Novel drug-like fluorenyl derivatives as selective butyrylcholinesterase and ß-amyloid inhibitors for the treatment of Alzheimer's disease.

Butyrylcholinesterase (BuChE) and amyloid ß (Aß) aggregation remain important biological target and mechanism in the search for effective treatment of Alzheimer's disease. Simultaneous inhibition thereof by the application of multifunctional agents may lead to improvement in terms of symptoms and causes of the disease. Here, we present the rational design, synthesis, biological evaluation and molecular modelling studies of novel series of fluorene-based BuChE and Aß inhibitors with drug-like characteristics and advantageous Central Nervous System Multiparameter Optimization scores. Among 17 synthesized and tested compounds, we identified 22 as the most potent eqBuChE inhibitor with IC50 of 38 nM and 37.4% of Aß aggregation inhibition at 10 µM. Based on molecular modelling studies, including molecular dynamics, we determined the binding mode of the compounds within BuChE and explained the differences in the activity of the two enantiomers of compound 22. A novel series of fluorenyl compounds meeting the drug-likeness criteria seems to be a promising starting point for further development as anti-Alzheimer agents.

New cyclopentaquinoline and 3,5-dichlorobenzoic acid hybrids with neuroprotection against oxidative stress for the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative brain disease. Thus, drugs including donepezil, rivastigmine, and galantamine are not entirely effective in the treatment of this multifactorial disease. The present study evaluates eight derivatives (3a-3h) as candidates with stronger anti-AD potential but with less side effects. Reactive oxygen species (ROS) assays were used to assess oxidative stress which involve in the neurodegeneration. The neuroprotective properties of 3e against oxidative stress were done in three experiments using MTT test. The anti-AD potential was determined based on their anticholinesterase inhibition ability, determined using Ellman's method, Aß aggregation potential according to thioflavin (Th) fluorescence assay, and their antioxidative and anti-inflammatory activities. Compound 3e exhibited moderate cholinesterase inhibition activity (AChE, IC50 = 0.131 µM; BuChE, IC50 = 0.116 µM; SI = 1.13), significant inhibition of Aß(1-42) aggregation (55.7%, at 5 µM) and acceptable neuroprotective activity. Extensive analysis of in vitro and in vivo assays indicates that new cyclopentaquinoline derivatives offer promise as candidates for new anti-AD drugs.

Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies.

The resolution of the three-dimensional structure of infectious prions at the atomic level is pivotal to understand the pathobiology of Transmissible Spongiform Encephalopathies (TSE), but has been long hindered due to certain particularities of these proteinaceous pathogens. Difficulties related to their purification from brain homogenates of disease-affected animals were resolved almost a decade ago by the development of in vitro recombinant prion propagation systems giving rise to highly infectious recombinant prions. However, lack of knowledge about the molecular mechanisms of the misfolding event and the complexity of systems such as the Protein Misfolding Cyclic Amplification (PMCA), have limited generating the large amounts of homogeneous recombinant prion preparations required for high-resolution techniques such as solid state Nuclear Magnetic Resonance (ssNMR) imaging. Herein, we present a novel recombinant prion propagation system based on PMCA that substitutes sonication with shaking thereby allowing the production of unprecedented amounts of multi-labeled, infectious recombinant prions. The use of specific cofactors, such as dextran sulfate, limit the structural heterogeneity of the in vitro propagated prions and makes possible, for the first time, the generation of infectious and likely homogeneous samples in sufficient quantities for studies with high-resolution structural techniques as demonstrated by the preliminary ssNMR spectrum presented here. Overall, we consider that this new method named Protein Misfolding Shaking Amplification (PMSA), opens new avenues to finally elucidate the three-dimensional structure of infectious prions.

Azobioisosteres of Curcumin with Pronounced Activity against Amyloid Aggregation, Intracellular Oxidative Stress, and Neuroinflammation.

Many (poly-)phenolic natural products, for example, curcumin and taxifolin, have been studied for their activity against specific hallmarks of neurodegeneration, such as amyloid-ß 42 (Aß42) aggregation and neuroinflammation. Due to their drawbacks, arising from poor pharmacokinetics, rapid metabolism, and even instability in aqueous medium, the biological activity of azobenzene compounds carrying a pharmacophoric catechol group, which have been designed as bioisoteres of curcumin has been examined. Molecular simulations reveal the ability of these compounds to form a hydrophobic cluster with Aß42, which adopts different folds, affecting the propensity to populate fibril-like conformations. Furthermore, the curcumin bioisosteres exceeded the parent compound in activity against Aß42 aggregation inhibition, glutamate-induced intracellular oxidative stress in HT22 cells, and neuroinflammation in microglial BV-2 cells. The most active compound prevented apoptosis of HT22 cells at a concentration of 2.5 µm (83 % cell survival), whereas curcumin only showed very low protection at 10 µm (21 % cell survival).

Dual Effect of Prussian Blue Nanoparticles on Aß40 Aggregation: ß-Sheet Fibril Reduction and Copper Dyshomeostasis Regulation.

Alzheimer's disease (AD), affecting almost 50 million individuals worldwide, is currently the first cause of dementia. Despite the tremendous research efforts in the last decade, only four supportive or palliative drugs, namely, acetylcholinesterase (AChE) inhibitors donepezil, galantamine, and rivastigmine and the glutamate NMDA receptor antagonist memantine, are currently available. New therapeutic strategies are becoming prominent, such as the direct inhibition of amyloid formation or the regulation of metal homeostasis. In the present report, the potential use of Prussian blue (PB), a drug that is in the World Health Organization Model List of Essential Medicines, in AD treatment is demonstrated. Both in vitro and in cellulo studies indeed suggest that PB nanoparticles (PBNPs) are capable of reducing the formation of typical amyloid-ß fibers (detected by thioflavin T fluorescence) and restoring the usual amyloid fibrillation pathway via chelation/sequestration of copper, which is found in high concentrations in senile plaques.

Peptidic Scaffolds To Reduce the Interaction of Cu(II) Ions with ß-Amyloid Protein.

Competitive Cu(II)-binding studies have been carried out between five decapeptides (both acyclic and cyclic), namely C-Asp, C-Asn, O-Asp, ODPro-Asp, and O-Asn, and the Aß(1-16) and Aß(1-40) fragments. Conformational constraints in such peptidic scaffolds affect their copper-binding affinity, which can be tuned. In the present study, the ability of these peptides to compete with Aß has been assessed in vitro, with the objective to examine whether such soft chelating agents may be used to lessen the deleterious interaction of Cu(II) with Aß. Fluorescence spectroscopy, electron paramagnetic resonance, and mass spectrometry data show that the more constrained peptide, i.e., cyclic C-Asp, which displays a Cu(II)-binding affinity comparable to that of Aß, is the only potential metal-protein attenuating compound (MPAC) candidate. In vitro aggregation studies with Aß(1-40) reveal that C-Asp can hamper the formation of copper-stabilized oligomeric Aß species, through capturing the metal ion prior to its interaction with monomeric Aß. The present study shows that (cyclic) peptides, preorganized for Cu(II) binding, may be applied for the development of potential copper-Aß attenuating compounds.

Thiosemicarbazone Derivatives as Inhibitors of Amyloid-ß Aggregation: Effect of Metal Coordination.

Three thiosemicarbazone derivatives, namely 4-(dimethylamino)benzaldehyde 4,4-dimethylthiosemicarbazone (HL1), 4-(dimethylamino)benzaldehyde thiosemicarbazone (HL2), and 4-(dimethylamino)benzaldehyde 4-methylthiosemicarbazone (HL3), have been synthesized and characterized. The three palladium(II) complexes 1-3 were prepared respectively from HL1, HL2, and HL3. The crystal structures of two coordination compounds, namely Pd(L2)2 (2) and Pd(L3)2 (3), were obtained, which showed the expected square-planar environment for the metal centers. The ligand HL3 and the Pd(II) complexes 1-3, which are stable in buffered solutions containing up to 5% DMSO, exhibit remarkable inhibitory properties against the aggregation of amyloid-ß, reducing the formation of fibrils. HL1, HL3, 2, and 3 display IC50 values (i.e., the concentrations required to reduce Aß fibrillation by 50%) below 1 µM, lower that of the reference compound catechin (IC50 = 2.8 µM). Finally, in cellulo studies with E. coli cells revealed that the palladium(II) compounds are significantly more efficient than the free ligands in inhibiting Aß aggregation inside bacterial inclusion bodies, thus illustrating a beneficial effect of metal coordination.

Multidirectional in vitro and in cellulo studies as a tool for identification of multi-target-directed ligands aiming at symptoms and causes of Alzheimer's disease.

Effective therapy of Alzheimer's disease (AD) requires treatment with a combination of drugs that modulate various pathomechanisms contributing to the disease. In our research, we have focused on the development of multi-target-directed ligands - 5-HT6 receptor antagonists and cholinesterase inhibitors - with disease-modifying properties. We have performed extended in vitro (FRET assay) and in cellulo (Escherichia coli model of protein aggregation) studies on their ß-secretase, tau, and amyloid ß aggregation inhibitory activity. Within these multifunctional ligands, we have identified compound 17 with inhibitory potency against tau and amyloid ß aggregation in in cellulo assay of 59% and 56% at 10 µM, respectively, hBACE IC50=4 µM, h5TH6 K i=94 nM, hAChE IC50=26 nM, and eqBuChE IC50=5 nM. This study led to the development of multifunctional ligands with a broad range of biological activities crucial not only for the symptomatic but also for the disease-modifying treatment of AD.

Pharmacophore Modeling and 3D-QSAR Study of Indole and Isatin Derivatives as Antiamyloidogenic Agents Targeting Alzheimer's Disease.

Thirty-six novel indole-containing compounds, mainly 3-(2-phenylhydrazono) isatins and structurally related 1H-indole-3-carbaldehyde derivatives, were synthesized and assayed as inhibitors of beta amyloid (Aß) aggregation, a hallmark of pathophysiology of Alzheimer's disease. The newly synthesized molecules spanned their IC50 values from sub- to two-digit micromolar range, bearing further information into structure-activity relationships. Some of the new compounds showed interesting multitarget activity, by inhibiting monoamine oxidases A and B. A cell-based assay in tau overexpressing bacterial cells disclosed a promising additional activity of some derivatives against tau aggregation. The accumulated data of either about ninety published and thirty-six newly synthesized molecules were used to generate a pharmacophore hypothesis of antiamyloidogenic activity exerted in a wide range of potencies, satisfactorily discriminating the 'active' compounds from the 'inactive' (poorly active) ones. An atom-based 3D-QSAR model was also derived for about 80% of 'active' compounds, i.e., those achieving finite IC50 values lower than 100 µM. The 3D-QSAR model (encompassing 4 PLS factors), featuring acceptable predictive statistics either in the training set (n = 45, q2 = 0.596) and in the external test set (n = 14, r2ext = 0.695), usefully complemented the pharmacophore model by identifying the physicochemical features mainly correlated with the Aß anti-aggregating potency of the indole and isatin derivatives studied herein.

On the Binding of Congo Red to Amyloid Fibrils.

Amyloids are characterized by their capacity to bind Congo red (CR), one of the most used amyloid-specific dyes. The structural features of CR binding were unknown for years, mainly because of the lack of amyloid structures solved at high resolution. In the last few years, solid-state NMR spectroscopy enabled the determination of the structural features of amyloids, such as the HET-s prion forming domain (HET-s PFD), which also has recently been used to determine the amyloid-CR interface at atomic resolution. Herein, we combine spectroscopic data with molecular docking, molecular dynamics, and excitonic quantum/molecular mechanics calculations to examine and rationalize CR binding to amyloids. In contrast to a previous assumption on the binding mode, our results suggest that CR binding to the HET-s PFD involves a cooperative process entailing the formation of a complex with 1:1 stoichiometry. This provides a molecular basis to explain the bathochromic shift in the maximal absorbance wavelength when CR is bound to amyloids.

Copper, dityrosine cross-links and amyloid-ß aggregation.

Copper is involved in Alzheimer's disease (AD) where it appears to affect the aggregation of amyloid-ß (Aß) and to catalyze the production of reactive oxygen species (ROS). Oxidative stress apparently produces Aß dimers that are covalently linked through two tyrosine residues. Such dityrosine cross-links are considered as potential markers of the disease and seem to be implicated in the pathological disorder. In the present study, pure o,o'-dityrosine (diY) was prepared enzymatically (with horseradish peroxidase; HRP), which was subsequently used to construct calibration lines aimed at quantifying nanomolar amounts of diY in reaction mixtures by fluorescence spectroscopy. Hence, diY concentrations down to 67 nM could be determined, which allowed to find that ca. 3% of dityrosine-bridged dimers of Aß(1-40) were produced after 3 days at 37 °C in the presence of copper and dihydrogen peroxide. These cross-linked dimers in the presence of copper(II) ions completely inhibit the typical aggregation of Aß, since ß sheets could not be detected applying the usual Thioflavin T (ThT) method. Furthermore, the use of a potent Cu(II) chelator, such as the ATCUN tripeptide, L-histidyl-L-alanyl-L-histidine (HAH), efficiently prevented the copper-mediated generation of ROS and the associated dityrosine-bridged Aß dimers, suggesting that such metal chelators may find future applications in the field of anti-AD drug design.

Identification of a novel cell death-inducing domain reveals that fungal amyloid-controlled programmed cell death is related to necroptosis.

Recent findings have revealed the role of prion-like mechanisms in the control of host defense and programmed cell death cascades. In fungi, HET-S, a cell death-inducing protein containing a HeLo pore-forming domain, is activated through amyloid templating by a Nod-like receptor (NLR). Here we characterize the HELLP protein behaving analogously to HET-S and bearing a new type of N-terminal cell death-inducing domain termed HeLo-like (HELL) and a C-terminal regulatory amyloid motif known as PP. The gene encoding HELLP is part of a three-gene cluster also encoding a lipase (SBP) and a Nod-like receptor, both of which display the PP motif. The PP motif is similar to the RHIM amyloid motif directing formation of the RIP1/RIP3 necrosome in humans. The C-terminal region of HELLP, HELLP(215-278), encompassing the motif, allows prion propagation and assembles into amyloid fibrils, as demonstrated by X-ray diffraction and FTIR analyses. Solid-state NMR studies reveal a well-ordered local structure of the amyloid core residues and a primary sequence that is almost entirely arranged in a rigid conformation, and confirm a ß-sheet structure in an assigned stretch of three amino acids. HELLP is activated by amyloid templating and displays membrane-targeting and cell death-inducing activity. HELLP targets the SBP lipase to the membrane, suggesting a synergy between HELLP and SBP in membrane dismantling. Remarkably, the HeLo-like domain of HELLP is homologous to the pore-forming domain of MLKL, the cell death-execution protein in necroptosis, revealing a transkingdom evolutionary relationship between amyloid-controlled fungal programmed cell death and mammalian necroptosis.

Signal transduction by a fungal NOD-like receptor based on propagation of a prion amyloid fold.

In the fungus Podospora anserina, the [Het-s] prion induces programmed cell death by activating the HET-S pore-forming protein. The HET-s ß-solenoid prion fold serves as a template for converting the HET-S prion-forming domain into the same fold. This conversion, in turn, activates the HET-S pore-forming domain. The gene immediately adjacent to het-S encodes NWD2, a Nod-like receptor (NLR) with an N-terminal motif similar to the elementary repeat unit of the ß-solenoid fold. NLRs are immune receptors controlling cell death and host defense processes in animals, plants and fungi. We have proposed that, analogously to [Het-s], NWD2 can activate the HET-S pore-forming protein by converting its prion-forming region into the ß-solenoid fold. Here, we analyze the ability of NWD2 to induce formation of the ß-solenoid prion fold. We show that artificial NWD2 variants induce formation of the [Het-s] prion, specifically in presence of their cognate ligands. The N-terminal motif is responsible for this prion induction, and mutations predicted to affect the ß-solenoid fold abolish templating activity. In vitro, the N-terminal motif assembles into infectious prion amyloids that display a structure resembling the ß-solenoid fold. In vivo, the assembled form of the NWD2 N-terminal region activates the HET-S pore-forming protein. This study documenting the role of the ß-solenoid fold in fungal NLR function further highlights the general importance of amyloid and prion-like signaling in immunity-related cell fate pathways.

Combined in Vitro Cell-Based/in Silico Screening of Naturally Occurring Flavonoids and Phenolic Compounds as Potential Anti-Alzheimer Drugs.

Alzheimer's disease (AD) is the main cause of dementia in people over 65 years. One of the major culprits in AD is the self-aggregation of amyloid-ß peptide (Aß), which has stimulated the search for small molecules able to inhibit Aß aggregation. In this context, we recently reported a simple, but effective in vitro cell-based assay to evaluate the potential antiaggregation activity of putative Aß aggregation inhibitors. In this work this assay was used together with docking and molecular dynamics simulations to analyze the anti-Aß aggregation activity of several naturally occurring flavonoids and phenolic compounds. The results showed that rosmarinic acid, melatonin, and o-vanillin displayed zero or low inhibitory capacity, curcumin was found to have an intermediate inhibitory potency, and apigenin and quercetin showed potent antiaggregation activity. Finally, the suitability of the combined in vitro cell-based/in silico approach to distinguish between active and inactive compounds was further assessed for an additional set of flavonols and dihydroflavonols.

PrionW: a server to identify proteins containing glutamine/asparagine rich prion-like domains and their amyloid cores.

Prions are a particular type of amyloids with the ability to self-perpetuate and propagate in vivo. Prion-like conversion underlies important biological processes but is also connected to human disease. Yeast prions are the best understood transmissible amyloids. In these proteins, prion formation from an initially soluble state involves a structural conversion, driven, in many cases, by specific domains enriched in glutamine/asparagine (Q/N) residues. Importantly, domains sharing this compositional bias are also present in the proteomes of higher organisms, thus suggesting that prion-like conversion might be an evolutionary conserved mechanism. We have recently shown that the identification and evaluation of the potency of amyloid nucleating sequences in putative prion domains allows discrimination of genuine prions. PrionW is a web application that exploits this principle to scan sequences in order to identify proteins containing Q/N enriched prion-like domains (PrLDs) in large datasets. When used to scan the complete yeast proteome, PrionW identifies previously experimentally validated prions with high accuracy. Users can analyze up to 10 000 sequences at a time, PrLD-containing proteins are identified and their putative PrLDs and amyloid nucleating cores visualized and scored. The output files can be downloaded for further analysis. PrionW server can be accessed at http://bioinf.uab.cat/prionw/.

Histidine-Rich Oligopeptides To Lessen Copper-Mediated Amyloid-ß Toxicity.

Brain copper imbalance plays an important role in amyloid-ß aggregation, tau hyperphosphorylation, and neurotoxicity observed in Alzheimer's disease (AD). Therefore, the administration of biocompatible metal-binding agents may offer a potential therapeutic solution to target mislocalized copper ions and restore metallostasis. Histidine-containing peptides and proteins are excellent metal binders and are found in many natural systems. The design of short peptides showing optimal binding properties represents a promising approach to capture and redistribute mislocalized metal ions, mainly due to their biocompatibility, ease of synthesis, and the possibility of fine-tuning their metal-binding affinities in order to suppress unwanted competitive binding with copper-containing proteins. In the present study, three peptides, namely HWH, HK(C) H, and HAH, have been designed with the objective of reducing copper toxicity in AD. These tripeptides form highly stable albumin-like complexes, showing higher affinity for Cu(II) than that of Aß(1-40). Furthermore, HWH, HK(C) H, and HAH act as very efficient inhibitors of copper-mediated reactive oxygen species (ROS) generation and prevent the copper-induced overproduction of toxic oligomers in the initial steps of amyloid aggregation in the presence of Cu(II) ions. These tripeptides, and more generally small peptides including the sequence His-Xaa-His at the N-terminus, may therefore be considered as promising motifs for the future development of new and efficient anti-Alzheimer drugs.

Natural Xanthones from Garcinia mangostana with Multifunctional Activities for the Therapy of Alzheimer's Disease.

Natural xanthones have diversity pharmacological activities. Here, a series of xanthones isolated from the pericarps of Garcinia mangostana Linn, named α-Mangostin, 8-Deoxygartanin, Gartanin, Garciniafuran, Garcinone C, Garcinone D, and γ-Mangostin were investigated. Biological screening performed in vitro and in Escherichia coli cells indicated that most of the xanthones exhibited significant inhibition of self-induced ß-amyloid (Aß) aggregation and also ß-site amyloid precursor protein-cleaving enzyme 1, acted as potential antioxidants and biometal chelators. Among these compounds, α-Mangostin, Gartanin, Garcinone C and γ-Mangostin showed better antioxidant properties to scavenge Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) free radical than Trolox, and potent neuroprotective effects against glutamate-induced HT22 cell death partly by up-regulating HO-1 protein level and then scavenging reactive oxygen species. Moreover, Gartanin, Garcinone C and γ-Mangostin could be able to penetrate the blood-brain barrier (BBB) in vitro. These findings suggest that the natural xanthones have multifunctional activities against Alzheimer's disease (AD) and could be promising compounds for the therapy of AD.

What makes a protein sequence a prion?

Typical amyloid diseases such as Alzheimer's and Parkinson's were thought to exclusively result from de novo aggregation, but recently it was shown that amyloids formed in one cell can cross-seed aggregation in other cells, following a prion-like mechanism. Despite the large experimental effort devoted to understanding the phenomenon of prion transmissibility, it is still poorly understood how this property is encoded in the primary sequence. In many cases, prion structural conversion is driven by the presence of relatively large glutamine/asparagine (Q/N) enriched segments. Several studies suggest that it is the amino acid composition of these regions rather than their specific sequence that accounts for their priogenicity. However, our analysis indicates that it is instead the presence and potency of specific short amyloid-prone sequences that occur within intrinsically disordered Q/N-rich regions that determine their prion behaviour, modulated by the structural and compositional context. This provides a basis for the accurate identification and evaluation of prion candidate sequences in proteomes in the context of a unified framework for amyloid formation and prion propagation.

Shogaol-huprine hybrids: dual antioxidant and anticholinesterase agents with ß-amyloid and tau anti-aggregating properties.

Multitarget compounds are increasingly being pursued for the effective treatment of complex diseases. Herein, we describe the design and synthesis of a novel class of shogaol-huprine hybrids, purported to hit several key targets involved in Alzheimer's disease. The hybrids have been tested in vitro for their inhibitory activity against human acetylcholinesterase and butyrylcholinesterase and antioxidant activity (ABTS.+, DPPH and Folin-Ciocalteu assays), and in intact Escherichia coli cells for their Aß42 and tau anti-aggregating activity. Also, their brain penetration has been assessed (PAMPA-BBB assay). Even though the hybrids are not as potent AChE inhibitors or antioxidant agents as the parent huprine Y and [4]-shogaol, respectively, they still exhibit very potent anticholinesterase and antioxidant activities and are much more potent Aß42 and tau anti-aggregating agents than the parent compounds. Overall, the shogaol-huprine hybrids emerge as interesting brain permeable multitarget anti-Alzheimer leads.

Amyloids in bacterial inclusion bodies.

Protein misfolding and aggregation into amyloid structures are associated with dozens of human diseases. Recent studies have provided compelling evidence for the existence of highly ordered, amyloid-like conformations in the insoluble inclusion bodies produced during heterologous protein expression in bacteria. Thus, amyloid aggregation seems to be an omnipresent process in both eukaryotic and prokaryotic organisms. Amyloid formation inside cell factories raises important safety concerns with regard to the toxicity and infectivity of recombinant proteins. Yet such findings also suggest that prokaryotic cells could be useful systems for studying how and why proteins aggregate in vivo, and they could also provide a biologically relevant background for screening therapeutic approaches to pathologic protein deposition.