Insights into antiamyloidogenic properties of the green tea extract (-)-epigallocatechin-3-gallate toward metal-associated amyloid-ß species.

Despite the significance of Alzheimer's disease, the link between metal-associated amyloid-ß (metal-Aß) and disease etiology remains unclear. To elucidate this relationship, chemical tools capable of specifically targeting and modulating metal-Aß species are necessary, along with a fundamental understanding of their mechanism at the molecular level. Herein, we investigated and compared the interactions and reactivities of the green tea extract, (-)-epigallocatechin-3-gallate [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate; EGCG], with metal [Cu(II) and Zn(II)]-Aß and metal-free Aß species. We found that EGCG interacted with metal-Aß species and formed small, unstructured Aß aggregates more noticeably than in metal-free conditions in vitro. In addition, upon incubation with EGCG, the toxicity presented by metal-free Aß and metal-Aß was mitigated in living cells. To understand this reactivity at the molecular level, structural insights were obtained by ion mobility-mass spectrometry (IM-MS), 2D NMR spectroscopy, and computational methods. These studies indicated that (i) EGCG was bound to Aß monomers and dimers, generating more compact peptide conformations than those from EGCG-untreated Aß species; and (ii) ternary EGCG-metal-Aß complexes were produced. Thus, we demonstrate the distinct antiamyloidogenic reactivity of EGCG toward metal-Aß species with a structure-based mechanism.

The ongoing search for small molecules to study metal-associated amyloid-ß species in Alzheimer's disease.

The development of a cure for Alzheimer's disease (AD) has been impeded by an inability to pinpoint the root cause of this disorder. Although numerous potential pathological factors have been indicated, acting either individually or mutually, the molecular mechanisms leading to disease onset and progression have not been clear. Amyloid-ß (Aß), generated from proteolytic processing of the amyloid precursor protein (APP), and its aggregated forms, particularly oligomers, are suggested as key pathological features in AD-affected brains. Historically, highly concentrated metals are found colocalized within Aß plaques. Metal binding to Aß (metal-Aß) generates/stabilizes potentially toxic Aß oligomers, and produces reactive oxygen species (ROS) in vitro (redox active metal ions; plausible contribution to oxidative stress). Consequently, clarification of the relationship between Aß, metal ions, and toxicity, including oxidative stress via metal-Aß, can lead to a deeper understanding of AD development. To probe the involvement of metal-Aß in AD pathogenesis, rationally designed and naturally occurring molecules have been examined as chemical tools to target metal-Aß species, modulate the interaction between the metal and Aß, and subsequently redirect their aggregation into nontoxic, off-pathway unstructured aggregates. These ligands are also capable of attenuating the generation of redox active metal-Aß-induced ROS to mitigate oxidative stress. One rational design concept, the incorporation approach, installs a metal binding site into a framework known to interact with Aß. This approach affords compounds with the simultaneous ability to chelate metal ions and interact with Aß. Natural products capable of Aß interaction have been investigated for their influence on metal-induced Aß aggregation and have inspired the construction of synthetic analogues. Systematic studies of these synthetic or natural molecules could uncover relationships between chemical structures, metal/Aß/metal-Aß interactions, and inhibition of Aß/metal-Aß reactivity (i.e., aggregation modes of Aß/metal-Aß; associated ROS production), suggesting mechanisms to refine the design strategy. Interdisciplinary investigations have demonstrated that the designed molecules and natural products control the aggregation pathways of metal-Aß species transforming their size/conformation distribution. The aptitude of these molecules to impact metal-Aß aggregation pathways, either via inhibition of Aß aggregate formation, most importantly of oligomers, or disaggregation of preformed fibrils, could originate from their formation of complexes with metal-Aß. Potentially, these molecules could direct metal-Aß size/conformational states into alternative nontoxic unstructured oligomers, and control the geometry at the Aß-ligated metal center for limited ROS formation to lessen the overall toxicity induced by metal-Aß. Complexation between small molecules and Aß/metal-Aß has been observed by nuclear magnetic resonance spectroscopy (NMR) and ion mobility-mass spectrometry (IM-MS) pointing to molecular level interactions, validating the design strategy. In addition, these molecules exhibit other attractive properties, such as antioxidant capacity, prevention of ROS production, potential blood-brain barrier (BBB) permeability, and reduction of Aß-/metal-Aß-induced cytotoxicity, making them desirable tools for unraveling AD complexity. In this Account, we summarize the recent development of small molecules, via both rational design and the selection and modification of natural products, as tools for investigating metal-Aß complexes, to advance our understanding of their relation to AD pathology.

Abnormal metal levels in the primary visual pathway of the DBA/2J mouse model of glaucoma.

The purpose of this study was to determine metal ion levels in central visual system structures of the DBA/2J mouse model of glaucoma. We used inductively coupled plasma mass spectrometry (ICP-MS) to measure levels of iron (Fe), copper (Cu), zinc (Zn), magnesium (Mg), manganese (Mn), and calcium (Ca) in the retina and retinal projection of 5-month (pre-glaucomatous) and 10-month (glaucomatous) old DBA/2J mice and age-matched C57BL/6J controls. We used microbeam X-ray fluorescence (µ-XRF) spectrometry to determine the spatial distribution of Fe, Zn, and Cu in the superior colliculus (SC), which is the major retinal target in rodents and one of the earliest sites of pathology in the DBA/2J mouse. Our ICP-MS experiments showed that glaucomatous DBA/2J had lower retinal Fe concentrations than pre-glaucomatous DBA/2J and age-matched C57BL/6J mice. Pre-glaucomatous DBA/2J retina had greater Mg, Ca, and Zn concentrations than glaucomatous DBA/2J and greater Mg and Ca than age-matched controls. Retinal Mn levels were significantly deficient in glaucomatous DBA/2J mice compared to aged-matched C57BL/6J and pre-glaucomatous DBA/2J mice. Regardless of age, the SC of C57BL/6J mice contained greater Fe, Mg, Mn, and Zn concentrations than the SC of DBA/2J mice. Greater Fe concentrations were measured by µ-XRF in both the superficial and deep SC of C57BL/6J mice than in DBA/2J mice. For the first time, we show direct measurement of metal concentrations in central visual system structures affected in glaucoma and present evidence for strain-related differences in metal content that may be specific to glaucomatous pathology.

Amyloid-ß-neuropeptide interactions assessed by ion mobility-mass spectrometry.

Recently, small peptides have been shown to modulate aggregation and toxicity of the amyloid-ß protein (Aß). As such, these new scaffolds may help discover a new class of biotherapeutics useful in the treatment of Alzheimer's disease. Many of these inhibitory peptide sequences have been derived from natural sources or from Aß itself (e.g., C-terminal Aß fragments). In addition, much earlier work indicates that tachykinins, a broad class of neuropeptides, display neurotrophic properties, presumably through direct interactions with either Aß or its receptors. Based on this work, we undertook a limited screen of neuropeptides using ion mobility-mass spectrometry to search for similar such peptides with direct Aß binding properties. Our results reveal that the neuropeptides leucine enkephalin (LE) and galanin interact with both the monomeric and small oligomeric forms of Aß(1-40) to create a range of complexes having diverse stoichiometries, while some tachyknins (i.e., substance P) do not. LE interacts with Aß more strongly than galanin, and we utilized ion mobility-mass spectrometry, molecular dynamics simulations, gel electrophoresis/Western blot, and transmission electron microscopy to study the influence of this peptide on the structure of Aß monomer, small Aß oligomers, as well as the eventual formation of Aß fibrils. We find that LE binds selectively within a region of Aß between its N-terminal tail and hydrophobic core. Furthermore, our data indicate that LE modulates fibril generation, producing shorter fibrillar aggregates when added in stoichiometric excess relative to Aß.

Misfolded proteins in Alzheimer's disease and type II diabetes.

This tutorial review presents descriptions of two amyloidogenic proteins, amyloid-ß (Aß) peptides and islet amyloid polypeptide (IAPP), whose misfolding propensities are implicated in Alzheimer's disease (AD) and type II diabetes, respectively. Protein misfolding diseases share similarities, as well as some unique protein-specific traits, that could contribute to the initiation and/or development of their associated conditions. Aß and IAPP are representative amyloidoses and are used to highlight some of the primary considerations for studying misfolded proteins associated with human diseases in this review. Among these factors, their physiological formation, aggregation, interactions with metal ions and other protein partners, and toxicity are presented. Small molecules that target and modulate the metal-Aß interaction and neurotoxicity are included to illustrate one of the current approaches for uncovering the complexities of protein misfolding at the molecular level.

Reactivity of diphenylpropynone derivatives toward metal-associated amyloid-ß species.

In Alzheimer's disease (AD), metal-associated amyloid-ß (metal-Aß) species have been suggested to be involved in neurotoxicity; however, their role in disease development is still unclear. To elucidate this aspect, chemical reagents have been developed as valuable tools for targeting metal-Aß species, modulating the interaction between the metal and Aß, and subsequently altering metal-Aß reactivity. Herein, we report the design, preparation, characterization, and reactivity of two diphenylpropynone derivatives (DPP1 and DPP2) composed of structural moieties for metal chelation and Aß interaction (bifunctionality). The interactions of these compounds with metal ions and Aß species were confirmed by UV-vis, NMR, mass spectrometry, and docking studies. The effects of these bifunctional molecules on the control of in vitro metal-free and metal-induced Aß aggregation were investigated and monitored by gel electrophoresis and transmission electron microscopy (TEM). Both DPP1 and DPP2 showed reactivity toward metal-Aß species over metal-free Aß species to different extents. In particular, DPP2, which contains a dimethylamino group, exhibited greater reactivity with metal-Aß species than DPP1, suggesting a structure-reactivity relationship. Overall, our studies present a new bifunctional scaffold that could be utilized to develop chemical reagents for investigating metal-Aß species in AD.

Development of bifunctional stilbene derivatives for targeting and modulating metal-amyloid-ß species.

Amyloid-ß (Aß) peptides and their metal-associated aggregated states have been implicated in the pathogenesis of Alzheimer's disease (AD). Although the etiology of AD remains uncertain, understanding the role of metal-Aß species could provide insights into the onset and development of the disease. To unravel this, bifunctional small molecules that can specifically target and modulate metal-Aß species have been developed, which could serve as suitable chemical tools for investigating metal-Aß-associated events in AD. Through a rational structure-based design principle involving the incorporation of a metal binding site into the structure of an Aß interacting molecule, we devised stilbene derivatives (L1-a and L1-b) and demonstrated their reactivity toward metal-Aß species. In particular, the dual functions of compounds with different structural features (e.g., with or without a dimethylamino group) were explored by UV-vis, X-ray crystallography, high-resolution 2D NMR, and docking studies. Enhanced bifunctionality of compounds provided greater effects on metal-induced Aß aggregation and neurotoxicity in vitro and in living cells. Mechanistic investigations of the reaction of L1-a and L1-b with Zn(2+)-Aß species by UV-vis and 2D NMR suggest that metal chelation with ligand and/or metal-ligand interaction with the Aß peptide may be driving factors for the observed modulation of metal-Aß aggregation pathways. Overall, the studies presented herein demonstrate the importance of a structure-interaction-reactivity relationship for designing small molecules to target metal-Aß species allowing for the modulation of metal-induced Aß reactivity and neurotoxicity.

Use of 73Ge NMR spectroscopy for the study of electronic interactions.

The lack of understanding of the structural and electronic factors that affect the often difficult to observe germanium resonance has been a major deterrent to studies of bonding interactions at germanium. We utilized the symmetrical system GeR 4 to determine what structural factors inherent in the R group affect the shape and position of the (73)Ge resonance. The (73)Ge resonances of symmetrical tetrakis germanium compounds of the type GeR 4 (R = alkyl, aryl), GeX 4 (X = F, Cl, Br, I), Ge(OR) 4 (R = alkyl, methoxyalkyl, dimethylaminoalkyl), Ge(NR 2) 4 (R = alkyl), and Ge(SR) 4 (R = alkyl, dimethylaminoalkyl) were examined for evidence of intramolecular coordination. Although many of these compounds have sharp resonances due to idealized tetrahedral symmetry with relatively long relaxation times, others have broad or no observable resonances due to fast quadrupolar relaxation. We hypothesize that the perturbation of symmetry by even weak Lewis interactions or conformational changes causes broadening of the resonance before the interaction can become sufficiently strong to cause the significant low-frequency shift generally associated with hypercoordination in most nuclei. Intermolecular coordination to GeCl 4 is believed to be responsible for the low-frequency shifts in (73)Ge resonances and the associated changes in peak widths in mixtures with bases such as tributylphosphine oxide (TBPO) and triethylphosphine oxide (TEPO). Adduct formation with these bases is confirmed by broad (31)P resonances that are resolved into five peaks at -40 degrees C. The exchange-broadened resonances due to the 1:1 and 1:2 TEPO adducts are also observed at -40 degrees C in the (73)Ge spectrum. Thus, relatively strong bonding to the germanium in GeCl 4 results in both low-frequency shifts and broadening of the resonance. The broad (73)Ge resonances that occur in some compounds may be in part due to exchange as well as quadrupolar relaxation.

Synthetic Flavonoids, Aminoisoflavones: Interaction and Reactivity with Metal-Free and Metal-Associated Amyloid-ß Species.

Metal ion homeostasis in conjunction with amyloid-ß (Aß) aggregation in the brain has been implicated in Alzheimer's disease (AD) pathogenesis. To uncover the interplay between metal ions and Aß peptides, synthetic, multifunctional small molecules have been employed to modulate Aß aggregation in vitro. Naturally occurring flavonoids have emerged as a valuable class of compounds for this purpose due to their ability to modulate both metal-free and metal-induced Aß aggregation. Although, flavonoids have shown anti-amyloidogenic effects, the structural moieties of flavonoids responsible for such reactivity have not been fully identified. In order to understand the structure-interaction-reactivity relationship within the flavonoid family for metal-free and metal-associated Aß, we designed, synthesized, and characterized a set of isoflavone derivatives, aminoisoflavones (1-4), that displayed reactivity (i.e., modulation of Aß aggregation) in vitro. NMR studies revealed a potential binding site for aminoisoflavones between the N-terminal loop and central helix on prefibrillar Aß different from the non-specific binding observed for other flavonoids. The absence or presence of the catechol group differentiated the binding affinities and enthalpy/entropy balance between aminoisoflavones and Aß. Furthermore, having a catechol group influenced the binding mode with fibrillar Aß. Inclusion of additional substituents moderately tuned the impact of aminoisoflavones on Aß aggregation. Overall, through these studies, we obtained valuable insights on the requirements for parity among metal chelation, intermolecular interactions, and substituent variation for Aß interaction.

Exploring the reactivity of flavonoid compounds with metal-associated amyloid-ß species.

Metal ions associated with amyloid-ß (Aß) peptides have been suggested to be involved in the development of Alzheimer's disease (AD), but this remains unclear and controversial. Some attempts to rationally design or select small molecules with structural moieties for metal chelation and Aß interaction (i.e., bifunctionality) have been made to gain a better understanding of the hypothesis. In order to contribute to these efforts, four synthetic flavonoid derivatives FL1-FL4 were rationally selected according to the principles of bifunctionality and their abilities to chelate metal ions, interact with Aß, inhibit metal-induced Aß aggregation, scavenge radicals, and regulate the formation of reactive oxygen species (ROS) were studied using physical methods and biological assays. The compounds FL1-FL3 were able to chelate metal ions, but showed limited solubility in aqueous buffered solutions. In the case of FL4, which was most compatible with aqueous conditions, its binding affinities for Cu(2+) and Zn(2+) (nM and µM, respectively) were obtained through solution speciation studies. The direct interaction between FL4 and Aß monomer was weak, which was monitored by NMR spectroscopy and mass spectrometry. Employing FL1-FL4, no noticeable inhibitory effect on metal-mediated Aß aggregation was observed. Among FL1-FL4, FL3, having 3-OH, 4-oxo, and 4'-N(CH(3))(2) groups, exhibited similar antioxidant activity to the vitamin E analogue, Trolox, and ca. 60% reduction in the amount of hydrogen peroxide (H(2)O(2)) generated by Cu(2+)-Aß in the presence of dioxygen (O(2)) and a reducing agent. Overall, the studies here suggest that although four flavonoid molecules were selected based on expected bifunctionality, their properties and metal-Aß reactivity were varied depending on the structure differences, demonstrating that bifunctionality must be well tuned to afford desirable reactivity.

Dual-function triazole-pyridine derivatives as inhibitors of metal-induced amyloid-ß aggregation.

Dysregulated metal ions are hypothesized to play a role in the aggregation of the amyloid-ß (Aß) peptide, leading to Alzheimer's disease (AD) pathology. In addition to direct effects on Aß aggregation, both Cu and Fe can catalyze the generation of reactive oxygen species (ROS), possibly contributing to significant neuronal toxicity. Therefore, disruption of metal-Aß interactions has become a viable strategy for AD therapeutic development. Herein, we report a new series of dual-function triazole-pyridine ligands [4-(2-(4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)ethyl)morpholine (L1), 3-(4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)propan-1-ol (L2), 2-(4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)acetic acid (L3), and 5-(4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)pentan-1-amine (L4)] that interact with the Aß peptide and modulate its aggregation in vitro. Metal chelation and Aß interaction properties of these molecules were studied by UV-vis, NMR spectroscopy and X-ray crystallography. In addition, turbidity and transmission electron microscopy (TEM) were employed to determine the anti-aggregation properties of L1-L4. All compounds demonstrated an ability to limit metal-induced Aß aggregation. Overall, our studies suggest the utility of the triazole-pyridine framework in the development of chemical reagents toward inhibitors for metal-triggered Aß aggregation.

Recent Development of Bifunctional Small Molecules to Study Metal-Amyloid-ß Species in Alzheimer's Disease.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease related to the deposition of aggregated amyloid-ß (Aß) peptides in the brain. It has been proposed that metal ion dyshomeostasis and miscompartmentalization contribute to AD progression, especially as metal ions (e.g., Cu(II) and Zn(II)) found in Aß plaques of the diseased brain can bind to Aß and be linked to aggregation and neurotoxicity. The role of metal ions in AD pathogenesis, however, is uncertain. To accelerate understanding in this area and contribute to therapeutic development, recent efforts to devise suitable chemical reagents that can target metal ions associated with Aß have been made using rational structure-based design that combines two functions (metal chelation and Aß interaction) in the same molecule. This paper presents bifunctional compounds developed by two different design strategies (linkage or incorporation) and discusses progress in their applications as chemical tools and/or potential therapeutics.