title: Biochemical and cell-based assays for characterization of BACE-1 inhibitors.
abstract: The deposition of beta-amyloid peptides (A beta42 and A beta40) in neuritic plaques is one of the hallmarks of Alzheimer's disease (AD). A beta peptides are derived from sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. BACE-1 has been shown to be the major beta-secretase and is a primary therapeutic target for AD. In this article, two novel assays for the characterization of BACE-1 inhibitors 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-P2' positions 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.
authors.affiliation: Department of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA.
authors.forename: Beth L
authors.initials: BL
authors.lastname: Pietrak
authors.forename: Ming-Chih
authors.initials: MC
authors.lastname: Crouthamel
authors.forename: Katherine
authors.initials: K
authors.lastname: Tugusheva
authors.forename: Janet E
authors.initials: JE
authors.lastname: Lineberger
authors.forename: Min
authors.initials: M
authors.lastname: Xu
authors.forename: Jillian M
authors.initials: JM
authors.lastname: DiMuzio
authors.forename: Thomas
authors.initials: T
authors.lastname: Steele
authors.forename: Amy S
authors.initials: AS
authors.lastname: Espeseth
authors.forename: Shawn J
authors.initials: SJ
authors.lastname: Stachel
authors.forename: Craig A
authors.initials: CA
authors.lastname: Coburn
authors.forename: Samuel L
authors.initials: SL
authors.lastname: Graham
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Vacca
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Shi
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Simon
authors.forename: Daria J
authors.initials: DJ
authors.lastname: Hazuda
authors.forename: Ming-Tain
authors.initials: MT
authors.lastname: Lai
mesh_terms: D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000235:genetics; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors*; D002402:Cathepsin D / Q000037:antagonists & inhibitors; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases; D006801:Humans; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D011480:Protease Inhibitors / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013449:Sulfonamides / Q000494:pharmacology*; D014162:Transfection
chemical_list: D016564:Amyloid beta-Protein Precursor; C504441:EVNFEVEF peptide; C504442:N-methyl-N-(3-((1-phenylethyl)aminocarbonyl)-5-((1-benzyl-4-cyclopropylamino-3-hydroxybut-2-yl)aminocarbonyl)phenyl) methanesulfonamide; C414614:OM99-2; D009842:Oligopeptides; D011480:Protease Inhibitors; D013449:Sulfonamides; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; D002402:Cathepsin D