Marianna Török

Structural and dynamic features of Alzheimer's Aβ peptide in amyloid fibrils studied by site-directed spin labeling

Electron paramagnetic resonance spectroscopy analysis of 19 spin-labeled derivatives of the Alzheimers amyloid β (Aβ) peptide was used to reveal structural features of amyloid fibril formation. In the fibril, extensive regions of the peptide show an in-register, parallel arrangement. Based on the parallel arrangement and side chain mobility analysis we find the amyloid structure to be mostly ordered and specific, but we also identify more dynamic regions (N and C termini) and likely turn or bend regions (around residues 23–26). Despite their different aggregation properties and roles in disease, the two peptides, Aβ40 and Aβ42, homogeneously co-mix in amyloid fibrils suggesting that they possess the same structural architecture.

Solid acid (superacid) catalyzed regioselective adamantylation of substituted benzenes

Adamantylation of substituted benzenes with 1-bromo-adamantane was catalyzed by solid acids including acidic ion exchange and ionomer resins, HY zeolite, sulfated zirconia and supported superacids on HY zeolite and SiO2. Adamantylation generally takes place in excellent yield giving predominantly para products without formation of byproducts. The reactions did not require the usual workup of Friedel-Crafts reactions as catalysts were simply filtered of. Cross-linked polystyrene resin sulfonic acid (Amberlyst) was found particularly suitable as besides its high catalytic activity, high regioselectivity was observed with almost exclusive formation ofp-adamantylated benzenes. AMI, PM3 and MNDO semiempirical calculations of heats of formation showed that of all regioisomers, the para isomer is the most stable. The temperature dependence of adamantylation was also investigated allowing the optimization ofp-substituted product in high yield and excellent selectivity. Lack of isomerization of 1-p-tolyla-damantane using solid (Amberlyst, Nafion-H) and liquid acids (neat and modified trifluoromethanesulfonic acid) indicates absence of product isomerization, while the intramolecular rearrangement of the intermediate arenium complex is still possible.

Effect of chirality of small molecule organofluorine inhibitors of amyloid self-assembly on inhibitor potency

The effect of enantiomeric trifluoromethyl-indolyl-acetic acid ethyl esters on the fibrillogenesis of Alzheimers amyloid beta (Abeta) peptide is described. These compounds have been previously identified as effective inhibitors of the Abeta self-assembly in their racemic form. Thioflavin-T Fluorescence Spectroscopy and Atomic Force Microscopy were applied to assess the potency of the chiral target compounds. Both enantiomers showed significant inhibition in the in vitro assays. The potency of the enantiomeric inhibitors appeared to be very similar to each other suggesting the lack of the stereospecific binding interactions between these small molecule inhibitors and the Abeta peptide.

Structure–Activity Relationships of Organofluorine Inhibitors of β-Amyloid Self-Assembly

A broad group of structurally diverse small organofluorine compounds were synthesized and evaluated as inhibitors of β‐amyloid (Aβ) self‐assembly. The main goal was to generate a diverse library of compounds with the same functional group and to observe general structural features that characterize inhibitors of Aβ oligomer and fibril formation, ultimately identifying structures for further focused inhibitor design. The common structural motifs in these compounds are CF3‐C‐OH and CF3‐C‐NH groups that were proposed to be binding units in our previous studies. A broad range of potential small‐molecule inhibitors were synthesized by combining various carbocyclic and heteroaromatic rings with an array of substituents, generating a total of 106 molecules. The compounds were tested by standard methods such as thioflavin‐T fluorescence spectroscopy for monitoring fibril formation, biotinyl Aβ1–42 single‐site streptavidin‐based assays for observing oligomer formation, and atomic force microscopy for morphological studies. These assays revealed a number of structures that show significant inhibition against either Aβ fibril or oligomer formation. A detailed analysis of the structure–activity relationship of anti‐fibril and ‐oligomer properties is provided. These data present further experimental evidence for the distinct nature of fibril versus oligomer formation and indicate that the interaction of the Aβ peptide with chiral small molecules is not stereospecific in nature.

Design, Synthesis and Biological Activity of Multifunctional α,β- Unsaturated Carbonyl Scaffolds for Alzheimer’s Disease

A series of compounds containing an α,β-unsaturated carbonyl moiety, such as chalcones and coumarins were designed, synthesized and tested in a variety of assays to assess their potential as anti-Alzheimers disease (AD) agents. The investigations included the inhibition of cholinesterases (AChE, BuChE), the inhibition of amyloid beta (Aβ) self-assembly and the disassembly of preformed Aβ oligomers. Several compounds showed excellent potential as multifunctional compounds for AD. Docking studies for 16 that performed well in all the assays gave a clear interpretation of various interactions in the gorge of AChE. Based on the results, the long-chain coumarin scaffold appears to be a promising structural template for further AD drug development.

Diaryl hydrazones as multifunctional inhibitors of amyloid self-assembly.

The design and application of an effective, new class of multifunctional small molecule inhibitors of amyloid self-assembly are described. Several compounds based on the diaryl hydrazone scaffold were designed. Forty-four substituted derivatives of this core structure were synthesized using a variety of benzaldehydes and phenylhydrazines and characterized. The inhibitor candidates were evaluated in multiple assays, including the inhibition of amyloid β (Aβ) fibrillogenesis and oligomer formation and the reverse processes, the disassembly of preformed fibrils and oligomers. Because the structure of the hydrazone-based inhibitors mimics the redox features of the antioxidant resveratrol, the radical scavenging effect of the compounds was evaluated by colorimetric assays against 2,2-diphenyl-1-picrylhydrazyl and superoxide radicals. The hydrazone scaffold was active in all of the different assays. The structure-activity relationship revealed that the substituents on the aromatic rings had a considerable effect on the overall activity of the compounds. The inhibitors showed strong activity in fibrillogenesis inhibition and disassembly, and even greater potency in the inhibition of oligomer formation and oligomer disassembly. Supporting the quantitative fluorometric and colorimetric assays, size exclusion chromatographic studies indicated that the best compounds practically eliminated or substantially inhibited the formation of soluble, aggregated Aβ species, as well. Atomic force microscopy was also applied to monitor the morphology of Aβ deposits. The compounds also possessed the predicted antioxidant properties; approximately 30% of the synthesized compounds showed a radical scavenging effect equal to or better than that of resveratrol or ascorbic acid.

Molecular Docking of Enzyme Inhibitors: A Computational Tool for Structure-Based Drug Design

Molecular docking is a frequently used method in structure‐based rational drug design. It is used for evaluating the complex formation of small ligands with large biomolecules, predicting the strength of the bonding forces and finding the best geometrical arrangements. The major goal of this advanced undergraduate biochemistry laboratory exercise is to illustrate the importance and application of this tool. Students carry out the computational modeling of the interaction of acetylcholinesterase and its inhibitor, tacrine, and learn about the concepts of protein structure, enzyme‐inhibitor interactions, intermolecular forces, and role of molecular design in drug‐development.

Sulfonamides as multifunctional agents for Alzheimer's disease.

Sulfonamide linker-based inhibitors with extended linear structure were designed and synthesized with the aim of producing multifunctional agents against several processes involved in the pathology of Alzheimers disease (AD). The potency of the compounds were assessed in the inhibition of Aβ self-assembly (fibril and oligomer formation), in modulating cholinesterase (AChE, BuChE) activity, and scavenging free radicals. Several compounds exhibited promising Aβ self-assembly and cholinesterase inhibition and in parallel, showed good free radical scavenging properties. The investigation of the scaffold described in this study resulted in the identification of three compounds (14, 19 and 26) as promising leads for the further design of multifunctional drug candidates for AD.

Pharmacophore Modeling, Virtual and In Vitro Screening for Acetylcholinesterase Inhibitors and their Effects on Amyloid-β Self- Assembly

One of the most promising methods of unveiling the pharmacology of marketed drugs is to screen them against new biological targets. In an attempt to find inhibitors for acetylcholinesterase (AChE), the Drug Bank Database and natural alkaloids with other known medicinal values were screened through a four-point pharmacophore built in this study. The development of the pharmacophore was based on a structurally diverse set of reported AChE inhibitors and was validated using a separate set of known inhibitors. The developed pharmacophore indicated that the presence of one H-acceptor motif, one H-donor motif, one positively charged group and one aromatic ring is needed for AChE inhibition. Selected hits were further investigated by molecular docking and in vitro testing. The assays revealed that the majority of these compounds showed reasonable inhibition, indicating that the developed pharmacophore can indeed reliably screen molecules for potential AChE inhibitors. It appears that several commercially available marketed drugs have further potential as AChE inhibitors. To extend our study the same compounds have been tested in the fibrillogenesis inhibition of amyloidβ (Aβ) peptide to explore the possibility of their dual-function therapeutic activity in Alzheimers disease.

Introduction to Spin Label Electron Paramagnetic Resonance Spectroscopy of Proteins.

An undergraduate laboratory exercise is described to demonstrate the biochemical applications of electron paramagnetic resonance (EPR) spectroscopy. The β93 cysteine residue of hemoglobin is labeled by the covalent binding of 3‐maleimido‐proxyl (5‐MSL) and 2,2,5,5‐tetramethyl‐1‐oxyl‐3‐methyl methanethiosulfonate (MTSL), respectively. The excess spin label is removed by gel‐exclusion chromatography. Changes in the mobility of the reporter groups attached to the protein are monitored by EPR spectroscopy. While the spectral parameters of the rigidly attached 5‐MSL provide information on the rotation of the whole spin labeled protein, MTSL bound by a more flexible linkage describes the local environment of the cysteine residue in the interior of the protein structure. Students can study the known crystal structure of hemoglobin in comparison to the results they obtain by analyzing the EPR spectra. Overall, the exercise introduces them to laboratory techniques such as protein labeling, gel filtration, EPR spectroscopy, as well as familiarizes them with the online Protein Data Bank as a research resource and PyMOL software as a structure visualization tool.