Targeting BACE with small inhibitory nucleic acids - a future for Alzheimer's disease therapy?

beta-Secretase, a beta-site amyloid precursor protein (APP) cleaving enzyme (BACE), participates in the secretion of beta-amyloid peptides (Abeta), the major components of the toxic amyloid plaques found in the brains of patients with Alzheimer's disease (AD). According to the amyloid hypothesis, accumulation of Abeta is the primary influence driving AD pathogenesis. Lowering of Abeta secretion can be achieved by decreasing BACE activity rather than by down-regulation of the APP substrate protein. Therefore, beta-secretase is a primary target for anti-amyloid therapeutic drug design. Several approaches have been undertaken to find an effective inhibitor of human beta-secretase activity, mostly in the field of peptidomimetic, non-cleavable substrate analogues. This review describes strategies targeting BACE mRNA recognition and its down-regulation based on the antisense action of small inhibitory nucleic acids (siNAs). These include antisense oligonucleotides, catalytic nucleic acids - ribozymes and deoxyribozymes - as well as small interfering RNAs (siRNAs). While antisense oligonucleotides were first used to identify an aspartyl protease with beta-secretase activity, all the strategies now demonstrate that siNAs are able to inhibit BACE gene expression in a sequence-specific manner, measured both at the level of its mRNA and at the level of protein. Moreover, knock-down of BACE reduces the intra- and extracellular population of Abeta40 and Abeta42 peptides. An anti-amyloid effect of siNAs is observed in a wide spectrum of cell lines as well as in primary cortical neurons. Thus targeting BACE with small inhibitory nucleic acids may be beneficial for the treatment of Alzheimer's disease and for future drug design.     

Computer-aided discovery of novel non-peptide inhibitors against amyloid-beta (Aβ) peptide aggregation for treating Alzheimer's disease

A non-peptide inhibitor that is metabolically stable, orally active and capable of crossing the blood–brain barrier has been a popular option for treating Alzheimer's disease (AD). To identify novel non-peptide inhibitors for AD drug development, a structure-based pharmacophore model (SBPM) was developed using the representative docked conformation of the recently discovered peptide inhibitor PGKLVYA in the potential binding site on the Aβ(17–42) protofibril. The best SBPM, consisting of two hydrophobic, one hydrogen bond donor, and one positive ionisable feature, was further validated using ligand pharmacophore mapping studies. The well-validated SBPM was then used as the 3D query in virtual screening to identify potential hits from the National Cancer Institute database. These hits were subsequently filtered by toxicity prediction and molecular docking, and their binding stabilities and affinities were validated by 20-ns molecular dynamics simulations and molecular mechanics Poisson–Boltzmann surface area analysis, respectively. Finally, two Hits (NSC35984 and NSC102747) were identified as potential leads, which exhibited higher binding stability and affinity towards Aβ compared with PGKVYA. Our results also suggest that these two Hits have the ability to prevent Aβ adopting toxic β-sheet structures, and can be easily synthesised and have structural novelty, indicating that they are promising candidates for treating AD.     

Metabolite profiling of Neptunia oleracea and correlation with antioxidant and α-glucosidase inhibitory activities using 1H NMR-based metabolomics

Neptunia oleracea is a plant consumed as vegetable and used as a traditional herb to treat several ailments. This study evaluated metabolite variations among N. oleracea leaf and stem subjected to air drying (AD), freeze drying (FD) and oven drying (OD) using proton nuclear magnetic resonance (¹H NMR) based metabolomics. The correlation was also studied for the metabolite content with total phenolic content (TPC), DPPH free radical scavenging and α-glucosidase inhibitory activities. A total of 18 metabolites were identified from N. oleracea extracts, including 10 primary metabolites, 5 flavonoids and 3 phenolic acids using NMR. Ultra-high performance liquid chromatography tandem mass spectrometry analysis (UHPLC-MS/MS) confirmed the presence of the secondary metabolites and revealed the flavonoid derivatives present. All the identified phenolics are first reported from this plant. Multivariate data analysis (MVDA) showed strong correlation between the metabolites with the antioxidant and α-glucosidase inhibitory activities of FD N. oleracea leaves. The compounds suggested to be responsible for the high activity of FD leaves include vitexin-2-O-rhamnoside, catechin, caffeic acid, gallic acid and derivatives of quercetin, kaempferol and myricetin. This study demonstrates that FD N. oleracea leaves are a potential natural source for antioxidant and α-glucosidase inhibitors.     

BACE1 gene variants do not influence BACE1 activity, levels of APP or Aβ isoforms in CSF in Alzheimer's disease

The BACE1 gene encodes the beta-site APP-cleaving enzyme 1 and has been associated with Alzheimer's disease (AD). BACE1 is the most important β-secretase responsible for the generation of Alzheimer-associated amyloid β-proteins (Aβ) and may play a role in the amyloidogenic process in AD. We hypothesized that BACE1 gene variants might influence BACE1 activity or other markers for APP metabolism in the cerebrospinal fluid (CSF) and thereby contribute to the development of AD. We genotyped a Swedish sample of 269 AD patients for the rs638405 single nucleotide polymorphism (SNP) in the BACE1 gene and correlated genotype data to a broad range of amyloid-related biomarkers in CSF, including BACE1 activity, levels of Aβ₄₀, Aβ_42, α- and β-cleaved soluble APP (α-sAPP and β-sAPP), as well as markers for Alzheimer-type axonal degeneration, i.e., total-tau and phospho-tau₁₈₁. Gene variants of BACE1 were neither associated with amyloid-related biomarkers, nor with markers for axonal degeneration in AD.     

Lead optimization studies towards the discovery of novel carbamates as potent AChE inhibitors for the potential treatment of Alzheimer’s disease

The optimization of our previous lead compound 1 (AChE IC₅₀ = 3.31 μM) through synthesis and pharmacology of a series of novel carbamates is reported. The synthesized compounds were evaluated against mouse brain AChE enzyme using the colorimetric method described by Ellman et al. The three compounds 6a (IC₅₀ = 2.57 μM), 6b (IC₅₀ = 0.70 μM) and 6i (IC₅₀ = 2.56 μM) exhibited potent in vitro AChE inhibitory activities comparable to the drug rivastigmine (IC₅₀ = 1.11 μM). Among them, the compound 6b has been selected as possible optimized lead for further neuropharmacological studies. In addition, the AChE–carbamate Michaelis complexes of these potent compounds including rivastigmine and ganstigmine have been modeled using covalent docking protocol of GOLD and important direct/indirect interactions contributing to stabilization of the AChE–carbamate Michaelis complexes have been investigated.     

Dual-target-directed 1,3-diphenylurea derivatives: BACE 1 inhibitor and metal chelator against Alzheimer’s disease

Dual-target-directed 1,3-diphenylurea derivatives were designed by hybridizing BACE 1 inhibitor 1 with metal chelator LR-90. A database consisted of 1,3-diphenylurea derivatives was built and screened by the pharmacophore model (Hypo 1) of BACE 1 inhibitor. Based on the predicted results, 11 compounds (6a–d, 9a–g) with favorable Fitvalues were selected, synthesized and evaluated for their BACE 1 inhibitory activities, which showed that the predicted results were in good agreement with the experimental values. Besides, the synthesized compounds also displayed the ability to chelate metal ions. The most effective BACE 1 inhibitor 9f (27.85 ± 2.46 μmol/L) was selected for further receptor-binding studies, the result of which indicated that an essential hydrogen bonds was formed between the urea group of 9f and the catalytic aspartate Asp228.     

Cathepsins S, B and L with aminopeptidases display β-secretase activity associated with the pathogenesis of Alzheimer's disease

β-site APP-cleaving enzyme (BACE1) cleaves the wild type (WT) β-site very slowly (k(cat)/K(m): 46.6 m(-1) s(-1)). Therefore we searched for additional β-secretases and identified three cathepsins that split the WT β-site much faster. Human cathepsin S cleaves the WT β-site (k(cat)/K(m): 54 700 m(-1) s(-1)) 1170-fold faster than BACE1 and cathepsins B and L are 440- and 74-fold faster than BACE1, respectively. These cathepsins split two bonds flanking the WT β-site (K-MD-A), where the K-M bond (85%) is cleaved more efficiently than the D-A bond (15%). Cleavage at the major K-M bond yields Aβ (amyloid β-peptide) extended by N-terminal Met that should be removed to generate Aβ initiated by Asp1. The activity of cytosol and microsomal aminopeptidases on relevant peptides revealed rapid removal of N-terminal Met but not N-terminal Asp. Brain aminopeptidases showed similar specificity. Thus, aminopeptidases would convert Aβ extended by Met into regular Aβ (Asp1) found in amyloid plaques. Earlier studies indicate that Aβ is likely produced in the endosome and lysosome system where cathepsins S, B and L are localized and cysteine cathepsin inhibitors reduce the level of Aβ in cells and animals. Taken together, cathepsins S, B and L deserve further evaluation as therapeutic targets to develop disease modifying drugs to treat Alzheimer's disease.     

Synthesis and biological evaluation of novel quinazoline-triazole hybrid compounds with potential use in Alzheimer’s disease

A library of twelve quinazoline-triazole hybrid compounds were designed, synthesized and evaluated as a novel class of acetylcholinesterase inhibitors to treat Alzheimer’s disease (AD). The biological assay results demonstrated the ability of several hybrid compounds to inhibit AChE enzyme (IC₅₀ range = 0.2–83.9 µM). To understand the high potential activity of these compounds, molecular docking simulations were performed to get better insights into the mechanism of binding of quinazoline-triazole hybrid compounds. As expected, compounds 8a and 9a-b bind to both catalytic anionic site (CAS) and peripheral anionic site (PAS) in the active site of AChE enzyme, which implicates that these compounds could act as dual binding site inhibitors. These compounds were not cytotoxic and they also displayed appropriated physicochemical as well as pharmacokinetic profile to be developed as novel anti-AD drug candidates.     

Novel multi-target compounds in the quest for new chemotherapies against Alzheimer’s disease: An experimental and theoretical study

The lack of any effective therapy along with the aging world population anticipates a growth of the worldwide incidence of Alzheimer’s disease (AD) to more than 100 million cases by 2050. Accumulation of extracellular amyloid-β (Aβ) plaques, intracellular tangles in the brain, and formation of reactive oxygen species (ROS) are the major hallmarks of the disease. In the amyloidogenic process, a β-secretase, known as BACE 1, plays a fundamental role in the production of Aβ fragments, and therefore, inhibition of such enzymes represents a major strategy for the rational design of anti-AD drugs. In this work, a series of four multi-target compounds (1–4), inspired by previously described ionophoric polyphenols, have been synthesized and studied. These compounds have been designed to target important aspects of AD, including BACE 1 enzymatic activity, Aβ aggregation, toxic concentrations of Cu²⁺ metal ions and/or ROS production. Two other compounds (5 and 6), previously reported by some of us as antimalarial agents, have also been studied because of their potential as multi-target species against AD. Interestingly, compounds 3 and 5 showed moderate to good ability to inhibit BACE 1 enzymatic activity in a FRET assay, with IC₅₀′s in the low micromolar range (4.4?±?0.3 and 1.7?±?0.3?μM, respectively), comparable to other multi-target species, and showing that the observed activity was in part due to a competitive binding of the compounds at the active site of the enzyme. Theoretical docking calculations overall agreed with FRET assay results, displaying the strongest binding affinities for 3 and 5 at the active site of the enzyme. In addition, all compounds selectively interacted with Cu²⁺ metal ions forming 2:1 complexes, inhibited the production of Aβ-Cu²⁺ catalyzed hydroxyl radicals up to a ～100% extent, and scavenged AAPH-induced peroxyl radical species comparably to resveratrol, a compound used as reference in this work. Our results also show good anti-amyloidogenic ability: compounds 1–6 inhibited both the Cu²⁺-induced and self-induced Aβ(1–40) fibril aggregation to an extent that ranged from 31% to 77%, while they disaggregated pre-formed Aβ(1–40) mature fibrils up to a 37% and a 69% extent in absence and presence of Cu²⁺, respectively. Cytotoxicity was additionally studied in Tetrahymena thermophila and HEK293 cells, and compared to that of resveratrol, showing that compounds 1–6 display lower toxicity than that of resveratrol, a well-known non-toxic polyphenol.     

Dysfunction of TGF-β1 signaling in Alzheimer's disease: perspectives for neuroprotection

Alzheimer’s disease (AD) is a neurodegenerative disorder that affects about 35 million people worldwide. Current drugs for AD only treat the symptoms and do not interfere with the underlying pathogenic mechanisms of the disease. AD is characterized by the presence of β-amyloid (Aβ) plaques, neurofibrillary tangles, and neuronal loss. Identification of the molecular determinants underlying Aβ-induced neurodegeneration is an essential step for the development of disease-modifying drugs. Recently, an impairment of the transforming growth factor-β1 (TGF-β1) signaling pathway has been demonstrated to be specific to the AD brain and, particularly, to the early phase of the disease. TGF-β1 is a neurotrophic factor responsible for the initiation and maintenance of neuronal differentiation and synaptic plasticity. The deficiency of TGF-β1 signaling is associated with Aβ pathology and neurofibrillary tangle formation in AD animal models. Reduced TGF-β1 signaling seems to contribute both to microglial activation and to ectopic cell-cycle re-activation in neurons, two events that contribute to neurodegeneration in the AD brain. The neuroprotective features of TGF-β1 indicate the advantage of rescuing TGF-β1 signaling as a means to slow down the neurodegenerative process in AD.     

Peel of araticum fruit (Annona crassiflora Mart.) as a source of antioxidant compounds with α-amylase, α-glucosidase and glycation inhibitory activities

Annona crassiflora Mart., whose fruit is popularly known as araticum, is a member of the Annonaceae family found in the Brazilian Cerrado. Although this plant has several medicinal uses, its bioactive molecules are not fully understood. A bioguided assay was performed to identify the main bioactive compounds of A. crassiflora fruit peel from the ethanol extract fractions with antioxidant capacity and α-amylase, α-glucosidase and glycation inhibitory activities. Ethyl acetate and n-butanol fractions showed, respectively, higher antioxidant capacity (DPPH IC₅₀ 1.5 ± 0.1 and 0.8 ± 0.1 μg mL⁻¹, ORAC 3355 ± 164 and 2714 ± 79 μmol trolox eq/g, and FRAP 888 ± 16 and 921 ± 9 μmol trolox eq/g) and inhibitory activities against α-amylase (IC₅₀ 4.5 ± 0.8 and 1.7 ± 0.3 μg mL⁻¹), α-glucosidase (IC₅₀ 554.5 ± 158.6 and 787.8 ± 140.6 μg mL⁻¹) and glycation (IC₅₀ 14.3 ± 3.3 and 16.0 ± 4.2 μg mL⁻¹), and lower cytotoxicity, compared to the other fractions and crude ethanol extract. The HPLC-ESI-MS/MS analysis identified various biomolecules known as potent antioxidants, such as chlorogenic acid, (epi)catechin, procyanidins, caffeoyl-hexosides, quercetin-glucosides and kaempferol. The fruit peel of A. crassiflora, a specie from Cerrado, the Brazilian Savanna, provided a source of antioxidant compounds with properties to block carbohydrate digestive enzymes and formation of glycation products. Thus, there is potential to use the by-products of araticum in order to identify and isolate phytochemicals for application in nutraceutical supplements, food additives and pharmaceuticals products.     

Diamide amino-imidazoles: a novel series of γ-secretase inhibitors for the treatment of Alzheimer's disease

The synthesis and structure-activity relationship (SAR) of a novel series of di-substituted imidazoles, derived from modification of DAPT, are described. Subsequent optimization led to identification of a highly potent series of inhibitors that contain a β-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aβ in guinea pigs. The therapeutic index between Aβ reductions and changes in B-cell populations were studied for compound 10h.     

Insights into the inhibitory mechanism of a resveratrol and clioquinol hybrid against Aβ42 aggregation and protofibril destabilization: A molecular dynamics simulation study

Amyloid-β (Aβ) peptide instinctively aggregate and form plaques in the brain of Alzheimer’s disease (AD) patients. At present, there is no cure or treatment for AD, and significant effort has, therefore, been made to discover potent drugs against AD. Previous studies reported that a resveratrol and clioquinol hybrid compound [(E)-5-(4-hydroxystyryl)quinolone-8-ol], C1, strongly inhibit Aβ₄₂ aggregation and disassemble preformed fibrils. However, the atomic level details of the inhibitory mechanism of C1 against Aβ₄₂ aggregation and protrofibril disassembly remains elusive. In this regard, molecular docking and molecular dynamics (MD) simulation of Aβ₄₂ monomer, Aβ₄₂ monomer–C1 complex, Aβ₄₂ protofibril, and Aβ₄₂ protofibril–C1 complex were performed in the present study. MD simulations highlighted that C1 bind in the central hydrophobic core (CHC) region, i.e., KLVFF (16–20) of Aβ₄₂ monomer, which plays a critical role in Aβ₄₂ aggregation. C1 promote the formation of native helical conformation in the Aβ₄₂ monomer and decrease the probability of D23–K28 salt bridge interaction that is critical in the formation of aggregation-prone β-sheet conformation. Further, C1 destabilize Aβ₄₂ protofibril structure by increasing the interchain distance between chains A–B, disrupting the salt–bridge interaction between D23–K28, and decreasing the number of backbone hydrogen bonds between chains A–B of the Aβ₄₂ protofibril structure. The insights into the underlying inhibitory mechanism of small molecules that display potential in vitro anti–aggregation activity against Aβ₄₂ will be beneficial for the rational design of more potent drug molecules against AD.

Communicated by Ramaswamy H. Sarma     

Design,synthesis and biological assessment of N-adamantyl,substituted adamantyl and noradamantyl phthalimidines for nitrite,TNF-α and angiogenesis inhibitory activities

A library of 15 novel and heretofore uncharacterized adamantyl and noradamantyl phthalimidines was synthesized and evaluated for neuroprotective and anti-angiogenic properties. Phthalimidine treatment in LPS-challenged cells effected reductions in levels of secreted TNF-α and nitrite relative to basal amounts. The primary SAR suggests nitration of adamantyl phthalimidines has marginal effect on TNF-α activity but promotes anti-nitrite activity; thioamide congeners retain anti-nitrite activity but are less effective reducing TNF-α. Site-specific nitration and thioamidation provided phthalimidine 24, effecting an 88.5% drop in nitrite concurrent with only a 4% drop in TNF-α. Notable anti-angiogenesis activity was observed for 20, 21 and 22.     

Structure guided P1' modifications of HEA derived β-secretase inhibitors for the treatment of Alzheimer's disease

The synthesis and SAR of a series of BACE-1 hydroxyethyl amine inhibitors containing substitutions on a spirocyclobutyl moiety is described. Selectivity against cathepsin D, a related aspartyl protease with potential off target toxicity, and improved microsomal stability is exemplified.     

Is prostaglandin a mediator for the inhibitory action of histamine,hydrocortisone, and isoproterenol?

We examined the inhibitory actions of prostaglandin E₂, histamine, isoproterenol, hydrocortisone, and interferon on lymphocyte mitogenesis. There was a high degree of intercorrelation between the amount of inhibition caused by prostaglandin E₂, histamine, isoproterenol, and hydrocortisone, but not interferon, in any given subject; that is if lymphocytes from a given subject were highly sensitive to inhibition by one of those agents, they were also sensitive to the other agents. The inhibitory actions of histamine, isoproterenol, or hydrocortisone could be partially blocked by adding prostaglandin synthetase inhibitors to the mitogen cultures. Preincubation of the lymphocytes for 18 hr prior to the addition of mitogens and inhibitors resulted in a loss in sensitivity to the inhibitors other than interferon. Removal of glass-adherent cells (the prostaglandin-producing cells) prior to culture lessened the inhibition caused by histamine and isoproterenol. The above data would suggest that these inhibitors may act via prostaglandin; however, all of these compounds actually decrease prostaglandin production in cultures of peripheral blood mononuclear cells. The implications of these findings are discussed.     

Presence of AVT-, α-MSH-, LHRH- and somatostatin-like compounds in the rat pineal gland and their relationship with the UMO5R pineal fraction

Using antibodies against AVT, alpha-MSH, LHRH and somatostatin, immunoreactive cells were detected in the rat pineal gland. All of these antibodies stain the same cells, which also react immunocytochemically when an antibody against the UMO5R sheep pineal fraction, a fraction that presents antigonadotropic properties in vivo, is used. Relatively more immunoreactive cells are present in the pineals of young rats than in the pineals of adult animals. Comparison of the results obtained with different potent antibodies against each of the peptides, and a study of the staining properties of the antibodies in the pineal after solid phase absorption to different peptides or to different sheep pineal fractions, led to the proposal that the immunoreactivity found in the rat pineal is not due to the presence of AVT, alpha-MSH, LHRH or somatostatin, but to a cross-reaction of each of these antibodies with (an) unidentified compound(s). This compound is synthetized in the pineal gland, as was demonstrated using cultured pineals. The UMO5R and the Prot. 4 fractions of the sheep pineal seem to be chemically related to this unknown compound, the possible endocrine nature of which is discussed.