Hopeahainol A binds reversibly at the acetylcholinesterase (AChE) peripheral site and inhibits enzyme activity with a novel higher order concentration dependence.

Natural product inhibitors of AChE are of interest both because they offer promise as inexpensive drugs for symptomatic relief in Alzheimer's disease and because they may provide insights into the structural features of the AChE catalytic site. Hopeahainol A is an uncharged polyphenol AChE inhibitor from the stem bark of Hopea hainanensis with a constrained, partially dearomatized bicyclic core. Molecular modeling indicates that hopeahainol A binds at the entrance of the long but narrow AChE active site gorge because it is too bulky to be accommodated within the gorge without severe distortion of the gorge as depicted in AChE crystal structures. We conducted inhibitor competition experiments in which AChE inhibition was measured with hopeahainol A together with either edrophonium (which binds at the base of the gorge) or thioflavin T (which binds to the peripheral or P-site near the gorge mouth). The results agreed with the molecular modeling and indicated that hopeahainol A at lower concentrations (<200 µM) bound only to the P-site, as hopeahainol A and thioflavin T were unable to form a ternary complex with AChE while hopeahainol A and edrophonium did form a ternary complex with essentially no competition between them. Inhibition increased to a striking extent at higher concentrations of hopeahainol A, with plots analogous to classic Dixon plots showing a dependence on hopeahainol A concentrations to the third- or fourth order. The inhibition at higher hopeahainol A concentrations was completely reversed on dilution and blocked by bound edrophonium. We hypothesize that bound hopeahainol A induces conformational changes in the AChE active site that allow binding of additional hopeahainol A molecules, a phenomenon that would be unprecedented for a reversible inhibitor that apparently forms no covalent bonds with AChE.

Antiparallel ß-Sheet Structure within the C-Terminal Region of 42-Residue Alzheimer's Amyloid-ß Peptides When They Form 150-kDa Oligomers.

Understanding the molecular structures of amyloid-ß (Aß) oligomers and underlying assembly pathways will advance our understanding of Alzheimer's disease (AD) at the molecular level. This understanding could contribute to disease prevention, diagnosis, and treatment strategies, as oligomers play a central role in AD pathology. We have recently presented a procedure for production of 150-kDa oligomeric samples of Aß(1-42) (the 42-residue variant of the Aß peptide) that are compatible with solid-state nuclear magnetic resonance (NMR) analysis, and we have shown that these oligomers and amyloid fibrils differ in intermolecular arrangement of ß-strands. Here we report new solid-state NMR constraints that indicate antiparallel intermolecular alignment of ß-strands within the oligomers. Specifically, 150-kDa Aß(1-42) oligomers with uniform (13)C and (15)N isotopic labels at I32, M35, G37, and V40 exhibit ß-strand secondary chemical shifts in 2-dimensional (2D) finite-pulse radiofrequency-driven recoupling NMR spectra, spatial proximities between I32 and V40 as well as between M35 and G37 in 2D dipolar-assisted rotational resonance spectra, and close proximity between M35 H(α) and G37 H(α) in 2D CHHC spectra. Furthermore, 2D dipolar-assisted rotational resonance analysis of an oligomer sample prepared with 30% labeled peptide indicates that the I32-V40 and M35-G37 contacts are between residues on different molecules. We employ molecular modeling to compare the newly derived experimental constraints with previously proposed geometries for arrangement of Aß molecules into oligomers.

A mass spectrometric approach for characterization of amyloid-ß aggregates and identification of their post-translational modifications.

Endogenous amyloid-ß (Aß) oligomeric aggregates have been proposed as toxic agents in Alzheimer's disease (AD). Knowledge of their structures not only may provide insight into the basis of their neurotoxicities but also may reveal new targets for therapeutic drugs and diagnostic tools. However, the low levels of these Aß oligomers have impeded structural characterization. Evidence suggests that the endogenous oligomers are covalently modified in vivo. In this report, we demonstrate an established mass spectrometry (MS) methodology called precursor ion mapping (PIM) that potentially may be applied to endogenous oligomer characterization. First, we illustrate the use of this PIM technique with a synthetic Aß(1-40) monomer sample that had been cross-linked with transglutaminase (TGase) and digested with pepsin. From PIM analysis of an Aß(4-13) MS/MS fragment, precursor ions were identified that corresponded to peptic fragments of three TGase cross-linked species: Aß(4-19)--(4-19), Aß(4-19)--(20-34), and Aß(1-19)--(20-34). Next, we demonstrate the applicability of the PIM technique to an endogenous Aß sample that had been purified and concentrated by immunoaffinity chromatography. Without pepsin digestion, we successfully identified the full length and C-terminally truncated monomeric Aß species 1-35 to 1-42, along with select methionine-oxidized counterparts. Because PIM focuses only on a subpopulation of ions, namely the related precursor ions, the resulting spectra are of increased specificity and sensitivity. Therefore, this methodology shows great promise for structural analysis and identification of post-translational modification(s) in endogenous Aß oligomers.