Diarylaminothiazoles (Series A) and Diarylureas (Series B) are Potent Modulators of γ-Secretase Activity. (A) Chemical structures of key molecules from Series A and Series B GSMs, including Compound 6, the ethylene amino derivative of Compound 3 that was immobilized onto an Affigel matrix and used as an affinity chromatography ligand. (B) Concentration response curves (CRCs) for lowering of Aβ₄₂ levels produced by SH-SY5Y-APP cells. IC₅₀ values were derived using four parameter fit non-linear regression analyses.

Opposing Effects of Series A GSMs on Levels of Aβ₄₂ and Aβ₄₀ Versus Effects on Levels of Aβ₃₈ and Aβ₃₇ Peptide Variants. (A) SELDI-TOF mass spectroscopic analyses of anti-Aβ_17-24 mAb (4G8) immunoprecipitates of conditioned medium from CHO-APPswe cells treated with either vehicle (top panel), the functional GSI BMS-299897 (1 μM), also referred to as SIB-3520 (second panel), or the allosteric GSMs (each at 1 μM), Compound 2, Compound 5 or Compound 3 (bottom three panels, respectively). Equal amounts of synthetic Aβ_1-28 peptide were spiked into aliquots of vehicle- and the various compound-treated media samples prior to performing the immunoprecipitations and was used as an internal standard. (B) Immunoblots of anti-Aβ_1-12 (mAb-B436) immunoprecipitates (IP/westerns) of conditioned medium of Tg 2576 MBCs treated with vehicle, the functional GSI BMS-299897 (1 μM), also referred to as SIB-3520, or the indicated concentrations of the allosteric GSM, Compound 2. (C) Immunoblots of anti-Aβ_1-12 (mAb-B436) immunoprecipitates (IP/westerns) of CHAPSO-solubilized extracts of plasma or brain pools (left panel) from either vehicle or Compound 2-treated (100 mg/kg p.o for five consecutive days) Tg 2576 mice; (right panel) SDS-solubilized extracts of representative individual hemibrains from either vehicle or Compound 2-treated Tg 2576 mice. Immunoprecipitates of conditioned medium of CHO-APPswe cells following treatment with Compound 2 (1 μM) were used as Aβ peptide variant standards.

Affinity Chromatography Using an Immobilized Series A GSM Binds Isolated Pen-2 and Quantitatively Recovers Pen-2 from Nonionic Detergent-solubilized Cellular Extracts. (A-F) Compound 6 specifically retains Pen-2 from Triton X-100/Tween-20 (1.0%/0.2%)-solubilized CHO-APPswe whole cell lysates. Following gentle agitation of solubilzed whole cell lysates (WCL) with either the non-modified Affigel matrix (-) or Compound 6-immobilized Affigel matrix, flow thru supernatants (Super) were collected and columns were washed with PBS (Wash) and eluted with sample dilution buffer containing 1.0 % SDS (Elute). Equivalent aliquots of each fraction were electrophoresed using 4-20% gradient gels, immunoblotted/probed with the following antibodies; (A) anti-Pen-2, (B) anti- PS1-NTF, (C) anti-PS1-CTF, (D) anti-nicastrin (NCT), (E) anti-APP-CTF and (F) anti-phospholipase C-γ (PLCγ). (G) Affigel 10 with immobolized Compound 6 (+) retains purified recombinant Pen-2 and Affigel 10 alone (-) does not. PS-Holo and APP-Holo correspond to to PS1-holoprotein and APP-holoprotein, respectively.

Dose-dependent Reduction of Aβ₄₂ Levels in Plasma and Brain in Tg 2576 Mice Following Oral Administration of the Series A GSM Compound 4. (A) Time course of plasma and brain exposures of Compound 4 in female C57Bl/6 mice (2-3 mo of age, n=6 per group) following once daily oral administration for 3 consecutive days of Compound 4 (50 mg/kg, p.o.) in F110 vehicle. Plasma and brains were collected at the indicated times post-dose on day 3 and concentrations of Compound 4 were measured in plasma and brain extracts by LC-MS/MS. Exposure is given as the integrated area under the curve (AUC) of the compound concentration vs. time curve in ng·hr/ml. Brain/plasma is the brain:plasma ratio of Compound 4 exposure. (B) Dose-dependent efficacy of Compound 4 (p.o.) for lowering Aβ₄₂ in plasma (left panel) and brain (right panel) in Tg 2576 mice (3-4 mo of age, n=10/dose) following three days of once-daily dosing expressed as a percentage of vehicle (80% PEG-400 v/v) control. (C) Brain concentrations of Compound 4 attained following each dose. Data are mean ± SEM. *p < 0.05 by t-test for brain measurements or ANOVA with Dunnett's post-hoc analysis for plasma measurements.

Differential Effects of a Series A GSM, on Levels of Aβ₄₂, Aβ₄₀ and Aβ_total Peptide Variants. Concentration-response curves showing effects of the Series A GSM, Compound 3 (A and B) or the functional GSI, BMS-299897 (C and D) on levels of Aβ₄₂, Aβ₄₀ and Aβ_total peptide variants produced by CHO-APPswe cells (A and C) or SH-SY5Y-APP cells (B and D).

Series A GSMs do not Inhibit NICD Formation from Notch (NΔE) or AICD Formation from APP-CTFs. (A) Immunoblots (anti-NICD) of conditioned medium from HEK-APP-NΔE cells treated with the indicated concentrations of either DAPT or the designated allosteric GSMs. (B) Parallel analysis of AICD production (left panel) and Aβ₄₂ and Aβ₄₀ peptide formation (right panel) from CHO-APPswe membranes treated with either vehicle (0.2% DMSO), DAPT (1μM) or the indicated concentrations of the allosteric GSM, Compound 3. Data are expressed as mean ± SEM (n ≥ 3).

Compound 4, a Prototypical Series A GSM that Potently Lowers Levels of Aβ₄₂ and Aβ₄₀, Concomitantly Increases Levels of Aβ₃₈ and Aβ₃₇ and does not Inhibit γ-Secretase-Mediated Generation of E-Caderin γ-CTFs (E-Cad/CTF-γ) or NICD Generation from Notch (NΔE). (A) CRCs for Compound 4 (left panel) or BMS-299897 (right panel) on various Aβ peptide levels in conditioned medium following treatment (18 h) of Tg 2576 MBCs. Data from each specific sandwich ELISA or MesoScale assay were plotted as percent solvent control (0.2% v/v DMSO). IC₅₀ or EC₅₀ values were determined using four parameter fit non-linear regression analysis. (B) MALDI-TOF mass spectrometry of anti-Aβ_17-24 mAb (4G8) immunoprecipitates of conditioned medium from CHO-APPswe cells after treatment (18 h) with either Compound 4 (1μM) or vehicle (0.2% v/v DMSO). (C) Concentration response curves for inhibition of Aβ₄₂ and Aβ₄₀ production in HeLa cell membrane (left panel) or TAP-purified protein reconstituted γ-secretase complex (right panel) in vitro γ-secretase assays. IC₅₀ values were derived using four parameter fit non-linear regression analyses. (D) Immunoblots of E-cad γ-CTFs and the GAPDH control protein following treatment of human A431 cells with either DAPT (1 μM) or the indicated concentrations of Compound 4 for 18 h. Proteolysis of E-cadherin is induced by staurosporine (1 μM) treatment of A431 cells for 6 h. (F) Immunoblots of NICD and the GAPDH control protein following treatment of HEK-APP-NΔE cells with either DAPT (1 μM) or the indicated concentrations of Compound 4. DMSO (0.2%v/v) is the vehicle control (Veh) and 0.5× sample volume from vehicle treated cells (1/2 Veh) was analyzed to quantify relative levels of E-cad γ-CTF immunoreactivity (E) or NICD immunoreactivity (G) using laser scanning densitometry.

Chronic Daily Exposure of Tg 2576 Mice to the Series A GSM Compound 4 for Seven Consecutive Months is Well Tolerated and Significantly Reduces Overall Amyloid Load and Aβ Deposition in Neuritic Plaques. (A) Weekly group averaged body weights of Tg 2576 mice (8 mo – 15 mo of age) fed (ad libitum) either normal rodent chow (Vehicle; n=20) or rodent chow containing an estimated dose of 50 mg/kg/day of Compound 4 (Compound 4; n=19) for 29 consecutive weeks. Data are mean ± SEM. (B) Brain Aβ peptide (Aβ₄₂, Aβ₄₀ and Aβ₃₈) concentrations (measured using Meso Scale triplex kits) in aqueous buffer soluble (DEA), denaturing detergent-extractable (SDS) and formic acid-extractable (FA) brain fractions from Tg 2576 mice fed either normal rodent chow or chow containing Compound 4. Data are expressed as mean (± SEM) of percentage of control levels in normal chow-fed mice (p ≤ 0.002 for each of the Aβ peptide variants in all extracts except for Aβ₃₈ in FA extract; p = 0.0441). (C) Representative photomicrographs of Campbell-Switzer silver stained sections showing neuritic plaque staining in the cerebral cortex and hippocampus from a normal chow-fed 15-mo old Tg 2576 mouse (left panel) and a 15-mo old Tg 2576 mouse fed Compound 4 (50 mg/kg/day)-containing chow (right panel) from 8 to 15 months of age. (D) Percentage of cerebral cortical area (left panel) and hippocampal area (right panel) occupied by neuritic plaques, quantified from 4 brain levels. Data are expressed as percent area (mm²) of individual mice from normal chow-fed (Vehicle) and Compound 4-containing chow-fed (Compound 4) Tg 2576 mice.