No abstract

Little is known about the precise mechanism of action of beta-sheet ligands, hampered by the notorious solubility problems involved with protein misfolding and amyloid formation. Recently the nucleation site for the pathogenic aggregation of the Alzheimer's peptide was identified as the KLVFF sequence in the central region of Abeta. A combination of two aminopyrazole ligands with di- or tripeptides taken from this key fragment now furnished water-soluble Abeta-specific ligands which allow model investigations in water. A detailed conformational analysis provides experimental evidence for an increased beta-sheet content induced in the peptide. Strong indications were also found for the peptide backbone recognition via hydrogen bonds plus hydrophobic contributions between aminopyrazole nuclei and Phe residues. The affinity of these new ligands toward the KKLVFF fragment is highly dependent on their sequence and composition from natural and artificial amino acids. Thus, for the first time, detailed insight is gained into the complexation of beta-sheet ligands with model peptides taken directly from Abeta.

A new concept is introduced for the rational design of ␤-sheet ligands, which prevent protein aggregation. Oligomeric acylated aminopyrazoles with a donor-acceptor-donor (DAD) hydrogen bond pattern complementary to that of a ␤-sheet efficiently block the solvent-exposed ␤-sheet portions in A␤-(1-40) and thereby prevent formation of insoluble protein aggregates. Density gradient centrifugation revealed that in the initial phase, the size of A␤ aggregates was efficiently kept between the trimeric and 15-meric state, whereas after 5 days an additional high molecular weight fraction appeared. With fluorescence correlation spectroscopy (FCS) exactly those two, i.e. a dimeric aminopyrazole with an oxalyl spacer and a trimeric head-to-tail connected aminopyrazole, of nine similar aminopyrazole ligands were identified as efficient aggregation retardants whose minimum energy conformations showed a perfect complementarity to a ␤-sheet. The concentration dependence of the inhibitory effect of a trimeric aminopyrazole derivative allowed an estimation of the dissociation constant in the range of 10 ؊5 M. Finally, electrospray ionization mass spectrometry (ESI-MS) was used to determine the aggregation kinetics of A␤-(1-40) in the absence and in the presence of the ligands. From the comparable decrease in A␤ monomer concentration, we conclude that these ␤-sheet ligands do not prevent the initial oligomerization of monomeric A␤ but rather block further aggregation of spontaneously formed small oligomers. Together with the results from density gradient centrifugation and fluorescence correlation spectroscopy it is now possible to restrict the approximate size of soluble A␤ aggregates formed in the presence of both inhibitors from 3-to 15-mers.

Self-association of aminopyrazole peptide hybrid 1 leads to stacked nanorosettes. This remarkable, well-ordered structure obeys the laws of nucleic acid self-assembly. In a strictly hierarchical process, formation of aminopyrazole "base" triplets via a hydrogen bond network is accompanied by pi-stacking with a second rosette and final dimerization of two double rosettes to a four-layer superstructure, stabilized by a six-fold half-crown alkylammonium lock. The final complex is soluble in organic as well as in aqueous solution. It was characterized in the solid state by X-ray crystallography, in water by NMR spectroscopy, and in silico by quantum chemical shift calculation. All these methods provide strong evidence for the same hexameric complex geometry. Its structural features bear striking similarity to nucleic acid architectures and their peptidic counterparts, especially alanyl-PNA. The whole self-assembly process is highly solvent- and temperature-dependent and occurs with a high degree of cooperativity--no intermediates are observed. Formation and dissociation of the nanorosette, however, are kinetically slow. The limitation to a hexameric aggregate can be explained by six sterically demanding valine residues, whose replacement by alanines may result in formation of infinite fibers.