(485ak) Comparative Study of Inhibition at Multiple Stages of Amyloid-β Self-Assembly Provides Mechanistic Insight

The ?amyloid cascade hypothesis', linking self-assembly of the amyloid-β protein (Aβ) to the pathogenesis of Alzheimer's disease (AD), has led to the emergence of inhibition of Aβ self-assembly as a therapeutic strategy for this currently unpreventable and devastating disease. The complexity of Aβ self-assembly, which involves multiple reaction intermediates related by nonlinear and interconnected nucleation and growth mechanisms, provides multiple points for inhibitor intervention. Several small molecules that inhibit the in vitro formation of amyloid fibrils have been identified; however, little insight has been garnered concerning the point at which these inhibitors intervene within the Aβ self-assembly pathway.

We have employed three distinct Aβ self-assembly assay formats to assess different mechanisms of Aβ self-assembly. The overall process of Aβ self-assembly is studied using an assay that monitors aggregate formation from monomeric protein, while later stages of assembly are investigated using two assays that isolate two distinct mechanisms of growth for soluble Aβ aggregation intermediates: elongation via monomer addition and lateral association. Aggregate formation and growth were evaluated using biophysical techniques, including fluorescence spectroscopy and dynamic light scattering, as well as transmission electron microscopy.

Using these assays, a julolidine derivative was identified as a novel inhibitor of Aβ self-assembly. This compound was shown to selectively inhibit the growth of soluble aggregates by lateral association, while having little effect on soluble aggregate elongation via monomer addition. Parallel inhibition of aggregate formation from monomeric protein implicates a role for lateral aggregate association in the overall extent of aggregate formation. Together, these results imply that this compound binds the lateral surface of a soluble on-pathway intermediate to prevent association with other aggregates required for continued assembly into mature fibrils. In addition, inhibition of soluble Aβ aggregate association exhibited an IC50 with a somewhat lower stoichiometric ratio than the IC50 determined for inhibition of aggregate formation from monomeric protein. This quantitative comparison suggests that a range of aggregate sizes may be recognized by this compound. Together, these findings demonstrate that the quantitative evaluation of inhibition at different mechanistic steps within the Aβ self-assembly process can provide insight into the mechanism of inhibitor action, which may facilitate optimization of effective inhibitor structures.