کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10533189 | 961855 | 2005 | 8 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Biochemical and cell-based assays for characterization of BACE-1 inhibitors
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
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چکیده انگلیسی
The deposition of β-amyloid peptides (Aβ42 and Aβ40) in neuritic plaques is one of the hallmarks of Alzheimer's disease (AD). Aβ peptides are derived from sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases. BACE-1 has been shown to be the major β-secretase and is a primary therapeutic target for AD. In this article, two novel assays for the characterization of BACE-1inhibitors are reported. The first is a sensitive 96-well HPLC biochemical assay that uses a unique substrate containing an optimized peptide cleavage sequence, NFEV, spanning from the P2-P2Ⲡpositions This substrate was processed by BACE-1 approximately 10 times more efficiently than was the widely used substrate containing the Swedish (NLDA) sequence. As a result, the concentration of the enzyme required for the assay can be as low as 100 pM, permitting the evaluation of inhibitors with subnanomolar potency. The assay has also been applied to related aspartyl proteases such as cathepsin D (Cat D) and BACE-2. The second assay is a homogeneous electrochemiluminescence assay for the evaluation of BACE-1 inhibition in cultured cells that assesses the level of secreted amyloid EV40_NF from HEK293T cells stably transfected with APP containing the novel NFEV sequence. To illustrate the use of these assays, the properties of a potent, cell-active BACE-1 inhibitor are described.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 342, Issue 1, 1 July 2005, Pages 144-151
Journal: Analytical Biochemistry - Volume 342, Issue 1, 1 July 2005, Pages 144-151
نویسندگان
Beth L. Pietrak, Ming-Chih Crouthamel, Katherine Tugusheva, Janet E. Lineberger, Min Xu, Jillian M. DiMuzio, Thomas Steele, Amy S. Espeseth, Shawn J. Stachel, Craig A. Coburn, Samuel L. Graham, Joseph P. Vacca, Xiao-Ping Shi, Adam J. Simon,