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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.

Expressed protein ligation is used to replace the conserved axial ligand methionine 121 from Pseudomonas aeruginosa (Az) with isostructural unnatural amino acid analogues, oxomethionine, difluoromethionine, trifluoromethionine, selenomethionine and norleucine. A linear correlation between the hydrophobicity of the axial ligand and the reduction potential of the copper ion is observed and a strong linear correlation between change in hydrophobicity and change in reduction potential is indicated.

Evolutionary links between type 1 blue copper (T1 Cu), type 2 red copper (T2 Cu), and purple CuA cupredoxins have been proposed, but the structural features and mechanism responsible for such links as well as for assembly of CuA sites in vivo are poorly understood, even though recent evidence demonstrated that the Cu(II) oxidation state plays an important role in this process. In this study, we examined the kinetics of Cu(II) incorporation into the CuA site of a biosynthetic CuA model, CuA azurin (CuAAZ) and found that both T1 Cu and T2 Cu intermediates form on the path to final CuA reconstitution in a pH-dependent manner, with slower kinetics and greater accumulation of the intermediates as the pH is raised from 5.0 to 7.0. While these results are similar to those observed previously in the native CuA center of nitrous oxide reductase, the faster kinetics of copper incorporation into CuAAz allowed us to use lower copper equivalents to reveal a new pathway of copper incorporation, including a novel intermediate that has not been reported in cupredoxins before, with intense electronic absorption maxima at ∼410 and 760 nm. We discovered that this new intermediate underwent reduction to Cu(I), and proposed that it is a Cu(II)-dithiolate species. Oxygen-dependence studies demonstrated that the T1 Cu species only formed in the presence of molecular oxygen, suggesting the T1 Cu intermediate is a one-electron oxidation product of a Cu(I) species. By studying CuAAZ variants where the Cys and His ligands are mutated, we have identffied the T2 Cu intermediate as a capture complex with Cys116 and the T1 Cu intermediate as a complex with Cys112 and His120. These results led to a unified mechanism of copper incorporation and new insights regarding the evolutionary link between all cupredoxin sites as well as the in vivo assembly of CuA centers. [ABSTRACT FROM AUTHOR]