The development of potent gamma-secretase inhibitors having substituted heterocycles attached to a benzobicyclo[4.2.1]nonane core is described. This work led to the identification of [6S,9R,11R]-2',3',4',5,5',6,7,8,9,10-decahydro-2-(5-(4-fluorophenyl)-1-methyipyrazol-3-yl)-5'-(2,2,2-trifluoroethyl)spiro[6,9-methanobenzocyclooctene-11,3'-[1,2,5]thiadiazole] 1',1'-dioxide (16), which has excellent in vitro potency (0.06 nM) and which reduced amyloid-beta in APP-YAC mice with an ED50 of 1 mg/kg (po). 16 had a good pharmacokinetic profile in three preclinical species.

As the population ages, there is a growing need for effective therapies for the treatment of neurological diseases. A limited number of therapeutics are currently available to improve cognitive function and research is limited by the need for in vivo models. Zebrafish have recently become a focus of neurobehavioral studies since larvae display neuropathological and behavioral phenotypes that are quantifiable and relate to those seen in man. Due to the small size of Zebrafish larvae, assays can be undertaken in 96 well plates and as the larvae can live in as little as 200 μl of fluid, only a few milligrams of compound are needed for screening. Thus in vivo analysis of the effects of compounds can be undertaken at much earlier stages in the drug discovery process. This review will look at the utility of the zebrafish in the study of neurological diseases and its role in improving the throughput of candidate compounds in in vivo screens.

There is a substantial body of evidence indicating that beta-amyloid peptides (Abeta) are critical factors in the onset and development of Alzheimer's disease (AD). One strategy for combating AD is to reduce or eliminate the production of Abeta through inhibition of the gamma-secretase enzyme, which cleaves Abeta from the amyloid precursor protein (APP). We demonstrate here that chronic treatment for 3 months with 3 mg/kg of the potent, orally bioavailable and brain-penetrant gamma-secretase inhibitor N-[cis-4-[(4-chlorophenyl)-sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) attenuates the appearance of amyloid plaques in the Tg2576 mouse. These reductions in plaques were also accompanied by a decrease in the level of reactive gliosis. The morphometric and histological measures agreed with biochemical analysis of Abeta(40) and Abeta(42) in the cortex. Interestingly, the volume of the plaques across treatment groups did not change, indicating that reducing Abeta levels does not significantly alter deposit growth once initiated. Furthermore, we demonstrate that these beneficial effects can be achieved without causing histopathological changes in the ileum, spleen, or thymus as a consequence of blockade of the processing of alternative substrates, such as the Notch family of receptors. This indicates that in vivo a therapeutic window between these substrates seems possible--a key concern in the development of this approach to AD. An understanding of the mechanisms whereby MRK-560 shows differentiation between the APP and Notch proteolytic pathway of gamma-secretase should provide the basis for the next generation of gamma-secretase inhibitors.

Habituation, where a response is reduced when exposed to a continuous stimulus is one of the simplest forms of non-associative learning and has been shown in a number of organisms from sea slugs to rodents. However, very little has been reported in the zebrafish, a model that is gaining popularity for high-throughput compound screens. Furthermore, since most of the studies involving learning and memory in zebrafish have been conducted in adults, we sought to determine if zebrafish larvae could display non-associative learning and whether it could be modulated by compounds identified in previous rodent studies. We demonstrated that zebrafish larvae (7 days post fertilization) exhibit iterative reduction in a startle response to a series of acoustic stimuli. Furthermore, this reduction satisfied criteria for habituation: spontaneous recovery, more rapid reductions in startle to shorter intertrial intervals and dishabituation. We then investigated the pathways mediating this behavior using established compounds in learning and memory. Administration of rolipram (PDE4 inhibitor), donepezil (acetylcholinesterase inhibitor), and memantine (N-methyl-D-aspartic acid (NMDA) receptor antagonist) all increased the acoustic startle response and decreased habituation in the larvae, similar to previous rodent studies. Further studies demonstrated that NMDA blocked the memantine response and the effect of donepezil was blocked by mecamylamine but not atropine suggesting that the donepezil response was mediated by nicotinic rather than muscarinic receptors. Zebrafish larvae possess numerous advantages for medium to high-throughput screening; the model described herein therefore offers the potential to screen for additional compounds for further study on cognition function.

The protease gamma-secretase plays a pivotal role in the synthesis of pathogenic amyloid-beta in Alzheimer's disease (AD). Here, we report a further extension to a series of cyclohexyl sulfone-based gamma-secretase inhibitors which has allowed the preparation of highly potent compounds which also demonstrate robust Abeta(40) lowering in vivo (e.g., compound 32, MED 1mg/kg p.o. in APP-YAC mice).

Amyloid-beta (Aβ) deposits are one of the hallmarks of the neuropathological degeneration observed in Alzheimer's disease (AD) and Aβ concentrations have been reported to vary in different brain regions of AD patients. Aβ is produced by the sequential cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase, respectively. Previous studies have shown that over-expression of the γ-secretase complex leads to increased γ-secretase proteolytic activity increasing Aβ production. However, it is not known whether brain regions with highest Aβ concentration also express relatively higher levels of γ-secretase activity. Accordingly, the relationship between Aβ levels and γ-secretase activity across brain regions was investigated and correlated in the brains of transgenic and non-transgenic rodents commonly used in AD research. The data demonstrated that Aβ levels do vary in different brain regions in both transgenic and non-transgenic mice but are not correlated with regional γ-secretase activity. Furthermore, this study demonstrated that while mutations in the APP and PS1 sequences affect the absolute Aβ levels this is not reflected in an increase in γ-secretase proteolytic activity. The data in the current paper indicate that this assay is able to measure the level of γ-secretase activity in rodent species. Using this methodology will aid our understanding of physiological γ-secretase function.

The 3,4-fused sulfamides, sulfonamides and sulfone have been identified as highly potent gamma-secretase inhibitors. Evaluation of the SAR of substitution within these series has allowed the identification of a range of compounds which significantly reduce brain A beta in transgenic mouse models and thus have potential as possible treatments for Alzheimer's disease.

Department of In Vivo Neuroscience (J.D.B., J.R.A.), Department of Molecular & Cellular Neuroscience (D.W.S., R.O., H.D.W., S.E., N.W., T.W.R., M.S.S.), Department of Chemistry (M.A.R., I.C., T.H.), Merck Sharp & Dohme, Neuroscience Research Centre, Eastwick Road, Harlow, Essex, CM20 2QR, UK. Department of Safety Assessment (C.B-L, P.A.L) Merck Sharp & Dohme Research Centre, Chibret, Route de Marsat, Riom, 63963 Clermont-Ferrand, France JPET Fast Forward. Published on November 10, 2006 as DOI:10.1124/jpet.106.114330

POTENTIALRISK FACTORFORDOPAMINERGIC NEURODEGENERATIONUNDERLYINGPARKINSON’SDISEASE M.Gerlach,K.-L.Double,F.Tribl and P.Riederer Division of Clinical Neurobiology, Department of Child and Adolescent Psychiatry and Psychotherapy, University of Würzburg, Germany, Prince of Wales Medical Research Institute, Sydney, Australia, Medical Proteome Center, University of Bochum, Germany, and Division of Clinical Neurochemistry, Department of Psychiatry and Psychotherapy, University of Würzburg, Germany

Plaques in the parenchyma of the brain containing A beta peptides are one of the hallmarks of Alzheimer's disease. These A beta peptides are produced by the final proteolytic cleavage of the amyloid precursor protein by the intramembraneous aspartyl protease gamma-secretase. Thus, one approach to lowering levels of A beta has been via the inhibition of the gamma-secretase enzyme. Here, we report a novel, bioavailable gamma-secretase inhibitor, N-[ cis-4[( 4-chlorophenyl) sulfonyl]-4-( 2,5-difluorophenyl) cyclohexyl]1,1,1-trifluoromethanesulfonamide ( MRK-560) that displayed oral pharmacokinetics suitable for once-a-day dosing. It was able to markedly reduce A beta in the brain and cerebrospinal fluid ( CSF) in the rat, with ED50 values of 6 and 10 mg/kg, respectively. Time-course experiments using MRK-560 demonstrated these reductions in A beta could be maintained for 24 h, and comparable temporal reductions in rat brain and CSF A beta(40) further suggested that these two pools of A beta are related. This relationship between the brain and CSF A beta was maintained when MRK-560 was dosed once a day for 2 weeks, and accordingly, when all the data for the dose-response curve and time courses were correlated, a strong association was observed between the brain and CSF A beta levels. These results demonstrate that MRK-560 is an orally bioavailable gamma-secretase inhibitor with the ability to markedly reduce A beta peptide in the brain and CSF of the rat and confirm the utility of the rat for assessing the effects of gamma-secretase inhibitors on central nervous system A beta( 40) levels in vivo.

Salzmann, J.; Anichtchik, O.; Best, J.; Richards, F.; Fleming, A.; Roach, A.a; Goldsmith, P.b Author Information

The body of evidence indicating that β-amyloid peptides (Aβ) are critical factors in the onset and development of Alzheimer's disease (AD) has become over whelming. Reducing the production of Aβ through inhibition of the secretase enzymes that cleave Aβ from the amyloid precursor protein (APP) is identified as a key point for therapeutic intervention and is being pursued aggressively. To demonstrate that chronic treatment with a γ secretase inhibitor attenuates the appearance of amyloid lesions in the Tg2576 mouse model of Alzheimer's disease (AD). Investigate any consequent changes in reactive gliosis and aberrant Tau phosphorylation. To examine peripheral target organs for evidence of pathology induced by activity at alternative substrates, such as the Notch family of receptors. Mice were treated for 91 days then brains were subjected to detailed morphometric and histological evaluation using a variety of procedures including immunohistochemistry, stereology and ligand binding. Biochemical measures of Aβ (40) and (42), reactive gliosis (GFAP) and aberrant tau phosphorylation were made from samples of cortex. Peripheral organs were collected for histopathological evaluation. The data indicate that the prolonged blockade of amyloid production by a γ secretase inhibitor will significantly and dose dependently decrease the rate at which amyloid deposits form, attenuating both vascular and parenchymal lesions to the same degree. The consistency of average deposit volume across treatment groups indicates that lowering amyloid concentrations at these levels does not significantly alter deposit growth once initiated. Reactive gliosis is reduced in concert with plaque pathology. Plaque numbers also correlate significantly with the appearance of aberrant tau phosphorylation. Further, we demonstrate that these beneficial effects can be achieved at levels of γ secretase inhibition which do not appear to induce histopathological changes in selected target organs as a consequence of activity at alternative substrates, such as the Notch family of receptors. This important data may help allay a key concern in the development of this therapeutic approach to AD.

A novel series of N-alkyl-substituted cyclic sulfamides were developed from a screening hit. Chemistries were developed which allowed surveys of N-alkyl groups and amines resulting in the identification of N-trifluoroethyl-substituted cyclic sulfamides with good in vitro and in vivo gamma-secretase activity. One compound with subnanomolar activity elicited a reduction in brain Abeta40 after oral dosing in APP-YAC mice.

A new series of gamma-secretase inhibitors was developed from an in-house screening hit based on a benzobicyclo[4.2.1]nonane core. Lead optimisation studies led to the development of a series of potent inhibitors and in vivo efficacy was demonstrated.

The efficacy of gamma-secretase inhibitors in vivo has, to date, been generally assessed in transgenic mouse models expressing increased levels of amyloid-beta (A beta) peptide thereby allowing the detection of changes in A beta production. However, it is not clear whether the in vivo potency of gamma-secretase inhibitors is independent of the level of amyloid precursor protein expression. In other words, does a gamma-secretase inhibitor have the same effect in nontransgenic physiological animals versus transgenic overexpressing animals? In the present study, an immunoassay has been developed which can detect A beta(40) in the rat brain, where concentrations are much lower than those seen in transgenic mice such as Tg2576 (c. 0.7 and 25 nM, respectively) and in cerebrospinal fluid (CSF, c. 0.3 nM). Using this immunoassay, the effects of the gamma-secretase inhibitor LY-411575 [N-2-[(2S)-2(3,5-difluorophenyl)-2-hydroxyethanoyl]-N-1-[(7S)-5-methyl-6- oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide] were assessed and robust dose-dependent reductions in rat brain and CSF A beta(40) levels were observed with ID50 values of 1.3 mg/kg for both brain and CSF. These values were comparable with those calculated for LY-411575 in transgenic mice. Time course experiments using LY-411575 demonstrated comparable temporal reductions in rat brain and CSF A beta(40), further suggesting these two pools of A beta are related. Accordingly, when all the data for the dose-response curve and time course were correlated, a strong association was observed between the brain and CSF A beta(40) levels. These data demonstrate the utility of the rat as a novel approach for assessing the effects of gamma-secretase inhibitors on central nervous system A beta(40) levels in vivo.

Many aspects of physiology and behavior are temporally organized into daily 24 hr rhythms, driven by an endogenous circadian clock. Studies in eukaryotes have identified a network of interacting genes forming interlocked autoregulatory feedback loops which underlie overt circadian organization in single cells [1, 2]. While in mammals the master oscillator resides in the suprachiasmatic nuclei of the hypothalamus [2], semiautonomous circadian oscillators also exist in peripheral tissues [3-5] and in immortalized fibroblasts, where rhythmicity is induced following a serum shock [6, 7]. We used this model system in combination with high-density cDNA microarrays to examine the magnitude and quality of clock control of gene expression in mammalian cells. Supported by application of novel bioinformatics tools, we find similar to2% of genes, including expected canonical clock genes, to show consistent rhythmic circadian expression across five independent experiments. Rhythmicity in most of these genes is novel, and they fall into diverse functional groups, highlighted by a predominance of transcription factors, ubiquitin-associated factors, proteasome components, and Ras/MAPK signaling pathway components. When grouped according to phase, 68% of the genes were found to peak during estimated subjective day, 32% during estimated subjective night, with a tendency to peak at a phase corresponding to anticipation of dawn or dusk.

Unique expression patterns of a large number of genes can now define cell specific signatures under a given context.Cell specific signatures will be invaluable to understanding differentiation, cell type specificity and the cellular biology of environmental responses.Expression signatures could greatly enhance drug development by increasing efficacy and decreasing toxicity of therapeutic compounds.We have used (Affymetrix) oligonucleotide microarrays to survey and compare the expression profiles of 7000 human genes from cells of various origins, including lymphocytes, umbilical vein endothelial cells (HUVEC), a breast adenocarcinoma cell line (MCF7) and a glioblastoma cell line (U373MG).Environmental context such as time in culture, state of differentiation, media composition, CO2 content and culture confluency may alter cell signatures.Chemical compound manipulation of cell lines provides one mechanism to dissect function at the gene expression level.For example, extended treatment of endothelial cells with vascular endothelial growth factor (VEGF) results in greater than two-fold changes in expression level of approximately 100 genes, while extended treatment of glioma cell lines with platelet derived growth factor (PDGF) results in greater that two-fold changes in 39 genes.Analysing expression data from multiple cell lines can also be used to identify similarities of expression profiles for all cell lines.We have observed that an average cell type expresses 2335 out of 7000 genes assayed (approxiamately 33 %).Sixty-four percent of the 2335 genes are expressed in endothelial, U373MG, and MCF7 cells, with a total of 630 expressed at similar levels in all three cell types despite variation in growth media.