P2-338: Substrate-targeting gamma-secretase modulators

Increased production and accumulation of the amyloid beta peptide 1–42 (Aβ42) has been linked to the development of Alzheimer's disease (AD). Thus, strategies to target Aβ42 are being pursued as therapeutic avenues to treat Alzheimer's disease (AD). Gamma-secretase modulators (GSMs), originally identified as a subset of non-steroidal anti-inflammatory drugs (NSAIDs), are one class of compounds that decrease Aβ42 production and have shown promise in cell culture and animal models. GSMs reduce the production of Aβ42, typically without decreasing Aβ40 levels, and do not inhibit cleavage of other γ-secretase substrates such as Notch. One GSM, tarenflurbil (R-flurbiprofen), is currently in Phase III clinical trials to evaluate its impact on cognition in patients with mild AD. Despite their therapeutic promise, the precise mechanism of GSMs has not been established. Previous publications have suggested that GSMs influence the production of Aβ peptides by targeting an allosteric site on the γ-secretase complex. However, to date no direct binding targets of GSMs have been reported that explain their biological activity. We synthesized biotinylated derivatives of the GSMs fenofibrate and flurbiprofen suitable for photoaffinity labeling experiments to identifying protein targets. GSM photoprobes retained modulatory activity but did not label the proteins in the core γ-secretase complex (presenilin, APH-1, PEN-2, and nicastrin). Instead, GSM photoprobes labeled APP, APP carboxyl terminal fragments, and Aβ. Substrate labeling was competed by other GSMs, and labeling of a synthetic APP γ-secretase substrate was more efficient than labeling of a synthetic Notch substrate. Peptide mapping studies localized the region of interaction to residues 28–36 of Aβ, a region critical for Aβ aggregation. Consistent with these data, we demonstrate that numerous compounds known to interact with this region of Aβ or Aβ amyloid act as GSMs, and that mutation of the GSM binding site in APP alters the sensitivity of the substrate to GSMs. These findings suggest that substrate targeting by GSMs mechanistically links two therapeutic actions, alteration in Aβ42 production and inhibition of Aβ aggregation, that may synergistically reduce Aβ deposition in AD.