Tracey Fiedler

The Amyloid-β Rise and γ-Secretase Inhibitor Potency Depend on the Level of Substrate Expression

The amyloid-β (Aβ) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-β precursor protein (APP) through consecutive proteolytic cleavages by β-site APP-cleaving enzyme and γ-secretase. Unexpectedly γ-secretase inhibitors can increase the secretion of Aβ peptides under some circumstances. This “Aβ rise” phenomenon, the same inhibitor causing an increase in Aβ at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Aβ rise depends on the β-secretase-derived C-terminal fragment of APP (βCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Aβ, known as “p3,” formed by α-secretase cleavage, did not exhibit a rise. In addition to the Aβ rise, low βCTF or C99 expression decreased γ-secretase inhibitor potency. This “potency shift” may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, γ-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Aβ under conditions of low substrate expression. The Aβ rise was also observed in rat brain after dosing with the γ-secretase inhibitor BMS-299897. The Aβ rise and potency shift are therefore relevant factors in the development of γ-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Aβ rise, including the “incomplete processing” and endocytic models, are discussed.

P-Glycoprotein Efflux and Other Factors Limit Brain Amyloid β Reduction by β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitors in Mice

Alzheimers disease (AD) is a progressive neurodegenerative disease. Amyloid β (Aβ) peptides are hypothesized to cause the initiation and progression of AD based on pathologic data from AD patients, genetic analysis of mutations that cause early onset forms of AD, and preclinical studies. Based on this hypothesis, β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) inhibitors are an attractive therapeutic approach for AD because cleavage of the APP by BACE1 is required to form Aβ. In this study, three potent BACE1 inhibitors are characterized. All three inhibitors decrease Aβ formation in cultured cells with IC50 values less than 10 nM. Analysis of APP C-terminal fragments by immunoblotting and Aβ peptides by mass spectrometry showed that these inhibitors decreased Aβ by inhibiting BACE1. An assay for Aβ1–40 in mice was developed and used to show that these BACE1 inhibitors decreased plasma Aβ1–40, but not brain Aβ1–40, in wild-type mice. Because these BACE1 inhibitors were substrates for P-glycoprotein (P-gp), a member of the ATP-binding cassette superfamily of efflux transporters, these inhibitors were administered to P-gp knockout (KO) mice. These studies showed that all three BACE1 inhibitors decreased brain Aβ1–40 in P-gp KO mice, demonstrating that P-gp is a major limitation for development of BACE1 inhibitors to test the amyloid hypothesis. A comparison of plasma Aβ1–40 and brain Aβ1–40 dose responses for these three compounds revealed differences in relative ED50 values, indicating that factors other than P-gp can also contribute to poor brain activity by BACE1 inhibitors.

Initial SAR studies on apamin-displacing 2-aminothiazole blockers of calcium-activated small conductance potassium channels.

An initial SAR study on a series of apamin-displacing 2-aminothiazole K(Ca)2 channel blockers is described. Potent inhibitors such as N-(4-methylpyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine (13) are disclosed, and for select members of the series, the relationship between the observed activity in a thallium flux, a binding and a whole-cell electrophysiology assay is presented.

Ex Vivo Occupancy of γ-Secretase Inhibitors Correlates with Brain β-Amyloid Peptide Reduction in Tg2576 Mice

Reduction of brain β-amyloid peptide (Aβ) synthesis by γ-secretase inhibitors is a promising approach for the treatment of Alzheimers disease. However, measurement of central pharmacodynamic effects in the Alzheimers disease patient will be a challenge. Determination of drug occupancy may facilitate the analysis of efficacy of γ-secretase inhibitors in a clinical setting. In this study, the relationship of γ-secretase site occupancy and brain Aβ40 reduction by γ-secretase inhibitors was examined in Tg2576 mice. [3H](2R,3S)-2-Isobutyl-N1-((S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-3-propylsuccinamide (IN973) was used as a γ-secretase radioligand, since it has been shown to bind to γ-secretase in rat, rhesus, and human brains with high affinity and specificity. We extended these findings by showing that [3H]IN973 bound to γ-secretase in Tg2576 brains with an affinity, specificity, and regional localization very similar to the other species. To quantify γ-secretase occupancy by γ-secretase inhibitors, an ex vivo binding assay was developed using [3H]IN973 and frozen brain sections from drug-treated mice. γ-Secretase occupancy and brain Aβ40 reduction were found to be highly correlated in animals dosed with either 2-[(1R)-1-[[4-chlorophenyl)-sulfonyl](2,5-difluorophenyl)amino] ethyl]-5-fluoro-benzenepropanoic acid (BMS-299897) or (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796) over a wide range of doses and times postdose, with the exception of the earliest times postdose. This lag in Aβ40 response to γ-secretase inhibition is probably related to the delayed clearance of previously produced Aβ40. The excellent correlation between brain Aβ40 and γ-secretase occupancy suggests that a positron emission tomography ligand for γ-secretase could be a valuable biomarker to determine whether γ-secretase inhibitors bind to their target in humans.

Ex vivo Occupancy of γ-Secretase Inhibitors Correlates with Brain Aβ Reduction in Tg2576 Mice

Reduction of brain β-amyloid peptide (Aβ) synthesis by γ-secretase inhibitors is a promising approach for the treatment of Alzheimers disease. However, measurement of central pharmacodynamic effects in the Alzheimers disease patient will be a challenge. Determination of drug occupancy may facilitate the analysis of efficacy of γ-secretase inhibitors in a clinical setting. In this study, the relationship of γ-secretase site occupancy and brain Aβ40 reduction by γ-secretase inhibitors was examined in Tg2576 mice. [3H](2R,3S)-2-Isobutyl-N1-((S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-3-propylsuccinamide (IN973) was used as a γ-secretase radioligand, since it has been shown to bind to γ-secretase in rat, rhesus, and human brains with high affinity and specificity. We extended these findings by showing that [3H]IN973 bound to γ-secretase in Tg2576 brains with an affinity, specificity, and regional localization very similar to the other species. To quantify γ-secretase occupancy by γ-secretase inhibitors, an ex vivo binding assay was developed using [3H]IN973 and frozen brain sections from drug-treated mice. γ-Secretase occupancy and brain Aβ40 reduction were found to be highly correlated in animals dosed with either 2-[(1R)-1-[[4-chlorophenyl)-sulfonyl](2,5-difluorophenyl)amino] ethyl]-5-fluoro-benzenepropanoic acid (BMS-299897) or (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796) over a wide range of doses and times postdose, with the exception of the earliest times postdose. This lag in Aβ40 response to γ-secretase inhibition is probably related to the delayed clearance of previously produced Aβ40. The excellent correlation between brain Aβ40 and γ-secretase occupancy suggests that a positron emission tomography ligand for γ-secretase could be a valuable biomarker to determine whether γ-secretase inhibitors bind to their target in humans.

Design, synthesis and evaluation of constrained tetrahydroimidazopyrimidine derivatives as antagonists of corticotropin-releasing factor type 1 receptor (CRF1R).

Several tetrahydroimidazopyrimidines were prepared using silver assisted cyclization as the key step. The binding affinities of compounds thus prepared were evaluated in vitro toward hCRF(1)R. Initial lead compound 16 (K(i)=32 nM) demonstrated modest putative anxiolytic effects in the mouse canopy test. Further optimization using parallel synthesis provided compounds with K(i)s <50 nM.

Preliminary SAR studies on non-apamin-displacing 4-(aminomethylaryl)pyrrazolopyrimidine KCa channel blockers

An exploratory SAR study on a series of potent, non-apamin-displacing 4-(aminomethylaryl)pyrazolopyrimidine K(Ca) channel blockers is described and their selectivity against K(Ca) channel subtypes is reported. The most potent analog, 5-chloro-N-(thiophen-2-ylmethyl)pyrazolo[1,5-a]pyrimidin-7-amine (24) displayed sub-micromolar activity in both a thallium flux and whole-cell electrophysiology assay and did not displace apamin in a competitive binding study.

P2-346: PGP efflux and other factors limit brain Aβ reduction by BACE1 inhibitors in mice

one protein located on the endoplasmic reticulum (ER) membrane, precisely at focal contacts between the ER and mitochondria (Hayashi & Su, Cell 131:596, 2007). The protein regulates the activity of different ER proteins, like IP3 receptors or ER stress sensors (GRP78/BiP, PERK, ATF-6). The 1 chaperone has the unique particularity to be sensitive to synthetic ligands, which therefore allow a very focal regulation of intracellular calcium homeostasis at ER and/or mitochondria contacts. As a consequence, 1 activators/agonists have been shown to induce acute modulation of transduction pathways, effective at the behavioral level. 1 Activators are indeed anti-amnesic, antidepressant and neuroprotective compounds. We validate new 1 activator compounds as neuroprotective agents against amyloid toxicity and analyze their mechanism of action. Methods: PRE-084 is a morpholine piperidine derivative acting as a high affinity and selective 1 activator. ANAVEX1-41 is a tetrahydro-furanmethanamine that shows high affinity for M1, M2, M4 muscarinic acetylcholine receptors and 1 protein. Both compounds are potent anti-amnesic drugs alleviating learning impairments observed in mice after the central (i.c.v.) injection of amyloid 25-35 peptide (A 25-35). Results: Central administration of A 25-35 induces within one week histological and biochemical changes, memory deficits, oxidative stress and ER stress in sensitive brain structures (hippocampus, cortex), highly reminiscent of the amyloid toxicity observed in Alzheimer’s disease. A 25-35 also provokes the induction of intracellular pro-apoptotic caspases and Bax-related proteins, markers of the induction of apoptosis. At the morphological level, A 25-35 induces a marked glial (astroglia, microglia) reaction and cell loss quantifiable in pyramidal layers of the hippocampus. Pre-administration of PRE-084 or ANAVEX1-41 prevents significantly all these pathological changes, showing that 1 activators are effective neuroprotectants. Part of the mechanism involves regulation of the expression of activity of IP3 receptors or ER stress sensors, in relation with the massive calcium overload induced by A 25-35. Conclusions: 1 Activators/agonists show potent neuroprotective and putatively disease-modifying activity against amyloid toxicity. Moreover, ANAVEX1-41 is active at 30-100 g/kg i.p., suggesting a cooperative action between muscarinic and 1 targets.