title: An improved cell-based method for determining the γ-secretase enzyme activity against both Notch and APP substrates.
abstract: γ-Secretase modulators (GSM), which reduce amyloidogenic Aβ(42) production while maintaining total Aβ levels, and Notch-sparing γ-secretase inhibitors (GSIs) are promising therapies for the treatment of Alzheimer's Disease (AD). To have a safety margin for therapeutic use, GSMs and GSIs need to allow Notch intracellular domain (NICD) production, while preventing neurotoxic Aβ peptide production. Typically, GSI and GSM effects on these substrates are determined using two different cell lines, one for the measurement of enzyme activity against each substrate. However, predicting selectivity for different substrates across cell systems may reduce the reliability of such ratios such that the in vitro data are not useful for predicting in vivo safety margins. This is especially concerning since the IC(50)'s of some GSIs vary depending upon the level of APP expression in a cell line. To circumvent this problem, we utilized the SUP-T1 cell line which expresses a truncated Notch receptor fragment that does not need sheddase cleavage to be a γ-secretase substrate. When combined with a sensitive method of measuring Aβ production, this assay system allows both substrates to be measured simultaneously, reducing the potential to calculate imprecise selectivity margins. To demonstrate the value of this system, known GSIs and GSMs were examined in the SUP-T1 dual substrate assay. IC(50)'s were determined for both substrates and the in vitro selectivity margin was calculated. These data suggest using a single cell line is a more accurate prediction of the fold difference between NICD inhibition and Aβ(42) lowering for therapeutically promising GSIs and GSMs.
authors.affiliation: Satori Pharmaceuticals, 281 Albany Street, Cambridge, MA 02139, USA. Tim.Mckee@satoripharma.com
authors.forename: Timothy D
authors.initials: TD
authors.lastname: McKee
authors.forename: Robyn M B
authors.initials: RM
authors.lastname: Loureiro
authors.forename: Jo Ann
authors.initials: JA
authors.lastname: Dumin
authors.forename: Vladislav
authors.initials: V
authors.lastname: Zarayskiy
authors.forename: Barbara
authors.initials: B
authors.lastname: Tate
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000494:pharmacology; D000544:Alzheimer Disease / Q000188:drug therapy / Q000201:enzymology / Q000378:metabolism; D053829:Amyloid Precursor Protein Secretases / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016564:Amyloid beta-Protein Precursor / Q000187:drug effects* / Q000378:metabolism; D001381:Azepines / Q000494:pharmacology; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D010069:Oxadiazoles / Q000494:pharmacology; D051880:Receptors, Notch / Q000187:drug effects* / Q000378:metabolism; D052616:Solid Phase Extraction; D013379:Substrate Specificity; D013449:Sulfonamides / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology
chemical_list: D016564:Amyloid beta-Protein Precursor; D001381:Azepines; C554092:BMS 708163; D004791:Enzyme Inhibitors; C484278:N2-((2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl)-N1-((7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo(b,d)azepin-7-yl)-L-alaninamide; D010069:Oxadiazoles; D051880:Receptors, Notch; D013449:Sulfonamides; D013876:Thiophenes; C539549:begacestat; D053829:Amyloid Precursor Protein Secretases; D000409:Alanine