Prevention of Alzheimer's Associated Aβ-Aggregation by Rationally Designed Non-Peptidic β-Sheet Ligands

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