Alzheimer's disease mutations in APP but not γ-secretase modulators affect epsilon-cleavage-dependent AICD production.

Pathological amino-acid substitutions in the amyloid precursor protein (APP) and chemical γ-secretase modulators affect the processing of APP by the γ-secretase complex and the production of the amyloid-beta peptide Aß42, the accumulation of which is considered causative of Alzheimer's disease. Here we demonstrate that mutations in the transmembrane domain of APP causing aggressive early-onset familial Alzheimer's disease affect both γ- and ε-cleavage sites, by raising the Aß42/40 ratio and inhibiting the production of AICD50-99, one of the two physiological APP intracellular domains (ICDs). This is in sharp contrast to γ-secretase modulators, which shift Aß42 production towards the shorter Aß38, but unequivocally spare the ε-site and APP- and Notch-ICDs production. Molecular simulations suggest that familial Alzheimer's disease mutations modulate the flexibility of the APP transmembrane domain and the presentation of its γ-site, modifying at the same time, the solvation of the ε-site.

Orally bioavailable and brain-penetrant pyridazine and pyridine-derived γ-secretase modulators reduced amyloidogenic Aß peptides in vivo.

Accumulation of amyloid ß (Aß) in brain is a pathological hallmark of Alzheimer's disease (AD). Aß is generated after sequential cleavage of its parental molecule, amyloid precursor protein (APP), by ß- and γ-secretases. Inhibition of γ-secretase activity is an effective approach for the reduction of Aß levels. Since γ-secretase targets many different substrates, selective inhibition of its cleavage of APP is believed to be critical in order to avoid undesirable side effects. γ-Secretase modulator (GSM) shifts the cleavage site on APP and production of amyloidogenic to non-amyloidogenic Aß fragments. Since GSMs only modulate and do not block cleavage of γ-secretase substrates, they are believed less likely to produce untoward adverse reactions. Here, we report in vivo Aß-lowering profiles of a pyridazine and a pyridine-derived GSM: GSM-C (Wan et al., 2011a) and GSM-D (Wan et al., 2011b). Both compounds reduced Aß40 and Aß42 productions, increased shorter Aß fragments, and had little effect on Notch signaling (∼100-fold selective). They had excellent oral bioavailability (97.8% for GSM-C, ∼100% for GSM-D) and good brain permeability (free brain to free blood AUC ratio of 0.41 and 1.10 for GSM-C and GSM-D, respectively). Oral administration of these compounds in both acute and sub-chronic conditions reduced Aß levels in plasma and brain in rats in a dose- and time-dependent manner. Therefore, GSM-C and GSM-D represent two GSMs that are orally bioavailable and brain-permeable. They could serve as excellent tools in the investigation of the role of Aß peptides in AD pathogenesis.

Focal expression of adeno-associated viral-mutant tau induces widespread impairment in an APP mouse model.

Adeno-associated virus serotype 6 (AAV6) viral vectors encoding mutant and normal tau were used to produce focal tau pathology. Two mutant forms of tau were used; the P301S tau mutation is associated with neurofibrillary tangle formation in humans, and the 3PO mutation leads to rapid tau aggregation and is associated with pathogenic phosphorylation and cytotoxicity in vitro. We show that adeno-associated viral injection into entorhinal cortex of normal and tau knockout animals leads to local overexpression of tau, and the presence of human tau in axons projecting to and emanating from the entorhinal cortex. Starting at 2 months and increasing by 6 months post-injection neurons expressing mutant tau developed hyperphosphorylated tau pathology, in addition to dystrophic neurites. There was neuronal loss in tau-expressing regions, which was similar in normal and in TASTPM mice injected with mutant tau. There was neuroinflammation around plaques, and in regions expressing mutant tau. We saw no evidence that mutant tau had affected amyloid-beta pathology or vice versa. Morris water maze behavioral tests demonstrated mild memory impairment attributable to amyloid-beta pathology at 2 and 4 months, with severe impairment at 6 months in animals receiving adeno-associated viral-3PO. Therefore, TASTPM mice injected with mutant tau displayed many of the main features characteristic of human Alzheimer's disease patients and might be used as a model to test new drugs to ameliorate clinical features of Alzheimer's disease.

Substrate determinants in the C99 juxtamembrane domains differentially affect γ-secretase cleavage specificity and modulator pharmacology.

The molecular mechanisms governing γ-secretase cleavage specificity are not fully understood. Herein, we demonstrate that extending the transmembrane domain of the amyloid precursor protein-derived C99 substrate in proximity to the cytosolic face strongly influences γ-secretase cleavage specificity. Sequential insertion of leucines or replacement of membrane-anchoring lysines by leucines elevated the production of Aß42, whilst lowering production of Aß40. A single insertion or replacement was sufficient to produce this phenotype, suggesting that the helical length distal to the ε-site is a critical determinant of γ-secretase cleavage specificity. Replacing the lysine at the luminal membrane border (K28) with glutamic acid (K28E) increased Aß37 and reduced Aß42 production. Maintaining a positive charge with an arginine replacement, however, did not alter cleavage specificity. Using two potent and structurally distinct γ-secretase modulators (GSMs), we elucidated the contribution of K28 to the modulatory mechanism. Surprisingly, whilst lowering the potency of the non-steroidal anti-inflammatory drug-type GSM, the K28E mutation converted a heteroaryl-type GSM to an inverse GSM. This result implies the proximal lysine is critical for the GSM mechanism and pharmacology. This region is likely a major determinant for substrate binding and we speculate that modulation of substrate binding is the fundamental mechanism by which GSMs exert their action.

Development and characterization of a novel membrane assay for full-length BACE-1 at pH 6.0.

ß-Site amyloid precursor protein cleaving enzyme-1 (BACE-1) is a transmembrane aspartic protease that mediates the initial cleavage of the amyloid precursor protein (APP), leading to the generation of amyloid-ß (Aß) peptides that are thought to be causative of Alzheimer's disease (AD). Consequently, inhibition of BACE-1 is an attractive therapeutic approach for the treatment of AD. In general, in vitro biochemical assays to monitor BACE-1 activity have used the extracellular domain of the protein that contains the catalytic active site. This form of BACE-1 is catalytically active at acidic pH and cleaves APP-based peptide substrates at the ß-site. However, this form of BACE-1 does not mimic the natural physiology of BACE-1 and shows minimal activity at pH 6.0, which is more representative of the pH within the intracellular compartments where BACE-1 resides. Moreover, high-throughput screens with recombinant BACE-1 at pH 4.5 have failed to identify tractable leads for drug discovery, and hence, BACE-1 inhibitor development has adopted a rational drug design approach. Here we describe the development and validation of a novel membrane assay comprising full-length BACE-1 with measurable activity at pH 6.0, which could be used for the identification of novel inhibitors of BACE-1.

The role of γ-secretase activating protein (GSAP) and imatinib in the regulation of γ-secretase activity and amyloid-ß generation.

γ-Secretase is a large enzyme complex comprising presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1 that mediates the intramembrane proteolysis of a large number of proteins including amyloid precursor protein and Notch. Recently, a novel γ-secretase activating protein (GSAP) was identified that interacts with γ-secretase and the C-terminal fragment of amyloid precursor protein to selectively increase amyloid-ß production. In this study we have further characterized the role of endogenous and exogenous GSAP in the regulation of γ-secretase activity and amyloid-ß production in vitro. Knockdown of GSAP expression in N2a cells decreased amyloid-ß levels. In contrast, overexpression of GSAP in HEK cells expressing amyloid precursor protein or in N2a cells had no overt effect on amyloid-ß generation. Likewise, purified recombinant GSAP had no effect on amyloid-ß generation in two distinct in vitro γ-secretase assays. In subsequent cellular studies with imatinib, a kinase inhibitor that reportedly prevents the interaction of GSAP with the C-terminal fragment of amyloid precursor protein, a concentration-dependent decrease in amyloid-ß levels was observed. However, no interaction between GSAP and the C-terminal fragment of amyloid precursor protein was evident in co-immunoprecipitation studies. In addition, subchronic administration of imatinib to rats had no effect on brain amyloid-ß levels. In summary, these findings suggest the roles of GSAP and imatinib in the regulation of γ-secretase activity and amyloid-ß generation are uncertain.

Effects of the selective 5-HT(7) receptor antagonist SB-269970 in animal models of psychosis and cognition.

The 5-hydroxytryptamine7 (5-HT7) receptor is a G-protein coupled receptor for serotonin that has been implicated in the pathophysiology of psychiatric and neurological disorders including anxiety, depression and schizophrenia. A number of studies have attempted to evaluate the potential role of the 5-HT7 receptor in schizophrenia by utilising genetic or pharmacological tools but to date these have provided conflicting results. Here we investigate the effect of a selective 5-HT7 receptor antagonist, SB-269970, in in vivo psychosis and cognition models and relate efficacy to brain exposures of the compound. SB-269970 significantly attenuated amphetamine-induced rearing and circling in rats. A similar effect was observed in an N-methyl d-aspartic acid (NMDA) receptor antagonist driven psychosis model, where SB-269970 significantly reversed phencyclidine-induced hyperlocomotion, rearing and circling; although the effect was not as robust as with the 5-HT2a receptor antagonist positive control, MDL100,907. SB-269970 also attenuated a temporal deficit in novel object recognition (NOR), indicative of an improvement in recognition memory. Pharmacokinetic analysis of plasma and brain samples taken after behavioural testing confirmed that efficacy was achieved at doses and pre-treatment times where receptor occupancy was substantial. These findings highlight the anti-psychotic and pro-cognitive potential of 5-HT7 receptor antagonists and warrant further studies to explore their therapeutic potential in schizophrenia.

Γ-secretase modulators do not induce Aß-rebound and accumulation of ß-C-terminal fragment.

γ-secretase inhibitors (GSIs) have been developed to reduce amyloid-ß (Aß) production for the treatment of Alzheimer's disease by inhibiting the cleavage of amyloid precursor protein (APP). However, cross-inhibitory activity on the processing of Notch can cause adverse reactions. To avoid these undesirable effects, γ-secretase modulators (GSMs) are being developed to selectively reduce toxic Aß production without perturbing Notch signaling. As it is also known that GSIs can cause a paradoxical increase of plasma Aß over the baseline after a transient reduction (known as Aß-rebound), we asked if GSMs would cause a similar rebound and what the potential mechanism might be. Our studies were performed with one GSI (LY-450139) and two chemically distinct GSMs. Although LY-450139 caused Aß-rebound as expected in rat plasma, the two GSMs did not. Inhibition of APP processing by LY-450139 induced an accumulation of γ-secretase substrates, α- and ß-C-terminal fragments of APP, but neither GSM caused such an accumulation. In conclusion, we discover that GSMs, unlike GSIs, do not cause Aß-rebound, possibly because of the lack of accumulation of ß-C-terminal fragments. GSMs may be superior to GSIs in the treatment of Alzheimer's disease not only by sparing Notch signaling but also by avoiding Aß-rebound.

Reduction in BACE1 decreases body weight, protects against diet-induced obesity and enhances insulin sensitivity in mice.

Insulin resistance and impaired glucose homoeostasis are important indicators of Type 2 diabetes and are early risk factors of AD (Alzheimer's disease). An essential feature of AD pathology is the presence of BACE1 (ß-site amyloid precursor protein-cleaving enzyme 1), which regulates production of toxic amyloid peptides. However, whether BACE1 also plays a role in glucose homoeostasis is presently unknown. We have used transgenic mice to analyse the effects of loss of BACE1 on body weight, and lipid and glucose homoeostasis. BACE1-/- mice are lean, with decreased adiposity, higher energy expenditure, and improved glucose disposal and peripheral insulin sensitivity than wild-type littermates. BACE1-/- mice are also protected from diet-induced obesity. BACE1-deficient skeletal muscle and liver exhibit improved insulin sensitivity. In a skeletal muscle cell line, BACE1 inhibition increased glucose uptake and enhanced insulin sensitivity. The loss of BACE1 is associated with increased levels of UCP1 (uncoupling protein 1) in BAT (brown adipose tissue) and UCP2 and UCP3 mRNA in skeletal muscle, indicative of increased uncoupled respiration and metabolic inefficiency. Thus BACE1 levels may play a critical role in glucose and lipid homoeostasis in conditions of chronic nutrient excess. Therefore strategies that ameliorate BACE1 activity may be important novel approaches for the treatment of diabetes.

Pyridine-derived γ-secretase modulators.

SAR of a novel series of pyridine-derived γ-secretase modulators is described. Compound 5 was found to be a potent modulator in vitro, which on further profiling, was found to decrease Aß42 and Aß40, and maintain (or increase) the levels of total Aß. Furthermore, representative compounds 1 and 5 demonstrated in vivo efficacy to lower Aß42 in the brain without altering Notch processing in the peripheral.

Pyridazine-derived γ-secretase modulators.

SAR of a novel series of pyridazine-derived γ-secretase modulators is described. Compound 25 was found to be a potent modulator in vitro, which on further profiling, was found to decrease Aß42 and Aß40, and maintain the levels of total Aß. Furthermore, 25 demonstrated excellent pharmacokinetic parameters as well as good CNS penetration in the rat.

Dynamics of Aß42 reduction in plasma, CSF and brain of rats treated with the γ-secretase modulator, GSM-10h.

BACKGROUND: Allosteric modulation of γ-secretase is an attractive therapeutic approach for the treatment of Alzheimer's disease. We recently identified a novel γ-secretase modulator, GSM-10h, which effectively lowers Aß42 production in cells and in amyloid precursor protein transgenic mice. OBJECTIVE: Here, we describe the in vivo characterization of GSM-10h in a model of endogenous Aß production. METHODS: Rats were administered orally with GSM-10h, and the effect on Aß levels in peripheral and central compartments was determined. In addition, the effect of GSM-10h on Notch processing was assessed. RESULTS: Acute administration of GSM-10h to rats causes a dose-dependent decrease in the level of Aß42 in plasma, CSF and brain, with little effect on the level of Aß40 in these compartments. The magnitude of Aß42 lowering in the CSF and brain was further enhanced upon sub-chronic administration of GSM-10h. No deleterious effect on Notch processing was evident in either of these studies. To further explore the dynamics of Aß42 reduction in peripheral and CNS compartments, a time course study was conducted. In all compartments, the decrease in Aß42 was greatest at 6 h after administration of GSM-10h. This decrease in Aß42 was maintained for 9-15 h, after which time Aß42 levels returned to baseline levels. Encouragingly, no rebound in Aß42 levels beyond baseline levels was observed. CONCLUSIONS: These findings support the γ-secretase modulator profile of GSM-10h, and highlight the utility of the rat for assessing the pre-clinical efficacy of γ-secretase modulators.

TASTPM mice expressing amyloid precursor protein and presenilin-1 mutant transgenes are sensitive to γ-secretase modulation and amyloid-ß42 lowering by GSM-10h.

BACKGROUND: Cleavage of the amyloid precursor protein (APP) by ß-site APP-cleaving enzyme and γ-secretase results in the generation of amyloid-ß (Aß) peptides that aggregate and deposit as senile plaques in brains of Alzheimer disease patients. Due to the fundamental role γ-secretase plays in the proteolysis of a number of proteins including Notch, pharmacological inhibition of γ-secretase has been associated with mechanism-based toxicities. Therefore, efforts have focussed on the modulation of γ-secretase activity to selectively decrease levels of Aß42 peptide while avoiding deleterious activity on Notch processing. OBJECTIVE: Here, we describe the in vitro and in vivo characterisation of a novel γ-secretase modulator, GSM-10h, and investigate the potential for shorter Aß peptides to induce neurotoxicity in rat primary cortical neurons. METHODS: The effect of GSM-10h on Aß levels was investigated in SH-SY5Y cells expressing mutant APP and in TASTPM mice expressing APP and presenilin-1 mutant transgenes. The effect of GSM-10h on Notch processing was also determined. RESULTS: In cells, GSM-10h decreased levels of Aß42 while concomitantly increasing levels of Aß38 in the absence of effects on Aß40 levels. In TASTPM mice, GSM-10h effectively lowered brain Aß42 and increased brain Aß38, with no effect on Notch signalling. Unlike Aß42, which causes neuronal cell death, neither Aß37 nor Aß38 were neurotoxic. CONCLUSIONS: These findings confirm GSM-10h exhibits the profile of a γ-secretase modulator. In addition, TASTPM mice are shown to be responsive to treatment with a γ-secretase modulator, thereby highlighting the utility of this bitransgenic mouse model in drug discovery efforts focussed on the development of γ-secretase modulators.

BACE-1 hydroxyethylamine inhibitors using novel edge-to-face interaction with Arg-296.

Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. Herein, is described a series of potent inhibitors based on an hydroxyethylamine (HEA) transition state mimetic template. These inhibitors interact with the non prime side of the enzyme using a novel edge-to-face interaction with Arg-296.

APP transgenic mouse models and their use in drug discovery to evaluate amyloid- lowering therapeutics.

A critical requirement in the development of Alzheimer's disease (AD) therapeutics is a demonstration of the in vivo efficacy of compounds in pre-clinical disease relevant models. One of the most frequently used models in AD research are transgenic mice overexpressing mutant forms of human amyloid precursor protein (APP) that are associated with early-onset familial AD. These mice exhibit an age-dependent accumulation and deposition of amyloid -peptide (A) as extracellular plaques in the brain, and thereby depict one of the key pathologies observed in the brains of AD patients. Although these mouse models do not recapitulate all the pathological features of AD, they have been invaluable in the development of therapeutic agents aimed at lowering A production, inhibiting A deposition or facilitating A clearance. Further development of these APP transgenic models led to the incorporation of transgenes for human mutant presenilins, resulting in an accelerated A deposition rate and human mutant tau protein leading to neurofibrillary tangle-like pathology. The latter was a major advance in the development of AD models, as it allowed researchers to investigate the interplay between the two key pathologies of AD. This review highlights how APP transgenic mouse models have successfully been used in drug discovery to support the progression of A lowering therapeutics to clinical trials to ultimately test the 'amyloid hypothesis' of AD.

Piperidine-derived gamma-secretase modulators.

This Letter details the SAR of a novel series of piperidine-derived gamma-secretase modulators. Compound 10h was found to be a potent modulator in vitro, which on further profiling, was found to decrease Abeta42, increase Abeta38 and have no effect on Abeta40 levels. Furthermore, 10h demonstrated excellent pharmacokinetic parameters in the mouse, rat and dog in addition to good CNS penetration in the mouse.

Second generation of BACE-1 inhibitors. Part 1: The need for improved pharmacokinetics.

Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. We have recently disclosed a series of transition-state mimetic BACE-1 inhibitors showing nanomolar potency in cell-based assays. Amongst them, GSK188909 (compound 2) had favorable pharmacokinetics and was the first orally bioavailable inhibitor reported to demonstrate brain amyloid lowering in an animal model. In this Letter, we describe the reasons that led us to favor a second generation of inhibitors for further in vivo studies.

Second generation of BACE-1 inhibitors part 2: Optimisation of the non-prime side substituent.

Our first generation of hydroxyethylamine transition-state mimetic BACE-1 inhibitors allowed us to validate BACE-1 as a key target for Alzheimer's disease by demonstrating amyloid lowering in an animal model, albeit at rather high doses. Finding a molecule from this series which was active at lower oral doses proved elusive and demonstrated the need to find a novel series of inhibitors with improved pharmacokinetics. This Letter describes the discovery of such inhibitors.

Second generation of BACE-1 inhibitors part 3: Towards non hydroxyethylamine transition state mimetics.

Our first generation of hydroxyethylamine BACE-1 inhibitors proved unlikely to provide molecules that would lower amyloid in an animal model at low oral doses. This observation led us to the discovery of a second generation of inhibitors having nanomolar activity in a cell-based assay and with the potential for improved pharmacokinetic profiles. In this Letter, we describe our successful strategy for the optimization of oral bioavailability and also give insights into the design of compounds with the potential for improved brain penetration.

Second generation of hydroxyethylamine BACE-1 inhibitors: optimizing potency and oral bioavailability.

BACE-1 inhibition has the potential to provide a disease-modifying therapy for the treatment of Alzheimer's disease. Optimization of a first generation of BACE-1 inhibitors led to the discovery of novel hydroxyethylamines (HEAs) bearing a tricyclic nonprime side. These derivatives have nanomolar cell potency and are orally bioavailable.