monitor product formation (Fig.4).

Fig. 3. Michaelis-Menton plot for ß-secretase with HiLyte Fluor^™488 / QXL^™520 FRET substrate. Initial velocities are plotted against substrate concentration.

Fig. 4. Assay kinetics. ß-secretase (1 U) was incubated with 20 mM of the HiLyte Fluor^™ 488/QXL^™ 520 FRET substrate. Fluorescent signal was continuously monitored at Ex/Em=488nm/ 520nm for 60 min.

Sensitivity of the assay was determined by using serial dilutions of the enzyme (Fig.5). FRET substrate was incubated with the indicated amount of ß-secretase 40 min at 37oC and fluorescence was measured using FlexStation 384II, Molecular Devices. The enzyme sensitivity of the assay was 0.03 mU/l ß-secretase.

Fig. 5. Secretase titration with HiLyte Fluor^™488/ QXL^™520 labeled FRET substrate.

To validate assay for inhibitor screening, this FRET substrate was incubated with enzyme in the presence of the secretase inhibitor provided in the kit. The inhibitor, the statine-based substrate analog KTEEISEVN-Sta-VAEF-NH₂, has been described previously in the literature.² Kinetic readings were taken every 5 min for 30 min at 37oC (FlexStation 384II, Molecular Devices). The calculated IC₅₀ was 5.62 nM (Fig.6).

Fig. 6. Inhibitor studies. To validate the assay for inhibitor screening, FRET substrate was incubated with enzyme in the presence of a secretase inhibitor KTEEISEVN-Sta-VAEF-NH₂ (cat# 23960).

Conclusions
We have developed a highly sensitive SensoLyte^™ 520 ß-secretase assay kit based on a HiLyte Fluor^™ 488/QXL^™ 520 FRET substrate. This homogeneous assay is capable of continuously monitoring the enzymatic reaction. The longer excitation and emission wavelengths of HiLyte Fluor^™ 488 keep interference from autofluorescence and absorbance of test compounds to a minimum. IC₅₀ values for the known inhibitor determined with SensoLyte^™ 520 ß-secretase assay kit were consistent with published data.

References
1. Mullan, M. et al. Nat.Genet. 1, 345 (1992).
2. Sinha, S. et al. Nature 402, 537 (1999).

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