Measurement of beta-amyloid peptides in specific cells using a photo thin-film transistor

The existence of beta-amyloid [Aβ] peptides in the brain has been regarded as the most archetypal biomarker of Alzheimer's disease [AD]. Recently, an early clinical diagnosis has been considered a great importance in identifying people who are at high risk of AD. However, no microscale electronic sensing devices for the detection of Aβ peptides have been developed yet. In this study, we propose an effective method to evaluate a small quantity of Aβ peptides labeled with fluorescein isothiocyanate [FITC] using a photosensitive field-effect transistor [p-FET] with an on-chip single-layer optical filter. To accurately evaluate the quantity of Aβ peptides within the cells cultured on the p-FET device, we measured the photocurrents which resulted from the FITC-conjugated Aβ peptides expressed from the cells and measured the number of photons of the fluorochrome in the cells using a photomultiplier tube. Thus, we evaluated the correlation between the generated photocurrents and the number of emitted photons. We also evaluated the correlation between the number of emitted photons and the amount of FITC by measuring the FITC volume using AFM. Finally, we estimated the quantity of Aβ peptides of the cells placed on the p-FET sensing area on the basis of the binding ratio between FITC molecules and Aβ peptides.     

Styryl-based compounds as potential in vivo imaging agents for beta-amyloid plaques

A group of styryl-based neutral compounds has been synthesized in this study for potential use as in vivo imaging agents for beta-amyloid plaques. Of 56 candidates, 14 compounds were found to label beta-amyloid plaques well on Alzheimer's disease (AD) human brain sections in vitro. The binding affinity to beta-amyloid fibrils was then determined by measuring the change in fluorescence intensity. Interestingly, we found that a class of quinaldine-styryl scaffold compounds displays specific binding to beta-amyloid fibrils. A representative compound, STB-8, was used in ex vivo and in vivo imaging experiments on an AD transgenic mouse model and demonstrated excellent blood-brain barrier (BBB) permeability and specific staining of the AD beta-amyloid plaques.     

Tacrine–Melatonin Hybrids as Multifunctional Agents for Alzheimer's Disease,with Cholinergic,Antioxidant, and Neuroprotective Properties

Tacrine–melatonin hybrids are potential multifunctional drugs for Alzheimer's disease that may simultaneously palliate intellectual deficits and protect the brain against both β‐amyloid peptide and oxidative stress. Molecular modeling studies show that they target both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. They are nontoxic and may be able to penetrate the CNS, according to in vitro PAMPA‐BBB assays.

     

6‐Methyluracil Derivatives as Bifunctional Acetylcholinesterase Inhibitors for the Treatment of Alzheimer's Disease

Novel 6‐methyluracil derivatives with ω‐(substituted benzylethylamino)alkyl chains at the nitrogen atoms of the pyrimidine ring were designed and synthesized. The numbers of methylene groups in the alkyl chains were varied along with the electron‐withdrawing substituents on the benzyl rings. The compounds are mixed‐type reversible inhibitors of cholinesterases, and some of them show remarkable selectivity for human acetylcholinesterase (hAChE), with inhibitory potency in the nanomolar range, more than 10 000‐fold higher than that for human butyrylcholinesterase (hBuChE). Molecular modeling studies indicate that these compounds are bifunctional AChE inhibitors, spanning the enzyme active site gorge and binding to its peripheral anionic site (PAS). In vivo experiments show that the 6‐methyluracil derivatives are able to penetrate the blood–brain barrier (BBB), inhibiting brain‐tissue AChE. The most potent AChE inhibitor, 3 d (1,3‐bis[5‐(o‐nitrobenzylethylamino)pentyl]‐6‐methyluracil), was found to improve working memory in scopolamine and transgenic APP/PS1 murine models of Alzheimer's disease, and to significantly decrease the number and area of β‐amyloid peptide plaques in the brain.     

Branched KLVFF tetramers strongly potentiate inhibition of beta-amyloid aggregation

The key pathogenic event in the onset of Alzheimer's disease (AD) is the aggregation of beta-amyloid (Abeta) peptides into toxic aggregates. Molecules that interfere with this process might act as therapeutic agents for the treatment of AD. The amino acid residues 16-20 (KLVFF) are known to be essential for the aggregation of Abeta. In this study, we have used a first-generation dendrimer as a scaffold for the multivalent display of the KLVFF peptide. The effect of four KLVFF peptides attached to the dendrimer (K(4)) on Abeta aggregation was compared to the effect of monomeric KLVFF (K(1)). Our data show that K(4) very effectively inhibits the aggregation of low-molecular-weight and protofibrillar Abeta(1-42) into fibrils, in a concentration-dependent manner, and much more potently than K(1). Moreover, we show that K(4) can lead to the disassembly of existing aggregates. Our data lead us to propose that conjugates that bear multiple copies of KLVFF might be useful as therapeutic agents for the treatment of Alzheimer's disease.     

The alpha-to-beta conformational transition of Alzheimer's Abeta-(1-42) peptide in aqueous media is reversible: a step by step conformational analysis suggests the location of beta conformation seeding

Current views of the role of beta-amyloid (Abeta) peptide fibrils range from regarding them as the cause of Alzheimer's pathology to having a protective function. In the last few years, it has also been suggested that soluble oligomers might be the most important toxic species. In all cases, the study of the conformational properties of Abeta peptides in soluble form constitutes a basic approach to the design of molecules with "antiamyloid" activity. We have experimentally investigated the conformational path that can lead the Abeta-(1-42) peptide from the native state, which is represented by an alpha helix embedded in the membrane, to the final state in the amyloid fibrils, which is characterized by beta-sheet structures. The conformational steps were monitored by using CD and NMR spectroscopy in media of varying polarities. This was achieved by changing the composition of water and hexafluoroisopropanol (HFIP). In the presence of HFIP, beta conformations can be observed in solutions that have very high water content (up to 99 % water; v/v). These can be turned back to alpha helices simply by adding the appropriate amount of HFIP. The transition of Abeta-(1-42) from alpha to beta conformations occurs when the amount of water is higher than 80 % (v/v). The NMR structure solved in HFIP/H2O with high water content showed that, on going from very apolar to polar environments, the long N-terminal helix is essentially retained, whereas the shorter C-terminal helix is lost. The complete conformational path was investigated in detail with the aid of molecular-dynamics simulations in explicit solvent, which led to the localization of residues that might seed beta conformations. The structures obtained might help to find regions that are more affected by environmental conditions in vivo. This could in turn aid the design of molecules able to inhibit fibril deposition or revert oligomerization processes.     

Role of Post-translational Modifications in Alzheimer's Disease

The global burden of Alzheimer's disease (AD) is growing. Valiant efforts to develop clinical candidates for treatment have continuously met with failure. Currently available palliative treatments are temporary and there is a constant need to search for reliable disease pathways, biomarkers and drug targets for developing diagnostic and therapeutic tools to address the unmet medical needs of AD. Challenges in drug-discovery efforts raise further questions about the strategies of current conventional diagnosis; drug design; and understanding of disease pathways, biomarkers and targets. In this context, post-translational modifications (PTMs) regulate protein trafficking, function and degradation, and their in-depth study plays a significant role in the identification of novel biomarkers and drug targets. Aberrant PTMs of disease-relevant proteins could trigger pathological pathways, leading to disease progression. Advancements in proteomics enable the generation of patterns or signatures of such modifications, and thus, provide a versatile platform to develop biomarkers based on PTMs. In addition, understanding and targeting the aberrant PTMs of various proteins provide viable avenues for addressing AD drug-discovery challenges. This review highlights numerous PTMs of proteins relevant to AD and provides an overview of their adverse effects on the protein structure, function and aggregation propensity that contribute to the disease pathology. A critical discussion offers suggestions of methods to develop PTM signatures and interfere with aberrant PTMs to develop viable diagnostic and therapeutic interventions in AD.     

Histone Deacetylase Inhibitors as Multitarget Ligands: New Players in Alzheimer's Disease Drug Discovery?

Histone deacetylase inhibitors (HDACIs) are responsible for controlling gene expression by modulating the acetylation status of histone proteins. Furthermore, they modulate the activity of cytoplasmic non-histone proteins. Due to the involvement of HDACs in neurodevelopment, memory formation, and cognitive processes, HDACIs have been suggested as innovative agents for the treatment of neurodegenerative disorders such as Alzheimer's disease (AD). Given their mechanisms of action and the complex nature of AD, HDACIs have been proposed for the design of novel multitarget ligands (MTLs). To this aim, the fragment responsible for HDAC inhibition has been coupled with other structures that are able to provide additional biological actions, such as antioxidant activity or the inhibition of phosphodiesterase 5, transglutaminase 2, and glycogen synthase kinase 3β. Herein we discuss recent efforts to design HDACI-based MTLs as potential disease-modifying entities.     

Turning Donepezil into a Multi-Target-Directed Ligand through a Merging Strategy

Thanks to the widespread use and safety profile of donepezil ( 1 ) in the treatment of Alzheimer's disease (AD), one of the most widely adopted multi-target-directed ligand (MTDL) design strategies is to modify its molecular structure by linking a second fragment carrying an additional AD-relevant biological property. Herein, supported by a proposed combination therapy of 1 and the quinone drug idebenone, we rationally designed novel 1 -based MTDLs targeting Aβ and oxidative pathways. By exploiting a bioisosteric replacement of the indanone core of 1 with a 1,4-naphthoquinone, we ended up with a series of highly merged derivatives, in principle devoid of the “physicochemical challenge” typical of large hybrid-based MTDLs. A preliminary investigation of their multi-target profile identified 9 , which showed a potent and selective butyrylcholinesterase inhibitory activity, together with antioxidant and antiaggregating properties. In addition, it displayed a promising drug-like profile.     

Tools of the Trade: Investigations into Design Strategies of Small Molecules to Target Components in Alzheimer's Disease

The growing prevalence of Alzheimer's disease (AD) has warranted the development of effective therapeutic methods. Current available drugs for AD (i.e., acetylcholinesterase (AChE) inhibitors and N‐methyl‐D ‐aspartate (NMDA) receptor antagonists) have only offered brief symptomatic relief. Considering that the numbers affected by AD are projected to substantially rise, long‐term strategies are urgently needed. The multiple series of small molecules to combat AD have been expanded, with current methods taking aim at factors, such as misfolded protein accumulation, metal ion dyshomeostasis, and oxidative stress. This concept article focuses on describing the design of compounds to target various components of AD and underlining recent advances that have been made.     

Ac‐LPFFD‐Th: A Trehalose‐Conjugated Peptidomimetic as a Strong Suppressor of Amyloid‐β Oligomer Formation and Cytotoxicity

The inhibition of amyloid formation is a promising therapeutic approach for the treatment of neurodegenerative diseases. Peptide‐based inhibitors, which have been widely investigated, are generally derived from original amyloid sequences. Most interestingly, trehalose, a nonreducing disaccharide of α‐glucose, is effective in preventing the aggregation of numerous proteins. We have determined that the development of hybrid compounds could provide new molecules with improved properties that might synergically increase the potency of their single moieties. In this work, the ability of Ac‐LPFFD‐Th, a C‐terminally trehalose‐conjugated derivative, to slow down the Aβ aggregation process was investigated by means of different biophysical techniques, including thioflavin T fluorescence, dynamic light scattering, ESI‐MS, and NMR spectroscopy. Moreover, we demonstrate that Ac‐LPFFD‐Th modifies the aggregation features of Aβ and protects neurons from Aβ oligomers' toxic insult.     

Novel Pyridine-Containing Sultones: Structure-Activity Relationship and Biological Evaluation as Selective AChE Inhibitors for the Treatment of Alzheimer's disease

Novel pyridine-containing sultones were synthesized and evaluated for their cholinesterase (ChE) inhibitory activity. Most of compounds showed selective acetylcholinesterase (AChE) inhibitory activity. The structure-activity relationship (SAR) showed: (i) the fused pyridine-containing sultones increase AChE inhibition, series B >series A ; (ii) for series A , the effect of the 4-substituent on AChE activity, p->m- or o-; (iii) for series B , a halophenyl group increase activity. Compound B4 (4-(4-chlorophenyl)-2,2-dioxide-3,4,5,6-tetrahydro-1,2-oxathiino[5,6-h]quinoline) was identified as a selective AChE inhibitor (IC₅₀=8.93 μM), and molecular docking studies revealed a good fit into TcAChE via hydrogen interactions between the δ-pyridylsultone scaffold with Asp72, Ser122, Phe288, Phe290 and Trp84. Compound B4 showed reversible and non-competitive (K_i=7.67 μM) AChE inhibition, nontoxicity and neuroprotective activity. In vivo studies confirmed that compound B4 could ameliorate the cognitive performance of scopolamine-treated C57BL/6 J mice, suggesting a significant benefit of AChE inhibition for a disease-modifying treatment of AD.     

An amphiphilic conjugate approach toward the design and synthesis of betulinic acid-polyphenol conjugates as inhibitors of the HIV-1 gp41 fusion core formation

Exploration of potent inhibitors of the HIV-1 gp41 fusion core formation is a promising strategy to discover small-molecule HIV-1 entry inhibitors for the treatment of HIV-1 infection. In this paper, a series of novel betulinic acid-polyphenol conjugates was designed, guided by molecular modeling of the binding of betulinic acid (BA) and phenolic galloyl/caffeoyl groups in the groove on the gp41 N-terminal heptad repeat (NHR) trimeric coiled coil. These conjugates were synthesized via conjugation of galloyl and caffeoyl groups with BA at the C-28 position. Their inhibitory activities of HIV gp41 six-helix bundle (6-HB) formation between the NHR peptide N36 and the C-terminal heptad repeat (CHR) peptide C34 were evaluated with size-exclusion HPLC. Conjugates bearing a galloyl group were found to exhibit four to sixfold higher inhibitory activities than that of parent compound BA, suggesting that they may be exploitable as HIV-1 fusion/entry inhibitors targeting gp41. The docking study on BA and its derivatives suggests that hydrophobic and hydrogen-bonding pockets exist in the groove of the gp41 NHR trimeric coiled coil and that a potent inhibitor should have amphiphilic structures to cooperatively interact with both pockets. This possibility was explored by incorporating both lipophilic and hydrophilic groups into the conjugates in a well-defined orientation to bind with both pockets in the gp41 NHR-trimer.     

Switch-peptides: design and characterization of controllable super-amyloid-forming host-guest peptides as tools for identifying anti-amyloid agents

Several amyloid-forming proteins are characterized by the presence of hydrophobic and highly amyloidogenic core sequences that play critical roles in the initiation and progression of amyloid fibril formation. Therefore targeting these sequences represents a viable strategy for identifying candidate molecules that could interfere with amyloid formation and toxicity of the parent proteins. However, the highly amyloidogenic and insoluble nature of these sequences has hampered efforts to develop high-throughput fibrillization assays. Here we describe the design and characterization of host-guest switch peptides that can be used for in vitro mechanistic and screening studies that are aimed at discovering aggregation inhibitors that target highly amyloidogenic sequences. These model systems are based on a host-guest system where the amyloidogenic sequence (guest peptide) is flanked by two beta-sheet-promoting (Leu-Ser)(n) oligomers as host sequences. Two host-guest peptides were prepared by using the hydrophobic core of Abeta comprising residues 14-24 (HQKLVFFAEDV) as the guest peptide with switch elements inserted within (peptide 1) or at the N and C termini of the guest peptide (peptide 2). Both model peptides can be triggered to undergo rapid self-assembly and amyloid formation in a highly controllable manner and their fibrillization kinetics is tuneable by manipulating solution conditions (for example, peptide concentration and pH). The fibrillization of both peptides reproduces many features of the full-length Abeta peptides and can be inhibited by known inhibitors of Abeta fibril formation. Our results suggest that this approach can be extended to other amyloid proteins and should facilitate the discovery of small-molecule aggregation inhibitors and the development of more efficacious anti-amyloid agents to treat and/or reverse the pathogenesis of neurodegenerative and systemic amyloid diseases.     

Design,Synthesis, and Biological Evaluation of Coumarin Derivatives Tethered to an Edrophonium‐like Fragment as Highly Potent and Selective Dual Binding Site Acetylcholinesterase Inhibitors

A large series of substituted coumarins linked through an appropriate spacer to 3‐hydroxy‐N,N‐dimethylanilino or 3‐hydroxy‐N,N,N‐trialkylbenzaminium moieties were synthesized and evaluated as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. The highest AChE inhibitory potency in the 3‐hydroxy‐N,N‐dimethylanilino series was observed with a 6,7‐dimethoxy‐3‐substituted coumarin derivative, which, along with an outstanding affinity (IC₅₀=0.236 nM ) exhibits excellent AChE/BChE selectivity (SI>300 000). Most of the synthesized 3‐hydroxy‐N,N,N‐trialkylbenzaminium salts display an AChE affinity in the sub‐nanomolar to picomolar range along with excellent AChE/BChE selectivities (SI values up to 138 333). The combined use of docking and molecular dynamics simulations permitted us to shed light on the observed structure–affinity and structure–selectivity relationships, to detect two possible alternative binding modes, and to assess the critical role of π–π stacking interactions in the AChE peripheral binding site.     

Spatiotemporal Control of Amyloid‐Like Aβ Plaque Formation Using a Multichannel Organic Electronic Device

We herein report on an iontronic device to drive and control Aβ1‐40 and Aβ1‐42 fibril formation. This system allows kinetic control of Aβ aggregation by regulation of H⁺ flows. The formed aggregates show both nanometer‐sized fibril structure and microscopic growth, thus mimicking senile plaques, at the H⁺‐outlet. Mechanistically we observed initial accumulation of Aβ1‐40 likely driven by electrophoretic migration which preceded nucleation of amyloid structures in the accumulated peptide cluster.

     

Selection of D-amino-acid peptides that bind to Alzheimer's disease amyloid peptide abeta1-42 by mirror image phage display

A mirror image phage display approach was used to identify novel and highly specific ligands for Alzheimer's disease amyloid peptide Abeta(1-42). A randomized 12-mer peptide library presented on M13 phages was screened for peptides with binding affinity for the mirror image of Abeta(1-42). After four rounds of selection and amplification the peptides were enriched with a dominating consensus sequence. The mirror image of the most representative peptide (D-pep) was shown to bind Abeta(1-42) with a dissociation constant in the submicromolar range. Furthermore, in brain tissue sections derived from patients that suffered from Alzheimer's disease, amyloid plaques and leptomeningeal vessels containing Abeta amyloid were stained specifically with a fluorescence-labeled derivative of D-pep. Fibrillar deposits derived from other amyloidosis were not labeled by D-pep. Possible applications of this novel and highly specific Abeta ligand in diagnosis and therapy of Alzheimer's disease are discussed.     

Structure–Activity Relationships and Binding Mode in the Human Acetylcholinesterase Active Site of Pseudo‐Irreversible Inhibitors Related to Xanthostigmine

Acetylcholinesterase inhibitors : We extended the AChE inhibitors SAR study to newly synthesized compounds based on the lead compound xantostigmine. Docking and molecular dynamics simulations were carried out to both define a new computational protocol, and to acquire a better understanding of the SAR data. These computations prompted us to evaluate two of the synthesized compounds as inhibitors of AChE‐induced Aβ aggregation.

     

Cholesterol: A Key in the Pathogenesis of Alzheimer's Disease

Herein we highlight recent advances in our understanding of the role of cholesterol in Alzheimer′s disease (AD). It has been proposed that cholesterol could enhance the risk of AD, and the interaction between cholesterol and amyloid‐β peptide 42 (Aβ42) has been studied extensively, yet until recently, the specific interaction mechanisms between them and how this affects Aβ42 aggregation had not yet been fully explored and had remained ambiguous. Vendruscolo and co‐workers addressed these issues in their recent article entitled “Cholesterol catalyses Aβ42 aggregation through a heterogeneous nucleation pathway in the presence of lipid membranes” (Habchi et al., Nat. Chem. 2018 , 10, 673). In this article, the authors revealed the mechanism behind cholesterol‐catalyzed Aβ42 aggregation, providing the potential to address the molecular origins of AD, thereby opening a new avenue for effective AD therapy.