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Background: A disease-modifying therapy for Alzheimer's disease (AD) is still an unmet clinical need. The formation of amyloid-β (Aβ) requires the initial cleavage of the amyloid-β protein precursor (AβPP) by BACE1 (beta-site AβPP cleaving enzyme 1), which is a prime therapeutic target for AD., Objective: We aimed to design and develop a selective BACE1 inhibitor suitable to AD treatment., Methods: The new BACE1 inhibitors consist on a chimeric peptide including a sequence related to the human Swedish mutant form of AβPP (AβPPswe) conjugated with the TAT carrier that facilitates cell membrane permeation and the crossing of the blood-brain barrier. Additionally to the chimeric peptide in the L-form, we developed a D-retroinverso chimeric peptide. The latter strategy, never used with BACE1 inhibitors, is considered to favor a significantly higher half-life and lower immunogenicity., Results: We found that both chimeric peptides inhibit recombinant BACE1 activity and decrease Aβ40/42 production in Neuro-2a (N2A) cells expressing AβPPswe without inducing cytotoxicity. The intraperitoneal administration of these peptides to 3xTg-AD mice decreased plasma and brain Aβ40/42 levels, as well as brain soluble AβPPβ production. Also, a reduction of insoluble Aβ was observed in the brain after chronic treatment. Noteworthy, the chimeric peptides selectively inhibited the AβPP-β cleavage relatively to the proteolysis of other BACE1 substrates such as close homologue of L1 (CHL1) and seizure-related gene 6 (SEZ6)., Conclusions: Overall these new BACE1 chimeric peptideshold promising potential as a selective disease-modifying therapy for AD.

BACE1 inhibitors hold potential as agents in disease-modifying treatment for Alzheimer's disease. BACE2 cleaves the melanocyte protein PMEL in pigment cells of the skin and eye, generating melanin pigments. This role of BACE2 implies that nonselective and chronic inhibition of BACE1 may cause side effects derived from BACE2. Herein, we describe the discovery of potent and selective BACE1 inhibitors using structure-based drug design. We targeted the flap region, where the shape and flexibility differ between these enzymes. Analysis of the cocrystal structures of an initial lead 8 prompted us to incorporate spirocycles followed by its fine-tuning, culminating in highly selective compounds 21 and 22. The structures of 22 bound to BACE1 and BACE2 revealed that a relatively high energetic penalty in the flap of the 22-bound BACE2 structure may cause a loss in BACE2 potency, thereby leading to its high selectivity. These findings and insights should contribute to responding to the challenges in exploring selective BACE1 inhibitors.

The excess accumulation of amyloid-β (Aβ) peptides derived from the sequential cleavage of amyloid precursor protein (APP) by secretases, is one of the toxic key events leading to neuronal loss in Alzheimer's disease (AD). Studies have shown that cholinergic activity may also be involved in the regulation of APP metabolism. In the current study, we have investigated the roles of toluidine blue O (TBO) and thionine (TH), newly recognized phenothiazine-derived cholinesterase inhibitors, on the metabolism of APP in Chinese hamster ovary cells stably expressing human APP751 and presenilin 1 (PS70 cells). We assessed the effects of both compounds on the levels of Aβ, soluble APP-α (sAPPα), intracellular APP and β-site APP-cleaving enzyme 1 (BACE1). After treatment of PS70 cells with TBO or TH without any side effect on cell viability, the levels of secreted Aβ40, Aβ42 and sAPPα were assayed by specific sandwich ELISAs while APP and BACE1 in cell lysates were analyzed using Western blot. The secreted Aβ40, Aβ42 and sAPPα in TBO- and TH-treated cells were found to be reduced in a dose-dependent manner compared to vehicle-treated cells. Results suggest that TH mitigated the Aβ pathology by lowering APP levels whereas reduced Aβ caused by TBO treatment seems to be the outcome of both less substrate availability and amyloidogenic APP processing. Taken together, our results represent the first report demonstrating that TBO and TH can affect amyloid metabolism in vitro., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Objective: To investigate the effect of β-sheet breaker peptide H102 on APP associated secretase in the hippocampus brain regions of APP/PS1 double transgenic mice(AD mice)., Methods: Thirty 6-month-old APP/PS1 double transgenic mice were randomly divided into AD group and H102 group, a group of C57BL/6J mice with the same age, number and background was set as controls( n =15). H102 (5.8 mg/kg) 5 μl was infused by intranasal administration to mice in H102 treatment group, and equal volume of blank solution of H102 was given to mice in control group and AD group. The ability of spatial reference memory was tested by Morris water maze after 30 days of treatment. And then immunohistochemistry tests and Western blot were used to detect the content of α-secretase (ADAM10, ADAM17), β-secretase (BACE1), γ-secretase (PS1, APH1a, PEN2) in the hippocampus brain regions., Results: Compared with the control group, the expression of BACE1, PS1, PEN-2 and APH1-a protein in the hippocampus of AD group were significantly increased, ADAM10, ADAM17 protein expression were significantly reduced ( P <0.05); Compared with the model group, H102 could significantly improve the spatial learning and memory ability of AD mice, significantly decreased the expression of BACE1, PS1, PEN-2 and APH1-a protein in the hippocampus, significantly increased the expression of ADAM10 and ADAM17 protein( P <0.05)., Conclusions: β sheet peptides blocked H102 can reduce the formation of Aβ in the hippocampus brain area, improve the activity of α-secretase in the hippocampus brain region, decrease the activity of β-and γ-secretase, improve the learning and memory ability of AD mice.

The deposition of amyloid-β (Aβ) oligomers in brain parenchyma has been implicated in the pathophysiology of Alzheimer's disease. Here we present a systems pharmacology model describing the changes in the amyloid precursor protein (APP) pathway after administration of three different doses (10, 30, and 125 mg/kg) of the β-secretase 1 (BACE1) inhibitor MBi-5 in cisterna magna ported rhesus monkeys. The time course of the MBi-5 concentration in plasma and cerebrospinal fluid (CSF) was analyzed in conjunction with the effect on the concentrations of the APP metabolites Aβ42, Aβ40, soluble β-amyloid precursor protein (sAPP) α, and sAPPβ in CSF. The systems pharmacology model contained expressions to describe the production, elimination, and brain-to-CSF transport for the APP metabolites. Upon administration of MBi-5, a dose-dependent increase of the metabolite sAPPα and dose-dependent decreases of sAPPβ and Aβ were observed. Maximal inhibition of BACE1 was close to 100% and the IC50 value was 0.0256 μM (95% confidence interval, 0.0137-0.0375). A differential effect of BACE1 inhibition on Aβ40 and Aβ42 was observed, with the Aβ40 response being larger than the Aβ42 response. This enabled the identification of an Aβ42 oligomer pool in the systems pharmacology model. These findings indicate that decreases in monomeric Aβ responses resulting from BACE1 inhibition are partially compensated by dissociation of Aβ oligomers and suggest that BACE1 inhibition may also reduce the putatively neurotoxic oligomer pool., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Accumulation of β-amyloid peptides (Aβ) and its progressive deposition into amyloid plaques are key events in the aetiology of Alzheimer's disease (AD). To date, AD treatment is symptomatic and consists of drugs treating the cognitive decline., Objective: Identifying molecules specifically targeting Aβ production or aggregation represents a huge challenge in the development of specific AD treatments. Several molecules reported as γ-secretase inhibitors or modulators have been evaluated, but so far none of them have proven to be selective or fully efficient. We have previously investigated the potential interest of plant extracts and we reported that Pterocarpus erinaceus stem-bark extract was active on Aβ release. Our aim here was to characterize the mechanisms by which this extract reduces Aβ levels., Methods: We tested P. erinaceus extract at non-toxic concentrations on cells expressing the human amyloid precursor protein (APP695) or its amyloidogenic β-cleaved C-terminal fragment (C99), as well as on neuronal cell lines. P. erinaceus extract was found to inhibit Aβ release. We further showed that this extract inhibited γ-secretase activity in cell-free and in vitro assays, strongly suggesting that P. erinaceus extract is a natural γ-secretase inhibitor. Importantly, this extract did not inhibit γ-secretase-dependent Notch intracellular domain release., Conclusion: P. erinaceus extract appears as a new potent γ-secretase inhibitor selective towards APP processing., (2013 S. Karger AG, Basel)

Despite great efforts to develop new therapeutic strategies against Alzheimer's disease (AD), the acetylcholinesterase inhibitors (AChEIs): donepezil, rivastigmine, and galantamine, have been used only as a palliative therapeutic approach. However, the pathogenesis of AD includes several factors such as cholinergic hypothesis, amyloid-β (Aβ) aggregation, and oxidative stress. For this reason, the design of compounds that target the genesis and progression of AD could offer a therapeutic benefit. We have designed a set of compounds (M-1 to M-5) with pharmacophore moieties to inhibit the release, aggregation, or toxicity of Aβ, act as AChEIs and have antioxidant properties. Once the compounds were designed, we analyzed their physicochemical parameters and performed docking studies to determine their affinity values for AChE, β-site amyloid-protein precursor cleaving enzyme 1 (BACE1), and the Aβ monomer. The best ligands, M-1 and M-4, were then synthesized, chemically characterized, and evaluated in vitro. The in vitro studies showed that these compounds inhibit AChE (M-1 Ki = 0.12 and M-4 Ki = 0.17 μM) and BACE1 (M-1 IC50 = 15.1 and M-4 IC50 = 15.4 nM). They also inhibit Aβ oligomerization and exhibit antioxidant activity. In addition, these compounds showed low cytotoxicity in microglial cells. For these reasons, they are promising for future use as drugs in AD mice transgenic models.

γ-Secretase modulators (GSM), which reduce amyloidogenic Aβ(42) production while maintaining total Aβ levels, and Notch-sparing γ-secretase inhibitors (GSIs) are promising therapies for the treatment of Alzheimer's Disease (AD). To have a safety margin for therapeutic use, GSMs and GSIs need to allow Notch intracellular domain (NICD) production, while preventing neurotoxic Aβ peptide production. Typically, GSI and GSM effects on these substrates are determined using two different cell lines, one for the measurement of enzyme activity against each substrate. However, predicting selectivity for different substrates across cell systems may reduce the reliability of such ratios such that the in vitro data are not useful for predicting in vivo safety margins. This is especially concerning since the IC(50)'s of some GSIs vary depending upon the level of APP expression in a cell line. To circumvent this problem, we utilized the SUP-T1 cell line which expresses a truncated Notch receptor fragment that does not need sheddase cleavage to be a γ-secretase substrate. When combined with a sensitive method of measuring Aβ production, this assay system allows both substrates to be measured simultaneously, reducing the potential to calculate imprecise selectivity margins. To demonstrate the value of this system, known GSIs and GSMs were examined in the SUP-T1 dual substrate assay. IC(50)'s were determined for both substrates and the in vitro selectivity margin was calculated. These data suggest using a single cell line is a more accurate prediction of the fold difference between NICD inhibition and Aβ(42) lowering for therapeutically promising GSIs and GSMs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A screen of a library of synthetic drugs and natural product extracts identified a botanical extract that modulates the processing of amyloid precursor protein (APP) in cultured cells to produce a lowered ratio of amyloid-beta peptide (1-42) (Aβ42) relative to Aβ40. This profile is of interest as a potential treatment for Alzheimer's disease. The extract, from the black cohosh plant (Actaea racemosa), was subjected to bioassay guided fractionation to isolate active components. Using a combination of normal-phase and reverse-phase chromatography, a novel triterpene monoglycoside, 1, was isolated. This compound was found to have an IC(50) of 100 nM for selectively reducing the production of amyloidogenic Aβ42 while having a much smaller effect on the production of Aβ40 (IC(50) 6.3 μM) in cultured cells overexpressing APP. Using IP-MS methods, this compound was found to modulate the pool of total Aβ produced by reducing the proportion of Aβ42 while increasing the relative amounts of shorter and less amyloidogenic Aβ37 and Aβ39. Concentrations of 1 sufficient to lower levels of Aβ42 substantially (up to 10 μM) did not significantly affect the processing of Notch or other aspects of APP processing. When 1 (10 μg) was administered to CD-1 normal mice intracerebroventricularly, the level of Aβ42 in brain was reduced. Assays for off-target pharmacology and the absence of overt signs of toxicity in mice dosed with compound 1 suggest a comparatively selective pharmacology for this triterpenoid. Compound 1 represents a new lead for the development of potential treatments for Alzheimer's disease via modulation of gamma-secretase.

Modulation of γ-secretase activity holds potential for the treatment of Alzheimer's disease. Most NSAID-derived γ-secretase modulators feature a carboxylic acid, which may impair blood-brain barrier permeation. The structure activity relationship of 33 carbazoles featuring diverse carboxylic acid isosteres or metabolic precursors thereof was established in a cellular amyloid secretion assay. The modulatory activity was observed for acidic moieties and metabolically labile esters only, which supports our hypothesis of an acid-lysine interaction to be relevant for this type of γ-secretase modulators., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Sequential processing of the β-amyloid precursor protein by β- and γ-secretase generates the amyloid β-peptide (Aβ), which is widely believed to play a causative role in Alzheimer disease. Selective lowering of the pathogenic 42-amino acid variant of Aβ by γ-secretase modulators (GSMs) is a promising therapeutic strategy. Here we report that mutations in presenilin (PS), the catalytic subunit of γ-secretase, display differential responses to non-steroidal anti-inflammatory drug (NSAID)-type GSMs and more potent second-generation compounds. Although many pathogenic PS mutations resisted lowering of Aβ(42) generation by the NSAID sulindac sulfide, the potent NSAID-like second-generation compound GSM-1 was capable of lowering Aβ(42) for many but not all mutants. We further found that mutations at homologous positions in PS1 and PS2 can elicit differential Aβ(42) responses to GSM-1, suggesting that a positive GSM-1 response depends on the spatial environment in γ-secretase. The aggressive pathogenic PS1 L166P mutation was one of the few pathogenic mutations that resisted GSM-1, and Leu-166 was identified as a critical residue with respect to the Aβ(42)-lowering response of GSM-1. Finally, we found that GSM-1-responsive and -resistant PS mutants behave very similarly toward other potent second-generation compounds of different structural classes than GSM-1. Taken together, our data show that a positive Aβ(42) response for PS mutants depends both on the particular mutation and the GSM used and that attenuated Aβ(42) responses to low potency GSMs can be overcome for many PS mutants by second generation GSMs.

Hypoxia which is mainly mediated by hypoxia-inducible factor 1 (HIF-1), can greatly contribute to the occurrence of Alzheimer's disease (AD) by increasing beta-site APP cleaving enzyme (BACE1) gene expression, protein level and beta-secretase activity, resulting in a significant generation of amyloid-beta (Abeta). Salidroside has been reported to have great neuroprotective effects. The aim of this study was to investigate the effects of salidroside on hypoxia-induced abnormal processing of the amyloid precursor protein (APP) in SH-SY5Y cells and its possible mechanism. Western blot analysis showed that 200muM of salidroside pretreatment significantly decreased BACE1 protein level and promoted the secretion of sAPPalpha in hypoxic condition. Salidroside had no effect on the level of APP, ADAM10 and ADAM17. ELISA analysis revealed that salidroside was able to inhibit the increase of beta-secretase activity and Abeta generation induced by hypoxia, with no effect on gamma-secretase activity. Notably, under hypoxia condition, mRNA of BACE1 and protein level of HIF-1alpha were decreased by salidroside pretreatment. These results demonstrated for the first time that salidroside was able to attenuate abnormal processing of amyloid precursor protein induced by hypoxia in SH-SY5Y cells, providing a new insight into prevention and treatment of Alzheimer's disease., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

The amyloid precursor protein (APP) is cleaved enzymatically by non-amyloidogenic and amyloidogenic pathways. alpha-Secretase cleaves APP within beta-amyloid protein (Abeta) sequence, resulting in the release of a secreted fragment of APP (sAPPalpha) and precluding Abeta generation. Cryptotanshinone (CTS), an active component of the medicinal herb Salvia miltiorrhiza, has been shown to improve learning and memory in several pharmacological models of Alzheimer's disease (AD). However, the effects of CTS on the Abeta plaque pathology and the APP processing in AD are unclear. Here we reported that CTS strongly attenuated amyloid plaque deposition in the brain of APP/PS1 transgenic mice. In addition, CTS significantly improved spatial learning and memory in APP/PS1 mice assessed by the Morris water maze testing. To define the exact molecular mechanisms involved in the beneficial effects of CTS, we investigated the effects of the CTS on APP processing in rat cortical neuronal cells overexpressing Swedish mutant human APP695. CTS was found to decrease Abeta generation in concentration-dependent (0-10muM) manner. Interestingly, the N-terminal APP cleavage product, sAPPalpha was markedly increased by CTS. Further study showed that alpha-secretase activity was increased by CTS. Taken together, our results suggested CTS improved the cognitive ability in AD transgenic mice and promoted APP metabolism toward the non-amyloidogenic products pathway in rat cortical neuronal cells. CTS shows a promising novel way for the therapy of AD.

In order to access beta-secretase (BACE1), and enzyme strongly implicated in the cause of Alzheimer's disease, inhibitors must possess sufficient lipophilicity to traverse two lipid bilayers. Current drug candidates, which are almost totally peptide-derived, are thus inefficient because cell permeability presents a serious limiting factor. In this study, lipophilic alkylated (C(10)-C(5)) flavanones from Sophora flavescens were examined for their inhibitory effects against beta-secretase. Lavandulyl flavanones (1, 2, 5, 6, and 8) showed potent beta-secretase inhibitory activities with IC(50)s of 5.2, 3.3, 8.4, 2.6, and 6.7microM, respectively, while no significant activity was observed in the corresponding hydrated lavandulyl flavanones (4 and 7) and prenylated flavanone (3). As we expected, lavandulyl flavanones reduced Abeta secretion dose-dependently in transfected human embryonic kidney (HEK-293) cells. In kinetic studies, all compounds screened were shown to be noncompetitive inhibitor.

Sequential cleavages of the beta-amyloid precursor protein cleaving enzyme 1 (BACE1) by beta-secretase and gamma-secretase generate the amyloid beta-peptides, believed to be responsible of synaptic dysfunction and neuronal cell death in Alzheimer's disease (AD). Levels of BACE1 are increased in vulnerable regions of the AD brain, but the underlying mechanism is unknown. Here we show that oxidative stress (OS) stimulates BACE1 expression by a mechanism requiring gamma-secretase activity involving the c-jun N-terminal kinase (JNK)/c-jun pathway. BACE1 levels are increased in response to OS in normal cells, but not in cells lacking presenilins or amyloid precursor protein. Moreover, BACE1 is induced in association with OS in the brains of mice subjected to cerebral ischaemia/reperfusion. The OS-induced BACE1 expression correlates with an activation of JNK and c-jun, but is absent in cultured cells or mice lacking JNK. Our findings suggest a mechanism by which OS induces BACE1 transcription, thereby promoting production of pathological levels of amyloid beta in AD.

One of the main neuropathological lesions observed at brain autopsy of Alzheimer's disease (AD) patients are the extracellular senile plaques mainly composed of amyloid-beta (Abeta) peptides. Abeta is generated by proteolytic processing of amyloid precursor protein (APP) via beta and gamma-secretases. The beta-secretase APP cleaving enzyme 1 (BACE1) has become a target of intense research aimed at blocking the enzyme activity. Recent studies showed that BACE1 is involved in processing other non-APP substrates, and that other proteases are involved in APP processing. We have recently established a novel approach to inhibit Abeta production via antibodies against the beta-secretase cleavage site of APP. These antibodies bind wild type and Swedish mutated APP expressed in transgenic mice brain tissues. The isolated antibodies do not bind any form of Abeta peptides. Antibody up-take experiments, using Chinese hamster ovary cells expressing wild-type APP, suggest that antibody internalization and trafficking are mediated via the endocytic pathway. Administration of antibodies to the cells growing media resulted in a considerable decrease in intracellular Abeta levels, as well as in the levels of the corresponding C-terminal fragment (C99). The relevance of intra-neuronal accumulation of mainly Abeta42 as an early event in AD pathogenesis suggests that this approach may be applicable as a novel therapeutic strategy in AD treatment.

Cerebral hypometabolism, oxidative stress and beta-amyloid peptide (Abeta) accumulation are key pathological events in Alzheimer's disease (AD). Beta-secretase (BACE, i.e., BACE1), a prerequisite for Abeta genesis, is elevated in sporadic AD. Recent studies show BACE upregulation in experimental conditions likely associated with energy insufficiency and/or oxidative stress. We investigated the effect of sublethal doses of mitochondrial respiratory inhibitors and potential endogenous oxidative substances on BACE expression in vivo using the retina as a model. Retinas were analyzed biochemically and anatomically 48 h following intraocular applications of mitochondrial complex I, II and IV inhibitors including rotenone, 3-nitropropionic acid and sodium azide, and plaque-containing oxidants including Fe(3+) and Abeta42 fibrils (Abeta42f). All agents caused elevations of BACE proteins and beta-site amyloid precursor protein (APP) cleavage product, beta-CTF, in retinal lysates in a dose-dependant manner. BACE activity and Abeta40 levels were also increased in agent-treated retinas relative to vehicle controls. BACE immunoreactivity in normal adult rat retina was present mostly in the plexiform layers, indicating a localization of the enzyme to synaptic terminals. No apparent change in laminar or cellular distribution of BACE labeling was detected in the experimental retinas. However, signs of neuronal stress including glial activation were observed in agent-treated retinas especially in high dosage groups. Our data suggest that mitochondrial respiratory inhibition and oxidative stress facilitate BACE expression in vivo. In addition, plaque constituents such as Fe(3+) and Abeta42f may participate in a self-enforcing cycle of amyloidogenesis via BACE upregulation.

Lipids play an important part as risk factors for Alzheimer's disease. This article summarizes the current understanding of the molecular mechanism by which amyloid-beta (Abeta) peptides regulate cholesterol and sphingomyelin metabolism, and how in return cholesterol and sphingomyelin regulate Abeta peptide production. An understanding of the physiological function of amyloid precursor protein processing and Abeta function is critical for the development of future therapeutic approaches, e.g. statin treatment., (Copyright 2006 S. Karger AG, Basel.)