Alzheimer’s disease (AD) is characterized by multiple, intertwined pathological features, including amyloid-β (Aβ) 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 Aβ 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 Aβ (metal–Aβ) species, suppresses Aβ aggregation in vitro, and diminishes toxicity induced by Aβ and metal-treated Aβ 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 Aβ and metal–Aβ suggests the synergy of incorporating structural components for both metal chelation and Aβ 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.

In the commonly used nucleation-dependent model of protein aggregation, aggregation proceeds only after a lag phase in which the concentration of energetically unfavorable nuclei reaches a critical value. The formation of oligomeric species prior to aggregation can be difficult to detect by current spectroscopic techniques. By using real-time 19F NMR along with other techniques, we are able to show that multiple oligomeric species can be detected during the lag phase of Aβ1-40 fiber formation, consistent with a complex mechanism of aggregation. At least 6 types of oligomers can be detected by 19F NMR. These include the reversible formation of large β-sheet oligomer immediately after solubilization at high peptide concentration; a small oligomer that forms transiently during the early stages of the lag phase; and 4 spectroscopically distinct forms of oligomers with molecular weights between ~30–100 kDa that appear during the later stages of aggregation. The ability to resolve individual oligomers and track their formation in real-time should prove fruitful in understanding the aggregation of amyloidogenic proteins and in isolating potentially toxic non-amyloid oligomers.

Alzheimer’s disease is characterized by the misfolding and self-assembly of the amyloidogenic protein amyloid-β (Aβ). The aggregation of Aβ leads to diverse oligomeric states, each of which may be potential targets for intervention. Obtaining insight into Aβ oligomers at the atomic level has been a major challenge to most techniques. Here, we use magic angle spinning recoupling 1H-1H NMR experiments to overcome many of these limitations. Using 1H-1H dipolar couplings as a NMR spectral filter to remove both high and low molecular weight species, we provide atomic-level characterization of a non-fibrillar aggregation product of the Aβ1-40 peptide using non-frozen samples without isotopic labeling. Importantly, this spectral filter allows the detection of the specific oligomer signal without a separate purification procedure. In comparison to other solid-state NMR techniques, the experiment is extraordinarily selective and sensitive. A resolved 2D spectra could be acquired of a small population of oligomers (6 micrograms, 7% of the total) amongst a much larger population of monomers and fibers (93% of the total). By coupling real-time 1H-1H NMR experiments with other biophysical measurements, we show that a stable, primarily disordered Aβ1-40 oligomer 5–15 nm in diameter can form and coexist in parallel with the well-known cross-β-sheet fibrils.

The aggregation of amyloidogenic proteins is infamous for being highly chaotic, with small variations in conditions sometimes leading to large changes in aggregation rates. Using the amyloidogenic protein IAPP (islet amyloid polypeptide protein, also known as amylin) as an example, we show that a part of this phenomenon may be related to the formation of micellelike oligomers at specific critical concentrations and temperatures. We show that pyrene fluorescence can sensitively detect micellelike oligomer formation by IAPP and discriminate between micellelike oligomers from fibers and monomers, making pyrene one of the few chemical probes specific to a prefibrillar oligomer. We further show that oligomers of this type reversibly form at critical concentrations in the low micromolar range and at specific critical temperatures. Micellelike oligomer formation has several consequences for amyloid formation by IAPP. First, the kinetics of fiber formation increase substantially as the critical concentration is approached but are nearly independent of concentration below it, suggesting a direct role for the oligomers in fiber formation. Second, the critical concentration is strongly correlated with the propensity to form amyloid: higher critical concentrations are observed for both IAPP variants with lower amyloidogenicity and for native IAPP at acidic pH in which aggregation is greatly slowed. Furthermore, using the DEST NMR technique, we show that the pathway of amyloid formation switches as the critical point is approached, with self-interactions primarily near the N-terminus below the critical temperature and near the central region above the critical temperature, reconciling two apparently conflicting views of the initiation of IAPP aggregation.

The aggregation of human islet amyloid polypeptide (hIAPP) has been linked to beta-cell death in type II diabetes. Zinc present in secretory granules has been shown to affect this aggregation. A combination of EXAFS, NMR, and AFM experiments shows that the influence of zinc is most likely due to the stabilization of prefibrillar aggregates of hIAPP.