Secretases as therapeutic targets for Alzheimer's disease

Accumulation of amyloid-β (Aβ) is widely accepted as the key instigator of Alzheimer’s disease (AD). The proposed mechanism is that accumulation of Aβ results in inflammatory responses, oxidative damages, neurofibrillary tangles and, subsequently, neuronal/synaptic dysfunction and neuronal loss. Given the critical role of Aβ in the disease process, the proteases that produce this peptide are obvious targets. The goal would be to develop drugs that can inhibit the activity of these targets. Protease inhibitors have proved very effective for treating other disorders such as AIDS and hypertension. Mutations in APP (amyloid-β precursor protein), which flanks the Aβ sequence, cause early-onset familial AD, and evidence has pointed to the APP-to-Aβ conversion as a possible therapeutic target. Therapies aimed at modifying Aβ-related processes aim higher up the cascade and are therefore more likely to be able to alter the progression of the disease. However, it is not yet fully known whether the increases in Aβ levels are merely a result of earlier events that were already causing the disease.     

Evidence for dimeric BACE-mediated APP processing

β-Secretase (BACE) is an aspartyl protease, which proteolytically processes amyloid precursor protein, making BACE an interesting pharmacological target in Alzheimer’s disease. To study the enzymatic function of BACE, we mutated either of the two aspartic acid residues in the active site of BACE. This rendered BACE functionally inactive without affecting the degree of glycosylation or endosomal localization. In contrast, substituting both active site aspartic acid residues produced a functionally inactive, endoplasmic reticulum-retained and partially glycosylated BACE. Interestingly, co-expression of the two single active site mutants partially restored β-site cleavage of amyloid precursor protein, and the restored activity was inhibited with similar dose-dependency and potency as wildtype BACE by a small molecule inhibitor raised against BACE. In sum, our data suggest that two different active site mutants can complement each other in a partially functional BACE dimer and mediate APP processing.     

Discovery of 4-aminomethylphenylacetic acids as γ-secretase modulators via a scaffold design approach

Starting from literature examples of nonsteroidal anti-inflammatory drugs (NSAIDs)-type carboxylic acid γ-secretase modulators (GSMs) and using a scaffold design approach, we identified 4-aminomethylphenylacetic acid 4 with a desirable γ-secretase modulation profile. Scaffold optimization led to the discovery of a novel chemical series, represented by 6b, having improved brain penetration. Further SAR studies provided analog 6q that exhibited a good pharmacological profile. Oral administration of 6q significantly reduced brain Aβ42 levels in mice and rats.     

Rational design and synthesis of aminopiperazinones as β-secretase (BACE) inhibitors

Aminopiperazinone inhibitors of BACE were identified by rational design. Structure based design guided idea prioritization and initial racemic hit 18a showed good activity. Modification in decoration and chiral separation resulted in the 40 nM inhibitor, (−)-37, which showed in vivo reduction of amyloid beta peptides. The crystal structure of 18a showed a binding mode driven by interaction with the catalytic aspartate dyad and distribution of the biaryl amide decoration towards S1 and S3 pockets.     

Discovery of fused 5,6-bicyclic heterocycles as γ-secretase modulators

We herein report the discovery of four series of fused 5,6-bicyclic heterocycles as γ-secretase modulators. Synthesis and SAR of these series are discussed. These compounds represent a new class of γ-secretase modulators that demonstrate moderate to good in vitro potency in inhibiting Aβ₄₂ production.     

A free radical-generating system regulates APP metabolism/processing

Oxidative stress, a risk factor in the pathophysiology of Alzheimer’s disease, is intimately associated with aging. We previously reported that the X-XOD free radical generating system acts as a modulator of lipid metabolism and a mild inducer of apoptotic death. Using the same cell model, the present study examines the metabolism/processing of the amyloid precursor protein (APP). Prior to inducing cell death, X-XOD promoted the secretion of α-secretase-cleaved soluble APP (sAPPα) and increased the level of APP carboxy-terminal fragments produced by α and γ secretase (αCTF and γCTF/AICD). In contrast, it reduced the activity of β-secretase and the level of secreted Aβ. The present results indicate that mild oxidative stress maintained throughout culturing regulates APP metabolism/processing in SK-N-MC human neuroblastoma cells.     

Impact of cholesterol level upon APP and BACE proximity and APP cleavage

Cleavage of APP by BACE is the first proteolytic step in the production of Amyloid β (Aβ, which accumulates in senile plaques in Alzheimer’s disease. BACE-cleavage of APP is thought to happen in endosomes. However, there are controversial data whether APP and BACE can already interact on the cell surface dependent on the cholesterol level. To examine whether APP and BACE come into close proximity on the cell surface in living cells, we employed a novel technique by combining time-resolved Förster resonance energy transfer (FRET) measurements with total internal reflection microscopy (TIRET microscopy). Our data indicate that BACE and APP come into close proximity within the cell, but probably not on the cell surface. To analyze the impact of alterations in cholesterol level upon BACE-cleavage, we measured sAPP secretion. Alteration of APP processing and BACE proximity by cholesterol might be explained by alterations in cell membrane fluidity.     

Amino-caprolactam γ-secretase inhibitors showing potential for the treatment of Alzheimer's disease

Herein we describe the structure-activity relationship (SAR) of amino-caprolactam analogs derived from amino-caprolactam benzene sulfonamide 1, highlighting affects on the potency of γ-secretase inhibition, selectivity for the inhibition of APP versus Notch processing by γ-secretase and selected pharmakokinetic properties. Amino-caprolactams that are efficacious in reducing the cortical Aβ_x-40 levels in FVB mice via a single 100 mpk IP dose are highlighted.     

Discovery of 2-methylpyridine-based biaryl amides as γ-secretase modulators for the treatment of Alzheimer’s disease

γ-Secretase modulators (GSMs) are potentially disease-modifying treatments for Alzheimer’s disease. They selectively lower pathogenic Aβ42 levels by shifting the enzyme cleavage sites without inhibiting γ-secretase activity, possibly avoiding known adverse effects observed with complete inhibition of the enzyme complex. A cell-based HTS effort identified the sulfonamide 1 as a GSM lead. Lead optimization studies identified compound 25 with improved cell potency, PKDM properties, and it lowered Aβ42 levels in the cerebrospinal fluid (CSF) of Sprague-Dawley rats following oral administration. Further optimization of 25 to improve cellular potency is described.     

Role of gangliosides in Alzheimer’s disease

One of the fundamental questions regarding the pathogenesis of Alzheimer’s disease (AD) is how the monomeric, nontoxic amyloid β-protein (Aβ) is converted to its toxic assemblies in the brain. A unique Aβ species was identified previously in an AD brain, which is characterized by its binding to the GM1 ganglioside (GM1). On the basis of the molecular characteristics of this GM1-bound Aβ (GAβ), it was hypothesized that Aβ adopts an altered conformation through its binding to GM1, and GAβ acts as a seed for Aβ fibrillogenesis in an AD brain. To date, various in vitro and in vivo studies of GAβ have been performed, and their results support the hypothesis. Using a novel monoclonal antibody specific to GAβ, it was confirmed that GAβ is endogenously generated in the brain. Regarding the role of gangliosides in the facilitation of Aβ assembly, it has recently been reported that region-specific deposition of hereditary variant-type Aβs is determined by local gangliosides in the brain. Furthermore, it is likely that risk factors for AD, including aging and the expression of apolipoprotein E4, alter GM1 distribution on the neuronal surface, leading to GAβ generation.     

Extracellular release of BACE1 holoproteins from human neuronal cells

BACE1 is a membrane-bound aspartyl protease involved in production of the Alzheimer's amyloid beta-protein. The BACE1 ectodomain is partially cleaved to generate soluble BACE1, but the physiological significance of this event is unclear. During our characterization of BACE1 shedding from human neuroblastoma SH-SY5Y cells stably expressing BACE1, we unexpectedly found that detectable amounts of BACE1 holoproteins were released extracellularly along with soluble BACE1. Treatment with the metalloprotease inhibitor, TAPI-1, inhibited BACE1 shedding but increased BACE1 holoprotein release. Soluble and full-length BACE1 were released in parallel, at least partly originating from the plasma membrane. Furthermore, the release of soluble BACE1, but not full-length BACE1, was increased by deletion of the C-terminal dileucine motif, indicating that dysregulated BACE1 sorting affects BACE1 shedding. These findings suggest that the release of BACE1 holoproteins may be a physiologically relevant cellular process.     

Inhibition of c-Jun N-terminal kinase increases apoE expression in vitro and in vivo

Apolipoprotein E (apoE), a ligand for the low-density lipoprotein receptor family, has been implicated in modulating glial inflammatory responses and the risk of neurodegeneration associated with Alzheimer’s disease. Glial cells activated by lipopolysaccharide (LPS) have decreased apoE levels and we recently showed that apoE itself can modulate the inflammatory response by reducing c-Jun N-terminal kinase (JNK) activation. Reduced JNK phosphorylation is vital to overcome the LPS-induced decrease in apoE expression, suggesting that JNK inhibition may be an effective way to increase apoE protein and protract its anti-inflammatory properties. This study investigates the impact of JNK inhibition on apoE production using two JNK inhibitors. Our work in primary glia and in vivo in mice injected with JNK inhibitor demonstrates that inhibition of JNK may be an effective way to increase apoE expression.     

Amyloid-β induced toxicity involves ganglioside expression and is sensitive to GM1 neuroprotective action

The effect of Aβ25-35 peptide, in its fibrillar and non-fibrillar forms, on ganglioside expression in organotypic hippocampal slice cultures was investigated. Gangliosides were endogenously labeled with D-[1-C¹⁴] galactose and results showed that Aβ25-35 affected ganglioside expression, depending on the peptide aggregation state, that is, fibrillar Aβ25-35 caused an increase in GM3 labeling and a reduction in GD1b labeling, whereas the non-fibrillar form was able to enhance GM1 expression. Interestingly, GM1 exhibited a neuroprotective effect in this organotypic model, since pre-treatment of the hippocampal slices with GM1 10 μM was able to prevent the toxicity triggered by the fibrillar Aβ25-35, when measured by propidium iodide uptake protocol. With the purpose of further investigating a possible mechanism of action, we analyzed the effect of GM1 treatment (1, 6, 12 and 24 h) upon the Aβ-induced alterations on GSK3β dephosphorylation/activation state. Results demonstrated an important effect after 24-h incubation, with GM1 preventing the Aβ-induced dephosphorylation (activation) of GSK3β, a signaling pathway involved in apoptosis triggering and neuronal death in models of Alzheimer’s disease. Taken together, present results provide a new and important support for ganglioside participation in development of Alzheimer’s disease experimental models and suggest a protective role for GM1 in Aβ-induced toxicity. This may be useful for designing new therapeutic strategies for Alzheimer’s treatment.     

Structure based design, synthesis and SAR of cyclic hydroxyethylamine (HEA) BACE-1 inhibitors

This Letter describes the de novo design of non-peptidic hydroxyethylamine (HEA) inhibitors of BACE-1 by elimination of P-gp contributing amide attachments. The predicted binding mode of the novel cyclic sulfone HEA core template was confirmed in a X-ray co-crystal structure. Inhibitors of sub-micromolar potency with an improved property profile over historic HEA inhibitors resulting in improved brain penetration are described.     

Searching for improved mimetic peptides inhibitors preventing conformational transition of amyloid-β42 monomer

A series of novel mimetic peptides were designed, synthesised and biologically evaluated as inhibitors of Aβ₄₂ aggregation. One of the synthesised peptidic compounds, termed compound 7 modulated Aβ₄₂ aggregation as demonstrated by thioflavin T fluorescence, acting also as an inhibitor of the cytotoxicity exerted by Aβ₄₂ aggregates. The early stage interaction between compound 7 and the Aβ₄₂ monomer was investigated by replica exchange molecular dynamics (REMD) simulations and docking studies. Our theoretical results revealed that compound 7 can elongate the helical conformation state of an early stage Aβ₄₂ monomer and it helps preventing the formation of β-sheet structures by interacting with key residues in the central hydrophobic cluster (CHC). This strategy where early “on-pathway” events are monitored by small molecules will help the development of new therapeutic strategies for Alzheimer’s disease.     

Protease digestion indicates that endogenous presenilin 1 is present in at least two physical forms

The membrane-bound protein complex γ-secretase is an intramembranous protease whose substrates are a number of type I transmembrane proteins including the β-amyloid precursor protein (APP). A presenilin molecule is thought to be the catalytic unit of γ-secretase and either of two presenilin homologues, PS1 or PS2, can play this role. Mutations in the presenilins, apparently leading to aberrant processing of APP, have been genetically linked to early-onset familial Alzheimer’s disease. To look for possible molecular heterogeneity in presenilin/γ-secretase we examined the ability of proteinase K (PK) to digest endogenously expressed presenilins in intact endoplasmic reticulum vesicles. We demonstrate the existence of two physically different forms of γ-secretase-associated PS1, one that is relatively PK-sensitive and one that is significantly more PK-resistant. A similarly PK-resistant form of PS2 was not observed. We speculate that the structural heterogeneity we observe may underlie, at least in part, previous observations indicating the physical and functional heterogeneity of γ-secretase. In particular, our results suggest that there are significant differences between γ-secretase complexes incorporating PS1 and PS2. This difference may underlie the more dominant role of PS1 in the generation of β-amyloid peptides and in familial Alzheimer’s disease.     

Two β-strands of RAGE participate in the recognition and transport of amyloid-β peptide across the blood brain barrier

Amyloid-β (Aβ) peptide is central to the development of brain pathology in Alzheimer disease (AD) patients. Association with receptors for advanced glycation end-products (RAGE) enables the transport of Aβ peptide from circulating blood to human brain, and also causes the activation of the NF-κB signaling pathway. Here we show that two β-strands of RAGE participate in the interaction with Aβ peptide. Serial deletion analysis of the RAGE V domain indicates that the third and eighth β-strands are required for interaction with Aβ peptide. Site-directed mutagenesis of amino acids located in the third and eighth β-strands abolish the interaction of RAGE with Aβ peptide. Wild-type RAGE activates the NF-κB signaling pathway in response to Aβ peptide treatment, while a RAGE mutant defective in Aβ binding does not. Furthermore, use of peptide for the third β-strand or a RAGE monoclonal antibody that targets the RAGE–Aβ interaction interface inhibited transport of the Aβ peptide across the blood brain barrier in a mice model. These results provide information crucial to the development of RAGE-derived therapeutic reagents for Alzheimer disease.     

A surface plasmon resonance-based biosensor with full-length BACE1 in a reconstituted membrane

A surface plasmon resonance (SPR) biosensor-based assay for membrane-embedded full-length BACE1 (β-site amyloid precursor protein cleaving enzyme 1), a drug target for Alzheimer’s disease, has been developed. It allows the analysis of interactions with the protein in its natural lipid membrane environment. The enzyme was captured via an antibody recognizing a C-terminal His6 tag, after which a lipid membrane was reconstituted on the chip using a brain lipid extract. The interaction between the enzyme and several inhibitors confirmed that the surface was functional. It had slightly different interaction characteristics as compared with a reference surface with immobilized ectodomain BACE1 but had the same inhibitor characteristic pH effect. The possibility of studying interactions with BACE1 under more physiological conditions than assays using truncated enzyme or conditions dictated by high enzyme activity is expected to increase our understanding of the role of BACE1 in Alzheimer’s disease and contribute to the discovery of clinically efficient BACE1 inhibitors. The strategy exploited in the current study can be adapted to other membrane-bound drug targets by selecting suitable capture antibodies and lipid mixtures for membrane reconstitution.     

Alzheimer’s disease and Notch signaling

Cleavage of the amyloid precursor protein (APP) by γ-secretase generates a neurotoxic amyloid β-peptide (Aβ) that is thought to be associated with the neurodegeneration observed in Alzheimer’s disease (AD) patients. Presenilin is the catalytic member of the γ-secretase proteolytic complex and mutations in presenilins are the major cause of early-onset familial Alzheimer’s disease. In addition to APP, γ-secretase substrates include Notch1 homologues, Notch ligands Delta and Jagged, and additional type I membrane proteins, raising concerns about mechanism-based toxicities that might arise as a consequence of inhibiting γ-secretase. Notch signaling is involved in tumorigenesis as well as in determining the fates of neural and nonneural cells during development and in adults. Alterations in proteolysis of the Notch by γ-secretase could be involved in the pathogenesis of AD. Inconsistently, several recent observations have indicated that enhanced Notch signaling and expression could be instrumental in neurodegeneration in AD. Therefore, detailed and precise study of Notch signaling in AD is important for elucidating diverse mechanisms of pathogenesis and potentially for treating and preventing Alzheimer’s disease.