Rapid and efficient screening of Alzheimer's disease β-amyloid inhibitors using label-free gold nanoparticles

Herein we report that a visual, label-free gold nanoparticle-based assay for rapid and efficient screening of Alzheimer's disease β-amyloid inhibitors.     

Synthesis, biological evaluation and molecular modeling of novel triazole-containing berberine derivatives as acetylcholinesterase and β-amyloid aggregation inhibitors

A series of novel triazole-containing berberine derivatives were synthesized via the azide-alkyne cycloaddition reaction. Their biological activity as inhibitors of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were evaluated. Among them, compound 16d, which featured a diisopropylamino substitution at the 4-position of triazole ring, was found to be a potent inhibitor of AChE, with IC(50) value of 0.044 μM. Compound 18d, which beares a butyl at the 4-position of the triazole ring, showed the highest potency of β-amyloid aggregation inhibition (77.9% at 20 μM). Molecular modeling studies indicated that the triazole moiety of berberine derivatives displayed a face-to-face π-π stacking interaction in a 'sandwich' form with the Trp84 (4.09 ?) and Phe330 (4.33 ?) in catalytic sites of AChE.     

Design,synthesis and biological evaluation of novel dual inhibitors of acetylcholinesterase and β-secretase

To explore novel effective drugs for the treatment of Alzheimer’s disease (AD), a series of dual inhibitors of acetylcholineterase (AChE) and β-secretase (BACE-1) were designed based on the multi-target-directed ligands strategy. Among them, inhibitor 28 exhibited good dual potency in enzyme inhibitory potency assay (BACE-1: IC₅₀ = 0.567 μM; AChE: IC₅₀ = 1.83 μM), and also showed excellent inhibitory effects on Aβ production of APP transfected HEK293 cells (IC₅₀ = 98.7 nM) and mild protective effect against hydrogen peroxide (H₂O₂)-induced PC12 cell injury. Encouragingly, intracerebroventricular injection of 28 into amyloid precursor protein (APP) transgenic mice caused a 29% reduction of Aβ_1–40 production. Therefore, 28 was demonstrated as a good lead compound for the further study and more importantly, the strategy of AChE and BACE-1 dual inhibitors might be a promising direction for developing novel drugs for AD patients.     

Synthesis and β-amyloid binding properties of rhenium 2-phenylbenzothiazoles

As a first step toward the development of ^99mTc PiB analogs, we have synthesized six neutral Re 2-phenylbenzothiazoles via pendant or integrated approach. These Re compounds bind to Aβ_1–40 fibrils with fairly good affinities (K_i = 10.0–88.6 nM) and have moderate lipophilicities (log P_C18 = 1.21–3.26). The Re compounds prepared via the integrated approach are smaller in size, and therefore their corresponding ^99mTc analogs would have a greater chance of crossing the blood-brain barrier well. For potential clinical applications, further optimization on the structure–activity relationship to obtain Re 2-phenylbenzothiazoles with higher binding affinities (<10 nM) might be needed. The integrated approach reported here to obtain neutral, compact and lipophilic Re 2-phenylbenzothiazoles could to be applied to other high affinity pharmacophores as well as to generate ^99mTc analogs that could hold promise for extending the use of Aβ imaging in living human brain to many more clinical settings because they could be used with SPECT.     

Cross interaction of β-amyloid peptide and prion protein fragments

Summary This article presents kinetic studies of cross interaction of β-amyloid peptide and prion protein fragments. Syntheses of three peptides (β25-35, β22-35 and PrP 109–126) were performed. Those peptides were used for aggregation studies in PBS and TRIS buffers using HPLC with DAD detector. Comparison of aggregation of peptides alone and in combination with other fragments was investigated. In all cases aggregation was faster in PBS than in TRIS solution. Obtained results suggest that β-amyloid peptide and prion protein may interact to form macromolecular complexes with different ability for aggregation.     

Structure-based design, synthesis, and biological evaluation of dihydroquinazoline-derived potent β-secretase inhibitors

Structure-based design, synthesis, and biological evaluation of a series of dihydroquinazoline-derived β-secretase inhibitors incorporating thiazole and pyrazole-derived P2-ligands are described. We have identified inhibitor 4f which has shown potent enzyme inhibitory (K(i)=13nM) and cellular (IC(50)=21nM in neuroblastoma cells) assays. A model of 4f was created based upon the X-ray structure of 3a-bound β-secretase. The model suggested possible interactions in the active site.     

Phytochrome-mediated synthesis of novel growth inhibitors,A-2α and β, and dwarfism in peas

Variations in the content of A-2α and β, novel endogenous growth inhibitors, andcis,trans- andtrans, trans-xanthoxins were determined in 6- or 7-d-old, dark-grown seedlings of peas (Pisum sativum L. cvs. Progress No. 9, dwarf, and Alaska, tall) under various treatments with red light (R), and compared with R-induced growth inhibition. After transfer of the plants to continuous R the contents of the A-2s in cv. Progress increased after a 20-min lag, and reached plateaus after 12 h, whereas they remained almost unchanged in darkness. Both the rates of increase of the A-2s and the plateau levels were proportional to the logarithm of the irradiance applied. After a 10-min R pulse, the contents of both A-2α and β increased with the same rapidity to reach peaks after 6 h, and then gradually decreased to the initial levels after about 24 h. The effect of R was shown to be phytochrome-dependent, being nullified by far-red light. The level of neithercis,trans- nortrans,trans-xanthoxin showed such a close correlation with growth inhibition, although both xanthoxins increased as a result of phytochrome action. It is highly suggestive that the A-2s, rather than the xanthoxins, are responsible for phytochrome-dependent growth inhibition in cv. Progress. In cv. Alaska, in contrast, R-induced increase of the A-2s was rapid but slight, and could not explain the transient growth inhibition, which was found to be as large as that in cv. Progress shortly after the onset of R. The large content of the A-2s in cv. Progress in the steady state under continuous R, compared with that in cv. Alaska, may explain the dwarfism of cv. Progress. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Thermodynamic and kinetic characterization of hydroxyethylamine β-secretase-1 inhibitors

Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting millions of people. β-Secretase-1 (BACE-1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form Aβ, is a well validated target for AD. Herein, the authors characterize 10 randomly selected hydroxyethylamine (HEA) BACE-1 inhibitors in terms of their association and dissociation rate constants and thermodynamics of binding using surface plasmon resonance (SPR). Rate constants of association (k_a) measured at 25 °C ranged from a low of 2.42 × 10⁴ M⁻¹ s⁻¹ to the highest value of 8.3 × 10⁵ M⁻¹ s⁻¹. Rate constants of dissociation (k_d) ranged from 1.09 × 10⁻⁴ s⁻¹ (corresponding to a residence time of close to three hours), to the fastest of 0.028 s⁻¹. Three compounds were selected for further thermodynamic analysis where it was shown that equilibrium binding was enthalpy driven while unfavorable entropy of binding was observed. Structural analysis revealed that upon ligand binding, the BACE-1flap folds down over the bound ligand causing an induced fit. The maximal difference between alpha carbon positions in the open and closed conformations of the flap was over 5 Å. Thus the negative entropy of binding determined using SPR analysis was consistent with an induced fit observed by structural analysis.     

Amino acids and β-aminopropionitrile as inhibitors of seed germination and growth

The germination of lettuce fruits and legume seeds was affected by the imbibition of solutions of certain amino acids. Seedling growth was inhibited more markedly than germination by these compounds. Non-protein amino acids were, as a group, more effective inhibitors of germination and seedling growth than were protein amino acids, with the exception of lysine. Anomalous results were obtained with β-aminopropionitrile.     

Structurally distinct toxicity inhibitors bind at common loci on β-amyloid fibril

The accumulation of aggregated β-Amyloid (Aβ) in the brain is a hallmark of Alzheimer's disease and is thought to play a role in the neurotoxicity associated with the disease. The mechanism by which Aβ aggregates induce toxicity is uncertain. Nonetheless, several small molecules have been found to interact with Aβ fibrils and to prevent their toxicity. In this paper we studied the binding of these known toxicity inhibitors to Aβ fibrils, as a means to explore surfaces or loci on Aβ aggregates that may be significant in the mechanism of action of these inhibitors. We believe knowledge of these binding loci will provide insight into surfaces on the Aβ fibrils important in Aβ biological activity. The program DOCK was used to computationally dock the inhibitors to an Aβ fibril. The inhibitors docked at two shared binding loci, near Lys28 and at the C-termini near Asn27 and Val39. The docking predictions were experimentally verified using lysine specific chemical modifications and Aβ fibrils mutated at Asn27. We found that both Congo red and Myricetin, despite being structurally different, bound at the same two sites. Additionally, our data suggests that three additional Aβ toxicity inhibitors may also bind in one of the sites. Identification of these common binding loci provides targets on the Aβ fibril surface that can be tested in the future for their role in Aβ biological activity.     

MEK inhibitors suppress β-amyloid production by altering the level of a β-C-terminal fragment of amyloid precursor protein in neuronal cells

β-Amyloid peptide (Aβ) is generated via sequential proteolysis of amyloid precursor protein (APP) by β- and γ-secretases. Cell-based screening experiments disclosed that the MEK (MAP kinase kinase) inhibitors, U0126 and PD184352, suppress Aβ secretion from human neuronal SH-SY5Y cells expressing Swedish mutant APP. These inhibitors did not affect the cellular levels of APP but significantly reduced those of the APP β-C-terminal fragment (β-CTF). Additionally, β-CTF levels were markedly reduced by these inhibitors in cells expressing the fragment in a γ-secretase-independent and proteasome-dependent manner. Our results suggest that MEK inhibitors reduce Aβ generation via secretase-independent alteration of β-CTF levels.     

Synthetic inhibitors of glucocerebroside β-glucosidase

The glucocerebrosidase of human placenta was studied with various potential inhibitors. Several compounds that resemble the lipoidal product of enzyme action, ceramide, proved to be excellent inhibitors, acting by mixed modes (primarily noncompetitively). These were N-decyl-dl-erythro-3-phenyl-2-amino-l, 3-propanediol and several p-substituted derivatives. These compounds were also highly effective in rat spleen toward glucocerebroside and p-nitrophenyl β-glucoside as substrates. The compounds were inactive toward the analogous enzyme, galactocerebrosidase of rat brain, and were slightly stimulatory toward the rat brain enzyme which makes galactocerebroside. Longer and shorter N-alkyl groups proved to be less effective. Decanoic acid amides of phenylaminopropanediol and related compounds proved to be relatively inert, although some were stimulatory. Deoxycorticosterone β-glucoside was a moderately effective noncompetitive inhibitor and is apparently hydrolyzed by a different glucosidase. p-Nitrophenyl β-glucoside was also a moderately effective inhibitor, acting by mixed modes. p-Chloromercuribenzenesulfonate was a good inhibitor, presumably acting on a sensitive cysteine residue. It is concluded that cerebrosidase contains two sensitive sites, one catalytic and the other allosteric, each containing an important anionic group and able to bind glucosides and ceramide-like compounds.     

Synthesis and biological evaluation of indole-chalcone derivatives as β-amyloid imaging probe

A series of chaclone derivatives containing an indole moiety were evaluated in competitive binding assays with Aβ_1-42 aggregates versus [¹²⁵I]IMPY. The affinity of these compounds ranged from 4.46 to >1008 nM, depending on the substitution on the phenyl ring. Fluorescent staining in vitro showed that one compound with a N,N-dimethylamino group intensely stained Aβ plaques within brain sections of AD transgenic mice. The radioiodinated probe [¹²⁵I]-(E)-3-(1H-indol-5-yl)-1-(4-iodophenyl)prop-2-en-1-one, [¹²⁵I]4, was prepared and autoradiography in sections of brain tissue from an animal model of AD showed that it labeled Aβ plaques specifically. However, experiments with normal mice indicated that [¹²⁵I]4 exhibited a low uptake into the brain in vivo (0.41% ID/g at 2 min). Additional chemical modifications of this indole-chalcone structure may lead to more useful imaging agents for detecting β-amyloid plaques in the brains of AD patients.     

Design,synthesis and evaluation of dual pharmacology β2-adrenoceptor agonists and PDE4 inhibitors

A novel series of formoterol–phthalazinone hybrids were synthesised and evaluated as dual pharmacology β₂-adrenoceptor agonists and PDE4 inhibitors. Most of the hybrids displayed high β₂-adrenoceptor agonist and moderate PDE4 inhibitory activities. The most potent compound, (R,R)-11c, exhibited agonist (EC₅₀ = 1.05 nM, pEC₅₀ = 9.0) and potent PDE4B2 inhibitory activities (IC₅₀ = 0.092 μM).     

Conformational Analysis of the β-amyloid Peptide Fragment, β(12-28)

Abstract NMR and CD spectroscopy have been used to examine the conformation of the peptide, β(12-28), (VHHQKLVFFAEDVGSNK) in aqueous and 60% TFE/40% H(2)0 solution at pH 2.4. In 60% TFE solution, the peptide is helical as confirmed by the CD spectrum and by the pattern of the NOE cross peaks detected in the NOESY spectrum of the peptide. In aqueous solution, the peptide adopts a more extended and flexible conformation. Broadening of resonances at low temperature, temperature-dependent changes in the chemical shifts of several of the CH(α) resonances and the observation of a number of NOE contacts between the hydrophobic side-chain protons of the peptide are indicative of aggregation in aqueous solution. The behavior of β(12-28) in 60% TFE and in aqueous solution are consistent with the overall conformation and aggregation behavior reported for the larger peptide fragment, β(1-28) and the parent β-amyloid peptide.     

Diastereoselective synthesis of fused cyclopropyl-3-amino-2,4-oxazine β-amyloid cleaving enzyme (BACE) inhibitors and their biological evaluation

The diastereoselective synthesis and structure activity relationship (SAR) of a series of fused cyclopropyl-3-amino-2,4-oxazine (2-oxa-4-azabicyclo[4.1.0]hept-3-en-3-amine)-containing BACE inhibitors is described. Through these efforts compound 2 was identified as a potent (cell IC₅₀ = 15?nM) BACE inhibitor with acceptable ADME properties. When tested in vivo, compound 2 demonstrated a significant reduction of brain and cerebral spinal fluid (CSF) Aβ₄₀ levels (46% and 66%, respectively) in a rat pharmacodynamic study and thus represents a suitable starting point for the further development of in vivo efficacious compounds for the treatment of Alzheimer's disease.     

Conformational Analysis of the β-amyloid Peptide Fragment, β(12–28)

Abstract

NMR and CD spectroscopy have been used to examine the conformation of the peptide, β(12–28), (VHHQKLVFFAEDVGSNK) in aqueous and 60% TFE/40% H₂0 solution at pH 2.4. In 60% TFE solution, the peptide is helical as confirmed by the CD spectrum and by the pattern of the NOE cross peaks detected in the NOESY spectrum of the peptide. In aqueous solution, the peptide adopts a more extended and flexible conformation. Broadening of resonances at low temperature, temperature-dependent changes in the chemical shifts of several of the CH_α resonances and the observation of a number of NOE contacts between the hydrophobic side-chain protons of the peptide are indicative of aggregation in aqueous solution. The behavior of β(12–28) in 60% TFE and in aqueous solution are consistent with the overall conformation and aggregation behavior reported for the larger peptide fragment, β(1–28) and the parent β-amyloid peptide.