Prevention of Alzheimer's Disease-associated Aβ Aggregation by Rationally Designed Nonpeptidic β-Sheet Ligands

Abstract

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.

Discovery and Structure Activity Relationship of Small Molecule Inhibitors of Toxic β-Amyloid-42 Fibril Formation

“…The pinhole diameter was 40 m. The instrument was calibrated with the dye rhodamine 6G with a known diffusion coefficient in water at 20°C of 2.8⅐10 Ϫ6 cm 2 /s. The A␤42 peptide was labeled N-terminally with the fluorescence dye Oregon green as described (36). Compounds were tested using preformed aggregates that were prepared freshly by diluting low concentrated (500 nM) DMSO-stock solutions of Oregon green-labeled A␤42 1:1 with deionized water.…”

“…Data evaluation was carried out by averaging the fluorescence fluctuations for every well and counting data points that deviated more than 5-fold from the mean fluorescence signal, i.e. every peak at a Z-score equal or higher than ϩ5 was counted as an A␤42 aggregate (36). Because the intensity of the fluorescence peaks is related to the number of dye molecules present in A␤42 aggregates, not only the number of peaks but also the product of number and height of peaks was evaluated.…”

“…Because the intensity of the fluorescence peaks is related to the number of dye molecules present in A␤42 aggregates, not only the number of peaks but also the product of number and height of peaks was evaluated. The results of every measurement were normalized to the values measured for the control samples (36).…”

“…An alternative approach is based on a rational design utilizing acylated 3-aminopyrazoles with a donor-acceptor-donor hydrogen bond pattern complementary to that of the ␤-sheet of A␤42 (35,36) (Fig. 1A).…”

“…1A). These compounds bearing 3-aminopyrazoles are either dimeric compounds, where the 3-aminopyrazole moieties are connected by a rigid oxalyl-linker, or oligomeric compounds, where the 3-aminopyrazole moieties are directly linked to each other by amide bonds (35,36).…”