There is evidence that binding of metal ions like Zn2+ and Cu2+ to amyloid beta‐peptides (Αβ) may contribute to the pathogenesis of Alzheimer’s disease. Cu2+ and Zn2+ form complexes with Αβ peptides in vitro; however, the published metal‐binding affinities of Αβ vary in an enormously large range. We studied the interactions of Cu2+ and Zn2+ with monomeric Αβ40 under different conditions using intrinsic Αβ fluorescence and metal‐selective fluorescent dyes. We showed that Cu2+ forms a stable and soluble 1 : 1 complex with Αβ40, however, buffer compounds act as competitive copper‐binding ligands and affect the apparent KD. Buffer‐independent conditional KD for Cu(II)‐Αβ40 complex at pH 7.4 is equal to 0.035 μmol/L. Interaction of Αβ40 with Zn2+ is more complicated as partial aggregation of the peptide occurs during zinc titration experiment and in the same time period (within 30 min) the initial Zn‐Αβ40 complex (KD = 60 μmol/L) undergoes a transition to a more tight complex with KD ∼ 2 μmol/L Competition of Αβ40 with ion‐selective fluorescent dyes Phen Green and Zincon showed that the KD values determined from intrinsic fluorescence of Αβ correspond to the binding of the first Cu2+ and Zn2+ ions to the peptide with the highest affinity. Interaction of both Zn2+ and Cu2+ ions with Αβ peptides may occur in brain areas affected by Alzheimer’s disease and Zn2+‐induced transition in the peptide structure might contribute to amyloid plaque formation.

Aggregation of amyloid‐β (Aβ) peptides is a central phenomenon in Alzheimer’s disease. Zn(II) and Cu(II) have profound effects on Aβ aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Aβ42 fibrillization and initiate formation of non‐fibrillar Aβ42 aggregates, and that the inhibitory effect of Zn(II) (IC50 = 1.8 μmol/L) is three times stronger than that of Cu(II). Medium and high‐affinity metal chelators including metallothioneins prevented metal‐induced Aβ42 aggregation. Moreover, their addition to preformed aggregates initiated fast Aβ42 fibrillization. Upon prolonged incubation the metal‐induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Aβ42. H13A and H14A mutations in Aβ42 reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross‐β core structure within region 10–23 of the amyloid fibril. Cu(II)‐Aβ42 aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)‐Aβ42 aggregates were non‐toxic. Disturbed metal homeostasis in the vicinity of zinc‐enriched neurons might pre‐dispose formation of metal‐induced Aβ aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal‐chelating therapies for Alzheimer’s disease is discussed in this light.

Aggregation of amyloid-β (Aβ) peptides is causatively linked to Alzheimer's disease (AD); thus, suppression of this process by small molecule inhibitors is a widely accepted therapeutic and preventive strategy for AD. Screening of the inhibitors of Aβ aggregation deserves much attention; however, despite intensive efforts, there are only a few high-throughput screening methods available, all of them having drawbacks related to the application of external fluorescent probes or artificial Aβ derivatives. We have developed a label-free MALDI MS-based screening test for inhibitors of Aβ₄₂ fibrillization that exhibits high sensitivity, speed, and automation possibilities suitable for high-throughput screening. The test was evaluated by transmission electron microscopy and compared with a fluorimetric thioflavin-based assay, where interference of a number of tested compounds with thioflavin T binding and/or fluorescence caused false-positive results. The MALDI MS-based method can significantly speed up in vitro screening of compound libraries for inhibitors of Aβ₄₂ fibrillization.