Different inhibitors of Aβ42-induced toxicity have distinct metal-ion dependency.

Oligomers of amyloid beta-protein (A beta) are thought to be the proximal toxic agents initiating the neuropathologic process in Alzheimer's disease (AD). Therefore, targeting the self-assembly and oligomerization of A beta has been an important strategy for designing AD therapeutics. In parallel, research into the metallobiology of AD has shown that Zn(2+ )can strongly modulate the aggregation of A beta in vitro and both promote and inhibit the neurotoxicity of A beta, depending on the experimental conditions. Thus, successful inhibitors of A beta self-assembly may have to inhibit the toxicity not only of A beta oligomers themselves but also of A beta-Zn2+ complexes. However, there has been relatively little research investigating the effects of A beta self-assembly and toxicity inhibitors in the presence of Zn2+. Our group has characterized previously a series of A beta 42 C-terminal fragments (CTFs), some of which have been shown to inhibit A beta oligomerization and neurotoxicity. Here, we asked whether three CTFs shown to be potent inhibitors of A beta 42 toxicity maintained their activity in the presence of Zn2+. Biophysical analysis showed that the CTFs had different effects on oligomer, beta-sheet, and fibril formation by A beta 42-Zn2+ complexes. However, cell viability experiments in differentiated PC-12 cells incubated with A beta 42-Zn2+ complexes in the absence or presence of these CTFs showed that the CTFs completely lost their inhibitory activity in the presence of Zn2+ even when applied at 10-fold excess relative to A beta 42. In light of these results, we tested another inhibitor, the molecular tweezer CLR01, which coincidentally had been shown to have a high affinity for Zn2+, suggesting that it could disrupt both A beta 42 oligomerization and A beta 42-Zn2+ complexation. Indeed, we found that CLR01 effectively inhibited the toxicity of A beta 42-Zn2+ complexes. Moreover, it did so at a lower concentration than needed for inhibiting the toxicity of A beta 42 alone. In agreement with these results, CLR01 inhibited beta-sheet and fibril formation in A beta 42-Zn2+ complexes. Our data suggest that, for the development of efficient therapeutic agents, inhibitors of A beta self-assembly and toxicity should be examined in the presence of relevant metal ions and that molecular tweezers may be particularly attractive candidates for therapy development.