Reactivity of Metal-Free and Metal-Associated Amyloid-ß with Glycosylated Polyphenols and Their Esterified Derivatives.

Both amyloid-ß (Aß) and transition metal ions are shown to be involved in the pathogenesis of Alzheimer's disease (AD), though the importance of their interactions remains unclear. Multifunctional molecules, which can target metal-free and metal-bound Aß and modulate their reactivity (e.g., Aß aggregation), have been developed as chemical tools to investigate their function in AD pathology; however, these compounds generally lack specificity or have undesirable chemical and biological properties, reducing their functionality. We have evaluated whether multiple polyphenolic glycosides and their esterified derivatives can serve as specific, multifunctional probes to better understand AD. The ability of these compounds to interact with metal ions and metal-free/-associated Aß, and further control both metal-free and metal-induced Aß aggregation was investigated through gel electrophoresis with Western blotting, transmission electron microscopy, UV-Vis spectroscopy, fluorescence spectroscopy, and NMR spectroscopy. We also examined the cytotoxicity of the compounds and their ability to mitigate the toxicity induced by both metal-free and metal-bound Aß. Of the polyphenols investigated, the natural product (Verbascoside) and its esterified derivative (VPP) regulate the aggregation and cytotoxicity of metal-free and/or metal-associated Aß to different extents. Our studies indicate Verbascoside represents a promising structure for further multifunctional tool development against both metal-free Aß and metal-Aß.

Inhibitory Activity Of Curcumin Derivatives Towards Metal-free And Metal-induced Amyloid-ß Aggregation.

When Alzheimer's disease (AD) progresses, several pathological features arise including accumulation of misfolded protein aggregates [e.g., amyloid-ß (Aß) plaques], metal ion dyshomeostasis, and oxidative stress. These characteristics are recently suggested to be interconnected through a potential factor, metal-associated Aß (metal-Aß) species. The role of metal-Aß species in AD pathogenesis remains unclear, however. To elucidate the contribution of metal-Aß species to AD pathology, as well as to develop small molecules as chemical tools and/or theranostic (therapeutic and diagnostic) agents for this disease, curcumin (Cur), a natural product from turmeric, and its derivatives have been studied towards both metal-free and metal-induced Aß aggregation. Although Cur has indicated anti-amyloidogenic activities and antioxidant properties, its biological use has been hindered due to low solubility and stability in physiologically relevant conditions. Herein, we report the reactivity of Cur and its derivatives (Gd-Cur, a potential multimodal Aß imaging agent; Cur-S, a water soluble derivative of Cur that has substitution at the phenolic hydroxyls) with metal-free Aß and metal-Aß species. Our results and observations indicate that Gd-Cur could modulate Cu(II)-triggered Aß aggregation more noticeably over metal-free or Zn(II)-induced analogues; however, Cur-S was not observed to noticeably modulate Aß aggregation with and without metal ions. Overall, our studies present information that could aid in optimizing the molecular scaffold of Cur for the development of chemical tools or theranostics for metal-Aß species.

Chelation-induced diradical formation as an approach to modulation of the amyloid-ß aggregation pathway.

Current approaches toward modulation of metal-induced Aß aggregation pathways involve the development of small molecules that bind metal ions, such as Cu(ii) and Zn(ii), and interact with Aß. For this effort, we present the enediyne-containing ligand (Z)-N,N'-bis[1-pyridin-2-yl-meth(E)-ylidene]oct-4-ene-2,6-diyne-1,8-diamine (PyED), which upon chelation of Cu(ii) and Zn(ii) undergoes Bergman-cyclization to yield diradical formation. The ability of this chelation-triggered diradical to modulate Aß aggregation is evaluated relative to the non-radical generating control pyridine-2-ylmethyl-(2-{[(pyridine-2-ylmethylene)-amino]-methyl}-benzyl)-amine (PyBD). Variable-pH, ligand UV-vis titrations reveal pKa = 3.81(2) for PyBD, indicating it exists mainly in the neutral form at experimental pH. Lipinski's rule parameters and evaluation of blood-brain barrier (BBB) penetration potential by the PAMPA-BBB assay suggest that PyED may be CNS+ and penetrate the BBB. Both PyED and PyBD bind Zn(ii) and Cu(ii) as illustrated by bathochromic shifts of their UV-vis features. Speciation diagrams indicate that Cu(ii)-PyBD is the major species at pH 6.6 with a nanomolar Kd, suggesting the ligand may be capable of interacting with Cu(ii)-Aß species. In the presence of Aß40/42 under hyperthermic conditions (43 °C), the radical-generating PyED demonstrates markedly enhanced activity (2-24 h) toward the modulation of Aß species as determined by gel electrophoresis. Correspondingly, transmission electron microscopy images of these samples show distinct morphological changes to the fibril structure that are most prominent for Cu(ii)-Aß cases. The loss of CO2 from the metal binding region of Aß in MALDI-TOF mass spectra further suggests that metal-ligand-Aß interaction with subsequent radical formation may play a role in the aggregation pathway modulation.

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.

Methyl Yellow: A Potential Drug Scaffold for Parkinson's Disease.

Parkinson's disease (PD) is an age-related neurodegenerative disease affecting movement. To date, there are no currently available therapeutic agents which can prevent or slow disease progression. Here, we evaluated an azobenzene derivative, methyl yellow (MY), as a potential drug scaffold for PD; its inhibitory activity toward monoamine oxidase B (MAO-B) as well as drug-like properties were investigated. The inhibitory effect of MY on MAO activity was determined by a MAO enzyme inhibition assay. In addition, the in vitro properties of MY as a drug candidate (e.g., blood-brain barrier (BBB) permeability, serum albumin binding, drug efflux through P-glycoprotein (P-gp), drug metabolism by P450, and mitochondrial toxicity) were examined. In vivo effectiveness of MY was also evaluated in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Parkinsonian mouse model. MY selectively inhibited MAO-B in a dose-dependent and reversible manner. MY was BBB-permeable, bound relatively weakly to serum albumin, was an unlikely substrate for both systems of P-gp and P450, and did not cause mitochondrial toxicity. Results from the MPTP Parkinsonian mouse model indicated that, upon treatment with MY, neurotoxicity induced by MPTP was mitigated. Investigations of MY demonstrate its inhibitory activity toward MAO-B, compliant properties for drug consideration, and its neuroprotective capability in the MPTP Parkinsonian mouse model. These data provide insights into potential use, optimization, and new design of azobenzene derivatives for PD treatment.

Cholesterol and metal ions in Alzheimer's disease.

Cholesterol and metal ions have been suggested to be associated with the onset and progression of Alzheimer's disease (AD). Moreover, recent findings have demonstrated a potential interconnection between these two factors. For example, (a) cholesterol has been shown to be misregulated in AD-afflicted brains, and the aberrant activity of proteins (particularly, apolipoprotein E (ApoE) and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGR)) has been linked to cholesterol-related AD exacerbation; (b) dyshomeostasis of metal ions associated with misfolded proteins (i.e., amyloid-ß (Aß) aggregates) found in the brains of AD patients is shown to promote oxidative stress leading to the malfunction of multiple proteins, including cytochrome c oxidase (CcO), and Cu/Zn superoxide dismutase (SOD1); (c) metal ion misregulation has also been observed to disrupt the activity of proteins (e.g., HMGR, low-density lipoproteins (LDL)), required for cholesterol production and regulation. Herein, we briefly discuss the potential involvement of cholesterol and metal ions in AD neuropathogenesis in both individual and interrelated manners.

Rational design of a structural framework with potential use to develop chemical reagents that target and modulate multiple facets of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by multiple, intertwined pathological features, including amyloid-ß (Aß) aggregation, metal ion dyshomeostasis, and oxidative stress. We report a novel compound (ML) prototype of a rationally designed molecule obtained by integrating structural elements for Aß aggregation control, metal chelation, reactive oxygen species (ROS) regulation, and antioxidant activity within a single molecule. Chemical, biochemical, ion mobility mass spectrometric, and NMR studies indicate that the compound ML targets metal-free and metal-bound Aß (metal-Aß) species, suppresses Aß aggregation in vitro, and diminishes toxicity induced by Aß and metal-treated Aß in living cells. Comparison of ML to its structural moieties (i.e., 4-(dimethylamino)phenol (DAP) and (8-aminoquinolin-2-yl)methanol (1)) for reactivity with Aß and metal-Aß suggests the synergy of incorporating structural components for both metal chelation and Aß interaction. Moreover, ML is water-soluble and potentially brain permeable, as well as regulates the formation and presence of free radicals. Overall, we demonstrate that a rational structure-based design strategy can generate a small molecule that can target and modulate multiple factors, providing a new tool to uncover and address AD complexity.

Tuning reactivity of diphenylpropynone derivatives with metal-associated amyloid-ß species via structural modifications.

A diphenylpropynone derivative, DPP2, has been recently demonstrated to target metal-associated amyloid-ß (metal-Aß) species implicated in Alzheimer's disease (AD). DPP2 was shown to interact with metal-Aß species and subsequently control Aß aggregation (reactivity) in vitro; however, its cytotoxicity has limited further biological applications. In order to improve reactivity toward Aß species and lower cytotoxicity, along with gaining an understanding of a structure-reactivity-cytotoxicity relationship, we designed, prepared, and characterized a series of small molecules (C1/C2, P1/P2, and PA1/PA2) as structurally modified DPP2 analogues. A similar metal binding site to that of DPP2 was contained in these compounds while their structures were varied to afford different interactions and reactivities with metal ions, Aß species, and metal-Aß species. Distinct reactivities of our chemical family toward in vitro Aß aggregation in the absence and presence of metal ions were observed. Among our chemical series, the compound (C2) with a relatively rigid backbone and a dimethylamino group was observed to noticeably regulate both metal-free and metal-mediated Aß aggregation to different extents. Using our compounds, cell viability was significantly improved, compared to that with DPP2. Lastly, modifications on the DPP framework maintained the structural properties for potential blood-brain barrier (BBB) permeability. Overall, our studies demonstrated that structural variations adjacent to the metal binding site of DPP2 could govern different metal binding properties, interactions with Aß and metal-Aß species, reactivity toward metal-free and metal-induced Aß aggregation, and cytotoxicity of the compounds, establishing a structure-reactivity-cytotoxicity relationship. This information could help gain insight into structural optimization for developing nontoxic chemical reagents toward targeting metal-Aß species and modulating their reactivity in biological systems.

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.

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.

Hydrogel nanoparticles with covalently linked coomassie blue for brain tumor delineation visible to the surgeon.

Delineation of tumor margins is a critical and challenging objective during brain cancer surgery. A tumor-targeting deep-blue nanoparticle-based visible contrast agent is described, which, for the first time, offers in vivo tumor-specific visible color staining. This technology thus enables color-guided tumor resection in real time, with no need for extra equipment or special lighting conditions. The visual contrast agent consists of polyacrylamide nanoparticles covalently linked to Coomassie Blue molecules (for nonleachable blue color contrast), which are surface-conjugated with polyethylene glycol and F3 peptides for efficient in vivo circulation and tumor targeting, respectively.

Effects of clioquinol on metal-triggered amyloid-beta aggregation revisited.

Amyloid-beta (Abeta) plaques are largely associated with the neuropathogenesis of Alzheimer's disease (AD). Metal ions such as Cu(II) and Zn(II) have been implicated as contributors to their formation and deposition. Metal chelators have been used to modulate metal-induced Abeta aggregation. The bidentate ligand clioquinol (CQ) presents an effective influence on metal-involved Abeta aggregation, which has been explained through its metal chelation and is generally monitored by fluorescence and turbidity assays in vitro. The studies herein, however, suggest that the effects of CQ on metal-driven Abeta aggregation may not be visualized accurately by both assays. Subsequently, the present work demonstrates that CQ is able to chelate metal ions from metal-Abeta species and to assist, in part, in the disaggregation of Abeta aggregates, but it could not completely hinder the progression of Abeta aggregation.