Acylguanidine inhibitors of beta-secretase: optimization of the pyrrole ring substituents extending into the S1' substrate binding pocket

The proteolytic enzyme beta-secretase (BACE-1) produces amyloid beta (Abeta) peptide, the primary constituent of neurofibrillary plaques, implicated in Alzheimer's disease, by cleavage of the amyloid precursor protein. A small molecule inhibitor of BACE-1, (diaminomethylene)-2,5-diphenyl-1H-pyrrole-1-acetamide (1, BACE-1 IC(50)=3.7 microM), was recently described, representing a new small molecule lead. Initial SAR investigation demonstrated the potential of accessing the nearby S(3) and S(1)(') substrate binding pockets of the BACE-1 enzyme by building substituents off one of the phenyl substituents and guanidinyl functional group. We report here the optimization of guanidinyl functional group substituents on 1, leading to potent submicromolar BACE-1 inhibitors.     

Hydrogen sulfide reduces mRNA and protein levels of β-site amyloid precursor protein cleaving enzyme 1 in PC12 cells

Hydrogen sulfide (H(2)S) is now identified as a new neuromodulator. Increasing evidence suggest that H(2)S may play an important role in the progression of Alzheimer's disease (AD). The aim of the present study is to investigate the effects of H(2)S on beta-site amyloid precursor protein cleaving enzyme 1 (BACE-1) expression and amyloid beta (Aβ) secretion in PC12 cells. The levels of BACE-1 mRNA were measured by quantitative polymerase chain reaction analysis. BACE-1 protein levels were assessed by Western blot. Cellular culture medium levels of Aβ1-42 were analyzed by ELISA. We found that sodium hydrosulfide (NaHS), a H(2)S donor, decreased BACE-1 mRNA and protein levels and Aβ1-42 release. Furthermore, NaHS promoted the phosphorylation of Akt and ERK but not JNK or p38 MAPK. However, the effects of NaHS on BACE-1 expression and Aβ1-42 secretion were abolished by inhibitors of phosphatidylinositol 3-kinase (PI3-K), but not of mitogen-activated protein kinase kinases (MEK). Our data indicate that H(2)S reduces BACE-1 expression in PC12 cells via activation of PI3-K/Akt signaling pathways. H(2)S releasing drugs may have therapeutic potential in AD patients.     

Flavonols and flavones as BACE-1 inhibitors: structure-activity relationship in cell-free, cell-based and in silico studies reveal novel pharmacophore features

Generation and accumulation of the amyloid beta peptide (Abeta) following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 (Beta-site APP Cleaving Enzyme-1, beta-secretase) and gamma-secretase is a main causal factor of Alzheimer's disease (AD). Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Abeta, is an attractive therapeutic approach for the treatment of AD. In this study, we discovered that natural flavonoids act as non-peptidic BACE-1 inhibitors and potently inhibit BACE-1 activity and reduce the level of secreted Abeta in primary cortical neurons. In addition, we demonstrated the calculated docking poses of flavonoids to BACE-1 and revealed the interactions of flavonoids with the BACE-1 catalytic center. We firstly revealed novel pharmacophore features of flavonoids by using cell-free, cell-based and in silico docking studies. These results contribute to the development of new BACE-1 inhibitors for the treatment of AD.     

Biochemical and cell-based assays for characterization of BACE-1 inhibitors

The deposition of beta-amyloid peptides (A beta42 and A beta40) in neuritic plaques is one of the hallmarks of Alzheimer's disease (AD). A beta peptides are derived from sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. BACE-1 has been shown to be the major beta-secretase and is a primary therapeutic target for AD. In this article, two novel assays for the characterization of BACE-1 inhibitors are reported. The first is a sensitive 96-well HPLC biochemical assay that uses a unique substrate containing an optimized peptide cleavage sequence, NFEV, spanning from the P2-P2' positions This substrate was processed by BACE-1 approximately 10 times more efficiently than was the widely used substrate containing the Swedish (NLDA) sequence. As a result, the concentration of the enzyme required for the assay can be as low as 100 pM, permitting the evaluation of inhibitors with subnanomolar potency. The assay has also been applied to related aspartyl proteases such as cathepsin D (Cat D) and BACE-2. The second assay is a homogeneous electrochemiluminescence assay for the evaluation of BACE-1 inhibition in cultured cells that assesses the level of secreted amyloid EV40_NF from HEK293T cells stably transfected with APP containing the novel NFEV sequence. To illustrate the use of these assays, the properties of a potent, cell-active BACE-1 inhibitor are described.     

Oral administration of a potent and selective non-peptidic BACE-1 inhibitor decreases beta-cleavage of amyloid precursor protein and amyloid-beta production in vivo

Generation and deposition of the amyloid beta (Abeta) peptide following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 and gamma-secretase is central to the aetiology of Alzheimer's disease. Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Abeta, is an attractive therapeutic approach for the treatment of Alzheimer's disease. We have designed a selective non-peptidic BACE-1 inhibitor, GSK188909, that potently inhibits beta-cleavage of APP and reduces levels of secreted and intracellular Abeta in SHSY5Y cells expressing APP. In addition, we demonstrate that this compound can effectively lower brain Abeta in vivo. In APP transgenic mice, acute oral administration of GSK188909 in the presence of a p-glycoprotein inhibitor to markedly enhance the exposure of GSK188909 in the brain decreases beta-cleavage of APP and results in a significant reduction in the level of Abeta40 and Abeta42 in the brain. Encouragingly, subchronic dosing of GSK188909 in the absence of a p-glycoprotein inhibitor also lowers brain Abeta. This pivotal first report of central Abeta lowering, following oral administration of a BACE-1 inhibitor, supports the development of BACE-1 inhibitors for the treatment of Alzheimer's disease.     

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.     

Flavonols and flavones as BACE-1 inhibitors: Structure–activity relationship in cell-free,cell-based and in silico studies reveal novel pharmacophore features

Generation and accumulation of the amyloid β peptide (Aβ) following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 (Beta-site APP Cleaving Enzyme-1, β-secretase) and γ-secretase is a main causal factor of Alzheimer's disease (AD). Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Aβ, is an attractive therapeutic approach for the treatment of AD. In this study, we discovered that natural flavonoids act as non-peptidic BACE-1 inhibitors and potently inhibit BACE-1 activity and reduce the level of secreted Aβ in primary cortical neurons. In addition, we demonstrated the calculated docking poses of flavonoids to BACE-1 and revealed the interactions of flavonoids with the BACE-1 catalytic center. We firstly revealed novel pharmacophore features of flavonoids by using cell-free, cell-based and in silico docking studies. These results contribute to the development of new BACE-1 inhibitors for the treatment of AD.     

BACE-1 inhibitors part 1: identification of novel hydroxy ethylamines (HEAs)

Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. Herein, is described the lead generation effort which resulted, with the support of X-ray crystallography, in the discovery of potent inhibitors based on a hydroxy ethylamine (HEA) transition-state mimetic. These inhibitors were capable of lowering amyloid production in a cell-based assay.     

Biological and computational evaluation of resveratrol inhibitors against Alzheimer’s disease

It has been reported that beta amyloid induces production of radical oxygen species and oxidative stress in neuronal cells, which in turn upregulates β-secretase (BACE-1) expression and beta amyloid levels, thereby propagating oxidative stress and increasing neuronal injury. A series of resveratrol derivatives, known to be inhibitors of oxidative stress-induced neuronal cell death (oxytosis) were biologically evaluated against BACE-1 using homogeneous time-resolved fluorescence (TRF) assay. Correlation between oxytosis inhibitory and BACE-1 inhibitory activity of resveratrol derivatives was statistically significant, supporting the notion that BACE-1 may act as pivotal mediator of neuronal cell oxytosis. Four of the biologically evaluated resveratrol analogs demonstrated considerably higher activity than resveratrol in either assay. The discovery of some “hits” led us to initiate detailed docking studies associated with Molecular Dynamics in order to provide a plausible explanation for the experimental results and understand their molecular basis of action.     

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.     

Abeta-induced BACE-1 cleaves N-terminal sequence of mPGES-2

We previously indicated that amyloid beta (Abeta) augments protein levels of beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) through oxidative stress. In this study, we revealed that BACE-1 is involved in the cleavage of membrane-bound prostaglandin E2 synthase-2 (mPGES-2) in its N-terminal portion, which, in turn, enhanced the generation of prostaglandin E2 (PGE2). PGE2 results in increased Abeta production, initiating a cell-injuring cycle. Using rat primary cortical neurons, a 48 h treatment with Abeta 1-42 (5 μM) resulted in the enhanced extracellular PGE2 levels up to about 1 ng/mL, which was attenuated by treatment with a BACE-1 inhibitor (200 nM). A synthetic peptide sequence of 20-amino acids that included the cleavage site of mPGES-2 (HTARWHL RAQDLHERS AAQLSLSS) was cleaved by recombinant BACE-1, confirmed using reverse-phase high-performance liquid chromatography. Cleaved or activated mPGES-2 augments the generation of PGE2. In addition, mPGES-2 was determined to be colocalized with BACE-1 and cyclooxygenase-2 in the perinuclear region in cells after exposure to Abeta. Exposure of neurons to PGE2 led to cell death, and Abeta production was enhanced by PGE2 (1 ng/mL, 48 h). Collectively, these results suggest that Abeta might cause neuroinflammation that aggravates Alzheimer’s disease pathogenesis.     

Developing β-secretase inhibitors for treatment of Alzheimer's disease

β-Secretase (memapsin 2; BACE-1) is the first protease in the processing of amyloid precursor protein leading to the production of amyloid-β (Aβ) in the brain. It is believed that high levels of brain Aβ are responsible for the pathogenesis of Alzheimer's disease (AD). Therefore, β-secretase is a major therapeutic target for the development of inhibitor drugs. During the past decade, steady progress has been made in the evolution of β-secretase inhibitors toward better drug properties. Recent inhibitors are potent, selective and have been shown to penetrate the blood-brain barrier to inhibit Aβ levels in the brains of experimental animals. Moreover, continuous administration of a β-secretase inhibitor was shown to rescue age-related cognitive decline in transgenic AD mice. A small number of β-secretase inhibitors have also entered early phase clinical trials. These developments offer some optimism for the clinical development of a disease-modifying drug for AD.     

The various aggregation states of beta-amyloid 1-42 mediate different effects on oxidative stress, neurodegeneration, and BACE-1 expression

The amyloid cascade hypothesis suggests that the insoluble and fibrillar form of beta-amyloid (A beta) may play a primary pathogenic role in Alzheimer disease at the molecular level. However, neither the rate of dementia nor the extent of neuronal change seems to correlate with the levels of amyloidotic plaques (i.e., aggregated/fibrillar A beta). Recent evidence suggests, however, that neurotoxicity may be exerted also by rather small soluble aggregates of A beta, including oligomers. To characterize the mechanisms underlying toxicity mediated by the various aggregation states of A beta peptides is then a major goal of research. In this work we investigated the effects of fibrillar, prefibrillar, and oligomeric A beta(1-42) on the induction of oxidative stress, cell death, and BACE-1 expression in NT2 neuronal cells. We found that prefibrillar and oligomeric A beta(1-42) resulted in a more dramatic increase in the oxidative stress markers 4-hydroxynonenal and hydrogen peroxide compared to fibrillar A beta(1-42). Moreover, increased oxidative stress levels also resulted in a more rapid and significant induction of both apoptotic and necrotic neuronal cell death. Accordingly, fibrillar A beta(1-42), but not the soluble nonfibrillar forms, was the only condition able to up-regulate BACE-1 expression and activity.     

Inhibiting β-secretase activity in Alzheimer's disease cell models with single-chain antibodies specifically targeting APP

The Amyloid-β (Aβ) peptide is produced from the amyloid precursor protein (APP) by sequential proteolytic cleavage of APP first by β-secretase and then by γ-secretase. β-Site APP cleaving enzyme-1 (BACE-1) is the predominant enzyme involved in β-secretase processing of APP and is a primary therapeutic target for treatment of Alzheimer's disease. While inhibiting BACE-1 activity has obvious therapeutic advantages, BACE-1 also cleaves numerous other substrates with important physiological activity. Thus, blanket inhibition of BACE-1 function may have adverse side effects. We isolated a single chain variable fragment (scFv) from a human-based scFv yeast display library that selectively inhibits BACE-1 activity toward APP by binding the APP substrate at the proteolytic site. We selected the iBSEC1 scFv, since it recognizes the BACE-1 cleavage site on APP but does not bind the adjacent highly antigenic N-terminal of Aβ, and thus it will target APP but not soluble Aβ. When added to 7PA2 cells, a mammalian cell line that overexpresses APP, the iBSEC1 scFv binds APP on the cell surface, reduces toxicity induced by APP overexpression, and reduces both intracellular and extracellular Aβ levels by around 50%. Since the iBSEC1 scFv does not contain the antibody F_c region, this construct does not pose the risk of exacerbating inflammation in the brain as faced with full-length monoclonal antibodies for potential therapeutic applications.     

Design and synthesis of novel 3,5-bis-N-(aryl/heteroaryl) carbamoyl-4-aryl-1,4-dihydropyridines as small molecule BACE-1 inhibitors

Alzheimer disease (AD) is a neuronal dementia for which no treatment has been consolidated yet. Major pathologic hallmark of AD is the aggregated extracellular amyloid-β plaques in the brains of disease sufferers. Aβ-peptide is a major component of amyloid plaques and is produced from amyloid precursor protein (APP) via the proteolysis action. An aspartyl protease known as β-site amyloid precursor protein cleaving enzyme (BACE-1) is responsible for this proteolytic action. Distinctive role of BACE-1 in AD pathogenesis has made it a validated target to develop anti-Alzheimer agents. Our structure-based virtual screening method led to the synthesis of novel 3,5-bis-N-(aryl/heteroaryl) carbamoyl-4-aryl-1,4-dihydropyridine BACE-1 inhibitors (6a–6p; in vitro hits). Molecular docking and DFT-based ab initio studies using B3LYP functional in association with triple-ζ basis set (TZV) proposed binding mode and binding energies of ligands in the active site of the receptor. In vitro BACE-1 inhibitory activities were determined by enzymatic fluorescence resonance energy transfer (FRET) assay. Most of the synthesized dihydropyridine scaffolds were active against BACE-1 while 6d, 6k, 6n and 6a were found to be the most potent molecules with IC₅₀ values of 4.21, 4.27, 4.66 and 6.78 μM, respectively. Superior BACE-1 inhibitory activities were observed for dihydropyridine derivatives containing fused/nonfused thiazole containing groups, possibly attributing to the additional interactions with S2–S3 subpocket residues. Relatively reliable correlation between calculated binding energies and experimental BACE-1 inhibitory activities was achieved (R² = 0.51). Moreover, compounds 6d, 6k, 6n and 6a exhibited relatively no calcium channel blocking activity with regard to nifedipine suggesting them as appropriate candidates for further modification(s) to BACE-1 inhibitory scaffolds.     

BACE-1 inhibition prevents the γ-secretase inhibitor evoked Aβ rise in human neuroblastoma SH-SY5Y cells

Background  

Accumulation of amyloid β-peptide (Aβ) in the plaques is one of the major pathological features in Alzheimer's disease (AD). Sequential cleavage of amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE-1) and γ-secretase results in the formation of Aβ peptides. Preventing Aβ formation is believed to attenuate AD progression and BACE-1 and γ-secretase are thus attractive targets for AD drug development.     

A non-amyloidogenic function of BACE-2 in the secretory pathway

beta-Site amyloid precursor protein cleavage enzyme (BACE)-1 and BACE-2 are members of a novel family of membrane-bound aspartyl proteases. While BACE-1 is known to cleave beta-amyloid precursor protein (betaAPP) at the beta-secretase site and to be required for the generation of amyloid beta-peptide (Abeta), the role of its homologue BACE-2 in amyloidogenesis is less clear. We now demonstrate that BACE-1 and BACE-2 have distinct specificities in cleavage of betaAPP in cultured cells. Radiosequencing of the membrane-bound C-terminal cleavage product revealed that BACE-2 cleaves betaAPP in the middle of the Abeta domain between phenylalanines 19 and 20, resulting in increased secretion of APPs-alpha- and p3-like products and reduced production of Abeta species. This cleavage can occur in the Golgi and later secretory compartments. We also demonstrate that BACE-1-mediated cleavage of betaAPP at Asp1 of the Abeta domain can occur as early as in the endoplasmic reticulum, while cleavage at Glu11 occurs in later compartments. These data indicate that the distinct specificities of BACE-1 and BACE-2 in their cleavage of betaAPP differentially affect the generation of Abeta.