In vitro antiamyloidogenic properties of 1,4-naphthoquinones

The aim of this study is to find out whether several 1,4-naphthoquinones (1,4-NQ) can interact with the amyloidogenic pathway of the amyloid precursor protein processing, particularly targeting at β-secretase (BACE), as well as at β-amyloid peptide (Aβ) aggregation and disaggregating preformed Aβ fibrils. Compounds bearing hydroxyl groups at the quinoid (2) or benzenoid rings (5, 6) as well as some 2- and 3-aryl derivatives (11-15) showed BACE inhibitory activity, without effect on amyloid aggregation or disaggregation. The halogenated compounds 8 and 10 were selective for the inhibition of amyloid aggregation. On the other hand, 1,4-naphthoquinone (1), 6-hydroxy-1,4-naphthoquinone (4) and 2-(3,4-dichlorophenyl)-1,4-naphthoquinone (26) did not show any BACE inhibitory activity but were active on amyloid aggregation and disaggregation preformed Aβ fibrils. Juglone (5-hydroxy-1,4-naphthoquinone (3), and 3-(p-hydroxyphenyl)-5-methoxy-1,4-napththoquinone (19) were active on all the three targets. Therefore, we suggest that 1,4-NQ derivatives, specially 3 and 19, should be explored as possible drug candidates or lead compounds for the development of drugs to prevent amyloid aggregation and neurotoxicity in Alzheimer’s disease.     

Design,synthesis and evaluation of curcumin-based fluorescent probes to detect Aβ fibrils

Amyloid β fibrillation is an early event in Alzheimer’s disease, so its detection is important to understand its roles in Alzheimer’s disease. Curcumin, which has poor water solubility, has been reported to have many pharmacological activities including potent anti-amyloid β fibril activity in Alzheimer’s disease. In this study, we found that curcumin analogues with the fluorescence property instead of non-inhibition of amyloid β fibrils. The development of new curcumin analogue, Me-CUR (9), as fluorescent switchable probe to detect amyloid β fibrils is described. Me-CUR (9) shows excellent fluorescence, especially higher than ThT (4), in the presence of amyloid β fibrils. These results suggest that Me-CUR (9) can become a useful in vitro amyloid fluorescence sensor for diagnosis of Alzheimer’s disease.     

Naphthalene-triazolopyrimidine hybrid compounds as potential multifunctional anti-Alzheimer’s agents

In an attempt to construct potential anti-Alzheimer’s agents Naphthalene-triazolopyrimidine hybrids were synthesized and screened in vitro against the two cholinesterases (ChE)s, amyloid β aggregation and for antioxidation activity. Single-crystal X-ray crystallography was utilized for crystal structure determination of one of the compounds. In vitro study of compounds revealed that most of the compounds are capable of inhibiting acetylcholinesterase and Butyrylcholinesterase activity. Particularly, the compounds 4e and 4d exhibited IC₅₀ values ranging from 8.6 to 14 nM against AChE lower than the standard drug Donepezil (IC₅₀ 49 nM). Best result was found for compound 4e with IC₅₀ of 8.6 nM (for AChE) and 150 nM (for BuChE). Selectivity upto that of Donepezil and even more was observed for 4a, 4c and 4h. Investigation by electron microscopy, transmission electron microscopy and ThT fluorescence assay unveils the fact that synthesized hybrids exhibit amyloid β self-aggregation inhibition. The compounds 4i and 4j revealed highest inhibitory potential, 85.46% and 72.77% at 50 μM respectively; above the standard Aβ disaggregating agent, Curcumin. Their antioxidation profile was also analyzed. Studies from DPPH free radical scavenging assay and ORAC assay depicts molecules to possess low antioxidation profile. Results suggest that triazolopyrimidines are potential candidate for Acetylcholinesterase (AChE), Butyrylcholinesterase (BuChE), and amyloid β aggregation inhibition. In silico ADMET profiling indicates drug-like properties with a very low toxic influence. Such synthesized compounds provide a strong vision for further development of potential anti-Alzheimer’s agents.     

Syntheses of tricyclic pyrones and pyridinones and protection of Aβ-peptide induced MC65 neuronal cell death

The SβC gene is conditionally expressed a 99-residue carboxy terminal fragment, C99, of amyloid precursor protein in MC65 cells and causes cell death. Consequently, MC65 cell line was used to identify inhibitors of toxicity related to intracellular amyloid β (Aβ) oligomers. Compounds that reduce the level of Aβ peptides, prevent Aβ aggregation, or eliminate existing Aβ aggregates may be used in the treatment of Alzheimer’s disease (AD). Previously, we found that a tricyclic pyrone (TP) molecule, compound 1, prevents MC65 cell death and inhibits Aβ aggregation. Hence various TPs containing heterocycle at C7 side chain and a nitrogen at position 2 or 5 were synthesized and their MC65 cell protective activities evaluated. TPs containing N3′-adenine moiety such as compounds 1 and 11 are most active with EC₅₀ values of 0.31 and 0.35 μM, respectively. EC₅₀ values of tricyclic N5-analog, pyranoisoquinolinone 13, and N2-analog, pyranopyridinone 20, are 2.49 and 1.25 μM, respectively, despite the lack of adenine moiety. Further investigation of tricyclic N2- and N5-analogs is warranted.     

Bacterial functional amyloids: Order from disorder

The discovery of intrinsic disorderness in proteins and peptide regions has given a new and useful insight into the working of biological systems. Due to enormous plasticity and heterogeneity, intrinsically disordered proteins or regions in proteins can perform myriad of functions. The flexibility in disordered proteins allows them to undergo conformation transition to form homopolymers of proteins called amyloids. Amyloids are highly structured protein aggregates associated with many neurodegenerative diseases. However, amyloids have gained much appreciation in recent years due to their functional roles. A functional amyloid fiber called curli is assembled on the bacterial cell surface as a part of the extracellular matrix during biofilm formation. The extracellular matrix that encases cells in a biofilm protects the cells and provides resistance against many environmental stresses. Several of the Csg (curli specific genes) proteins that are required for curli amyloid assembly are predicted to be intrinsically disordered. Therefore, curli amyloid formation is highly orchestrated so that these intrinsically disordered proteins do not inappropriately aggregate at the wrong time or place. The curli proteins are compartmentalized and there are chaperone-like proteins that prevent inappropriate aggregation and allow the controlled assembly of curli amyloids. Here we review the biogenesis of curli amyloids and the role that intrinsically disordered proteins play in the process.     

Design and synthesis of β-strand-fixed peptides inhibiting aggregation of amyloid β-protein

Aggregation of 42-residue amyloid β-protein (Aβ42) can be prevented by β-sheet breaker peptides (BSBps) homologous to LVFFA residues, which are included in a β-sheet region of Aβ42 aggregates. To enhance the affinity of BSBps to the Aβ42 aggregates, we designed and synthesized β-strand-fixed peptides (BSFps) whose side chains were cross-linked by ring closing metathesis. Conformation analysis verified that the designed peptides could be fixed in β-strand conformation. Among the synthesized pentapeptides, 1 and 12, whose side chains of 2nd and 4th residues were cross-linked, significantly inhibited the aggregation of Aβ42. This suggested that β-strand-fixation of BSBps could enhance their inhibitory activity against the Aβ42 aggregation. However, pentapeptides 1 and 12 had little effect on morphology of Aβ42 aggregates (fibrils) and neurotoxicity of Aβ42 against SH-SY5Y cells.     

Lodopyridones B and C from a marine sediment-derived bacterium Saccharomonospora sp.

HPLC-UV guided isolation of the culture broth of a marine bacterium Saccharomonospora sp. CNQ-490 has led to the isolation of two new natural products, lodopyridones B and C (1 and 2) along with the previously reported lodopyridone A (3). Their chemical structures were established from the interpretation of 2D NMR spectroscopic data and the comparison of NMR data with the lodopyridone A (3). Lodopyridones B and C (1 and 2) possess the thiazole, and chloroquinoline groups which are characteristic features of these molecules. Lodopyridones A–C show weak inhibitory activities on the β-site amyloid precursor protein cleaving enzyme 1 (BACE1).     

Synthesis,biological evaluation and molecular dynamic simulations of novel Benzofuran-tetrazole derivatives as potential agents against Alzheimer’s disease

A series of novel Benzofuran-tetrazole derivatives were successfully synthesised by integrating multicomponent Ugi-azide reaction with the molecular hybridization approach. Interestingly, a number of synthesized derivatives (5c, 5d, 5i, 5l, 5q and 5s) exhibited significant reduction of aggregation of “human” amyloid beta peptide, expressing on transgenic Caenorhabditis elegans (C. elegans) strain CL4176. Further, in silico docking results have evidenced the exquisite interaction of active compounds with the help of TcAChE–E2020 complex. These findings underscore the potential of these hybrids as lead molecules against Alzheimers’s disease.     

Quinones bearing non-steroidal anti-inflammatory fragments as multitarget ligands for Alzheimer’s disease

The anti-amyloid properties shared by several quinones inspired the design of a new series of hybrids derived from the multi-target drug candidate memoquin (1). The hybrids consist of a central benzoquinone core and a fragment taken from non-steroidal anti-inflammatory drugs, connected through polyamine linkers. The new hybrids retain the potent anti-aggregating activity of the parent 1, while exhibiting micromolar AChE inhibitory activities. Remarkably, 2, 4, (R)-6 and (S)-6 were Aβ aggregation inhibitors even more potent than 1. The balanced amyloid/cholinesterase inhibitory profile is an added value that makes the present series of compounds promising leads against Alzheimer’s disease.     

Structure activity relationship study of curcumin analogues toward the amyloid-beta aggregation inhibitor

Inhibition of the amyloid β aggregation process could possibly prevent the onset of Alzheimer’s disease. In this article, we report a structure–activity relationship study of curcumin analogues for anti amyloid β aggregation activity. Compound 7, the ideal amyloid β aggregation inhibitor in vitro among synthesized curcumin analogues, has not only potent anti amyloid β aggregation effects, but also water solubility more than 160 times that of curcumin. In addition, new approaches to improve water solubility of curcumin-type compounds are proposed.     

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.     

Structure-activity relationship of clovamide and its related compounds for the inhibition of amyloid β aggregation

Alzheimer’s disease (AD), a neurodegenerative disorder, is characterized by aggregation of amyloid β-protein (Aβ). Aβ aggregates through β-sheet formation and induces cytotoxicity against neuronal cells. Inhibition of Aβ aggregation by naturally occurring compounds is thus a promising strategy for the treatment of AD. We have already reported that caffeoylquinic acids and phenylethanoid glycosides, which possess two or more catechol moieties, strongly inhibited Aβ aggregation. Clovamide (1) containing two catechol moieties, isolated from cacao beans (Theobroma cacao L.), is believed to exhibit preventive effects on Aβ aggregation. To investigate the structure-activity relationship of clovamide (1) for the inhibition of Aβ aggregation, we synthesized 1 and related compounds 2–11 through reaction between l-DOPA, d-DOPA, l-tyrosine, or l-phenylalanine and caffeic acid, p-coumaric acid, or cinnamic acid, and compounds 12 and 13 were derived from 1. Among tested compounds 1–13, those containing one or two catechol moieties exhibited potent anti-aggregation activity, whereas the non-catechol-type related compounds showed little or no activity. This suggests that at least one catechol moiety is essential for inhibition of Aβ42 aggregation, and this activity increases depending on the number of catechol moieties. Consequently, clovamide (1) and its related compounds may be a promising therapeutic option for inhibiting Aβ-mediated pathology in AD.     

New phenylaniline derivatives as modulators of amyloid protein precursor metabolism

The chloroquinoline scaffold is characteristic of anti-malarial drugs such as chloroquine (CQ) or amodiaquine (AQ). These drugs are also described for their potential effectiveness against prion disease, HCV, EBV, Ebola virus, cancer, Parkinson or Alzheimer diseases. Amyloid precursor protein (APP) metabolism is deregulated in Alzheimer’s disease. Indeed, CQ modifies amyloid precursor protein (APP) metabolism by precluding the release of amyloid-beta peptides (Aβ), which accumulate in the brain of Alzheimer patients to form the so-called amyloid plaques. We showed that AQ and analogs have similar effects although having a higher cytotoxicity. Herein, two new series of compounds were synthesized by replacing 7-chloroquinolin-4-amine moiety of AQ by 2-aminomethylaniline and 2-aminomethylphenyle moieties. Their structure activity relationship was based on their ability to modulate APP metabolism, Aβ release, and their cytotoxicity similarly to CQ. Two compounds 15a, 16a showed interesting and potent effect on the redirection of APP metabolism toward a decrease of Aβ peptide release (in the same range compared to AQ), and a 3–10-fold increased stability of APP carboxy terminal fragments (CTFα and AICD) without obvious cellular toxicity at 100?µM.     

A new class of aggregation inhibitor of amyloid-β peptide based on an O-acyl isopeptide

Inhibition of amyloid β peptide (Aβ) aggregation is a potential therapeutic approach to treat Alzheimer’s disease. We report that an O-acyl isopeptide of Aβ1–42 (1) containing an ester bond at the Gly²⁵-Ser²⁶ moiety inhibits Aβ1–42 fibril formation at equimolar ratio. Inhibitory activity was retained by an N-Me-β-Ala²⁶ derivative (2), in which the ester of 1 was replaced with N-methyl amide to improve chemical stability at physiological pH. Inhibition was verified by fluorescence anisotropy, Western blot, and atomic force microscopy. This report suggests a new class of Aβ aggregation inhibitor based on modification of Aβ1–42 at Gly²⁵-Ser²⁶.     

Peptides derived from HIV-1 gp120 co-receptor binding domain form amyloid fibrils and enhance HIV-1 infection

Amyloid fibrils play important roles in HIV-1 infection. We found peptides derived from the HIV-1 gp120 co-receptor binding region, which are defined as enhancing peptides (EPs), could form amyloid fibrils and remarkably enhance HIV-1 infection. EPs bound to the virus and promoted the interaction between HIV-1 and target cells. The antiviral efficacy of antiretroviral drugs (ARVs) was substantially impaired in the presence of EPs. Epigallocatechin gallate (EGCG) could both inhibit the formation of fibrils composed of EPs and counteract the EP-mediated enhancement of HIV-1 infection. Our findings identify viral derived amyloid fibrils that hold potential for biochemical applications.Structured summary of protein interactionsEP1 and EP1 bind by fluorescence technology (View interaction)EP2 and EP2 bind by fluorescence technology (View interaction)EP3 and EP3 bind by fluorescence technology (View interaction)SEVI and SEVI bind by fluorescence technology (View interaction)EP1 and EP1 bind by transmission electron microscopy (View interaction)EP2 and EP2 bind by transmission electron microscopy (View interaction)EP3 and EP3 bind by transmission electron microscopy (View interaction)SEVI and SEVI bind by transmission electron microscopy (View interaction)     

Isolation of three new meroterpenoids and seven known compounds from Albatrellus yasudae and their Aβ-aggregation inhibitory activity

Alzheimer’s disease is a serious neurologic disorder that cannot be cured completely. In this study, we targeted compounds that inhibit amyloid-beta (Aβ) aggregation, based on the amyloid cascade hypothesis. Ten compounds (1–10) were isolated from CHCl₃ extracts of the mushroom Albatrellus yasudae using Aβ-aggregation inhibitory activity–guided separation. The structures of these compounds were elucidated from 1D and 2D NMR and MS spectral data. Compounds 1–3 were novel, whereas 4–10 were identified as the known compounds grifolin, grifolic acid, neogrifolin, confluentin, 2-hydroxyneogrifolin, daurichromenic acid, and a cerebroside derivative. Compounds 1–10 were tested for Aβ-aggregation inhibitory activity. Compounds 1, 3, 5, 6, 8, and 9 have potential as Aβ-aggregation inhibitory activity.     

Design and properties of [Met35(O)]Aβ42-lactam(Asp23/Lys28) possessing a lactam tether as a salt-bridge surrogate

A characteristic feature of higher-order structures of amyloid β peptide (Aβ) aggregates observed in Alzheimer disease is the salt-bridge between the side-chains of Asp²³ (carboxylate) and Lys²⁸ (ammonium). We synthesized an [Met³⁵(O)]Aβ42 possessing a covalently bound lactam tether as an Asp²³/Lys²⁸ salt-bridge surrogate (compound 3). The lactam tether of 3 markedly promoted the formation of stable protofibril-like species that exhibited amyloidogenic properties such as a cross-β-sheet structure and cytotoxicity. This finding is consistent with reports that the Asp²³/Lys²⁸ salt-bridge of Aβ42 is transiently formed in aggregation intermediates.     

Mechanistic analyses of the suppression of amyloid β42 aggregation by apomorphine

(R)-Apomorphine (1) has the potential to reduce the accumulation of amyloid β-protein (Aβ42), a causative agent of Alzheimer’s disease (AD). Although the inhibition of Aβ42 aggregation by 1 is ascribable to the antioxidative effect of its phenol moiety, its inhibitory mechanism at the molecular level remains to be fully elucidated. LC–MS and UV analyses revealed that 1 is autoxidized during incubation to produce an unstable o-quinone form (2), which formed a Michael adduct with Lys 16 and 28 of Aβ42. A further autoxidized form of 1 (3) with o-quinone and phenanthrene moieties suppressed Aβ42 aggregation comparable to 1, whereas treating 1 with a reductant, tris(2-carboxyethyl)phosphine diminished its inhibitory activity. ¹H-¹⁵N SOFAST-HMQC NMR studies suggested that 1 interacts with Arg5, His13,14, Gln15, and Lys16 of the Aβ42 monomer. These regions form intermolecular β-sheets in Aβ42 aggregates. Since 3 did not perturb the chemical shift of monomeric Aβ42, we performed aggregation experiments using 1,1,1,3,3,3-hexafluoro-2-propanol-treated Aβ42 to investigate whether 3 associates with Aβ42 oligomers. Compounds 1 and 3 delayed the onset of the oligomer-driven nucleation phase. Despite their cytotoxicity, they did not exacerbate Aβ42-mediated neurotoxicity in SH-SY5Y neuroblastoma cells. These results demonstrate that extension of the conjugated system in 1 by autoxidation can promote its planarity, which is required for intercalation into the β-sheet of Aβ42 nuclei, thereby suppressing further aggregation.     

Asn27 is Essential for the Neurotoxicity of Amyloid β(1–42) Peptide

In contrast to the general tendency of hydrophobicity-toxicity relationship of amyloid ?? peptide, we have previously found that the replacement of Asn²⁷ of amyloid ??(25?C35) peptide with Ala yielded a more hydrophobic but less toxic analog and that of Met³⁵ gave a less hydrophobic but more toxic one. To reveal the unique role of these two residues in the neurotoxicity of amyloid ??(1?C42) peptide, the major peptide constituent of amyloid plaques in human brain, we synthesized two analogs N27A and M35A in which Asn²⁷ and Met³⁵ of amyloid ??(1?C42) peptide was replaced with Ala, respectively. The former showed much weaker toxicity than the native peptide, while the latter showed almost an equivalent toxicity, indicating that the side chain amide group of Asn²⁷ has an essential role for the toxicity of amyloid ?? peptides.     

Discovery of novel 5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridine derivatives as γ-secretase modulators (Part 2)

γ-Secretase modulators (GSMs), which lower pathogenic amyloid beta (Aβ) without affecting the production of total Aβ or Notch signal, have emerged as a potential therapeutic agent for Alzheimer’s disease (AD). A novel series of 5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyridine derivatives was discovered and characterized as GSMs. Optimization of substituents at the 8-position of the core scaffold using ligand-lipophilicity efficiency (LLE) as a drug-likeness guideline led to identification of various types of high-LLE GSMs. Phenoxy compound (R)-17 exhibited especially high LLE as well as potent in vivo Aβ₄₂-lowering effect by single administration. Furthermore, multiple oral administration of (R)-17 significantly reduced soluble and insoluble brain Aβ₄₂, and ameliorated cognitive deficit in novel object recognition test (NORT) using Tg2576 mice as an AD model.