Cell-based assay for β-secretase activity

Oh, Myungsok; Kim, Sung Yun; Oh, Yeong Soo; Choi, Deog-Young; Sin, Hye Jin; Jung, Inhee Mook; Park, Woo Jin

doi:10.1016/j.ab.2003.08.036

Cited by 16 publications

(8 citation statements)

References 22 publications

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“…Such confirmation could be achieved if BACE1 inhibitors would slow disease progression in humans, and the search for such therapeutically active compounds is a major focus of AD drug discovery. Currently available test systems that measure cellular BACE1 activity, such as nonmammalian cells [49,50], allow only low throughput or are based on labor-and cost-intensive detection methods such as high-performance liquid chromatography (HPLC), ELISA, and Western blot [51,52]. In the current study, we have presented novel approaches to BACE1 activity screening that use the normal neuronal environment of the protease and allow high throughput at low cost.…”

Section: Discussionmentioning

confidence: 99%

Measurement of cellular β-site of APP cleaving enzyme 1 activity and its modulation in neuronal assay systems

Volbracht

Penzkofer

Mansson

et al. 2009

Analytical Biochemistry

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Amyloid-beta peptide (Abeta), a putatively causative agent of Alzheimer's disease (AD), is proteolytically derived from beta-amyloid precursor protein (APP). Here we describe cellular assays to detect the activity of the key protease beta-site of APP cleaving enzyme 1 (BACE1) based on an artificial reporter construct containing the BACE1 cleavage site of APP. These methods allow identification of inhibitors and indirect modulators of BACE1. In primary neuronal cultures transfected with human APP constructs (huAPP), Abeta production was modified by BACE1 inhibitors similarly to the production of endogenous murine Abeta in wild-type cells and to that of different transgenic neurons. To further improve the assay, we substituted the extracellular domain of APP by secreted alkaline phosphatase (SEAP). SEAP was easily quantified in the cell culture supernatants after cleavage of SEAP-APP by BACE1 or alpha-secretases. To render the assay specific for BACE1, the alpha-secretase cleavage site of SEAP-APP was eliminated either by site-directed mutagenesis or by substituting the transmembrane part of APP by the membrane domain of the erythropoietin receptor (EpoR). The pharmacology of these constructs was characterized in detail in HEK293 cells (human embryonic kidney cell line), and the SEAP-APP-EpoR construct was also introduced into primary murine neurons and there allowed specific measurement of BACE1 activity.

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Section: Discussionmentioning

confidence: 99%

Measurement of cellular β-site of APP cleaving enzyme 1 activity and its modulation in neuronal assay systems

Volbracht

Penzkofer

Mansson

et al. 2009

Analytical Biochemistry

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“…Apart from these assays, several other cellular assays intended for screening compounds for BACE-1 inhibition have been developed (14,22,23 ), but methods for convenient detection of BACE-1 activity in biological samples with sufficient specificity and sensitivity have not yet been established. In the present work we describe a convenient, rapid, and sensitive method for detection of BACE-1 activity in biological samples, based on immunocapture with a specific BACE-1-binding antibody and a sensitive 2-step detection method based on activation of an engineered proenzyme.…”

Section: Discussionmentioning

confidence: 99%

Detection of a Soluble Form of BACE-1 in Human Cerebrospinal Fluid by a Sensitive Activity Assay

Verheijen¹,

Huisman²,

Lent³

et al. 2006

Clinical Chemistry

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Background: Formation of deposits of the insoluble amyloid ␤-peptide is believed to be causally related with neurodegeneration in Alzheimer disease (AD). The ␤-peptide originates from a larger amyloid precursor protein (APP) by the action of proteolytic enzymes. The first proteolytic event leading to amyloid formation is the cleavage of APP by the membrane-bound aspartyl protease BACE-1, also known as memapsin-2. Inhibition of BACE-1 is thought to be a therapeutic approach to AD. Measuring BACE-1 activity in biological samples would be useful to elucidate the mechanism of AD and for development of AD drugs. Methods: We developed a sensitive and specific activity assay for BACE-1. The assay is based on a genetically engineered proenzyme that is specifically activated by BACE-

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“…Oh et.al. also utilized a cell based assay for BACE to identity peptide mimetic inhibitors using drosophila cells [42]. BACE1 and its significance to Alzheimers disease development has been extensively studied [43;44].…”

Section: Discussionmentioning

confidence: 99%

Identification of inhibitors using a cell-based assay for monitoring Golgi-resident protease activity

Coppola

Hamilton

Bhojani

et al. 2007

Analytical Biochemistry

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Non-invasive real time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease's natural milieu. We developed a protease-activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, upon protease mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV, and in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high throughput screening of 30,000 compound small molecule libraries, leading to identification of furin inhibitors. Further, this strategy was utilized to identify inhibitors of another Golgi protease, the β-site APP-cleaving enzyme (BACE). BACE cleavage of the amyloid precursor protein leads to formation of the Aβ peptide, a key event that leads to Alzheimer's disease. In conclusion, we describe a customizable, non-invasive technology for real time assessment of Golgi protease activity used to identify inhibitors of furin and BACE.

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Cell-based assay for β-secretase activity

Cited by 16 publications

References 22 publications

Measurement of cellular β-site of APP cleaving enzyme 1 activity and its modulation in neuronal assay systems

Measurement of cellular β-site of APP cleaving enzyme 1 activity and its modulation in neuronal assay systems

Detection of a Soluble Form of BACE-1 in Human Cerebrospinal Fluid by a Sensitive Activity Assay

Identification of inhibitors using a cell-based assay for monitoring Golgi-resident protease activity

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