Selective modulation of amyloid‐β peptide degradation by flurbiprofen, fenofibrate, and related compounds regulates Aβ levels

Abstract

Gamma‐secretase modulators (GSMs) include selected non‐steroidal anti‐inflammatory drugs such as flurbiprofen that selectively lowers the neurotoxic amyloid‐β peptide Aβ1–42. GSMs are attractive targets for Alzheimer’s disease, in contrast to ‘inverse GSMs,’ such as fenofibrate, which selectively increase the level of Aβ1–42. A methodology for screening of Aβ modulating drugs was developed utilizing an Aβ‐producing neuroblastoma cell line stably transfected with mutant human amyloid precursor protein, immunoprecipitation of Aβ peptides, and mass spectroscopic quantitation of Aβ1–37/Aβ1–38/Aβ1–40/Aβ1–42 using an Aβ internal standard. The unexpected conclusion of this work was that in this system, drug effects are independent of γ‐secretase. The methodology recapitulated reported results for modulation of Aβ by GSMs. However, control experiments in which exogenous Aβ1–40/Aβ1–42 was added (i) to drug‐treated wild‐type cells or (ii) to conditioned media from these wild‐type cells, gave comparable patterns of Aβ modulation. These results, suggesting that drugs modulate the ability of cell‐derived factors to degrade Aβ, was interrogated by adding protease inhibitors and performing molecular weight cut‐off fractionation. The results confirmed that modulation of Aβ1–40/Aβ1–42 was mediated by selective proteolysis. Treatment of N2a cells with flurbiprofen or fenofibric acid selectively enhanced Aβ1–42 clearance by extracellular proteolysis; treatment with HCT‐1026 or fenofibrate (esters of flurbiprofen and fenobric acid) inhibited clearance of Aβ1–40 and Aβ1–42.

Inhibition of Amyloidogenesis by Nonsteroidal Anti-inflammatory Drugs and Their Hybrid Nitrates

“…In contrast, fibric acid, the free acid form of 39 was reported to lower the levels of Aβ 1–42 . 16 A similar observation on Aβ 1–42 modulation was made herein with 1 and its ester, 38 . Whereas, the ester acted as a SARA, the free acid, 1 at 100 µM, selectively lowered Aβ 1–42 and elevated Aβ 1–37 relative to the vehicle control (Fig.…”

“…41–43 In order to study the effect of agents on amyloidogenesis in more detail, we determined the profile of Aβ fragment production from N2a.hAPP cells for selected compounds using an immunoprecipitation MALDI-TOF technique introduced in 2009. 16 This technique quantifies the amyloid fragments Aβ 1–37 , Aβ 1–38 , Aβ 1–40 , and Aβ 1–42 , using Aβ 1–43 as an internal standard, since this fragment is not a product of amyloidogenesis. In the N2a.hAPP amyloidogenesis assay, we observed that the known SARA 39 (200 µM) increased Aβ 1–42 levels threefold but had modest effects on Aβ 1–37 and Aβ 1–38 , although lowering the level of the latter fragment (Fig.…”

“…Further experiments showed that the modulation of Aβ 1–42 and Aβ 1–40 levels by SARA and SALA drugs was mediated by expression of metalloprotease activity induced by drug treatment of cells. 16 Levels of Aβ 1–42 and Aβ 1–40 were measured by the MALDI-TOF method after treatment of N2a cells with drugs for 24 h and transfer of the CM to a solution of Aβ 1–42 and Aβ 1–40 (Fig. 5).…”

“…Studies on this cell line demonstrated that NSAID SALAs stimulate metalloprotease degradation of Aβ 1–40 and Aβ 1–42 . 16 The enhanced proteolysis of Aβ is currently seen as a preferred drug discovery strategy. 17, 18 …”

The roles of GxxxG motif and gamma-secretase components in APP processing

“…A recent study provided another mode of action for so-called secretase modulators 48 . While developing a methodology for screening drugs that can decrease Aβ levels, Abdul-Hay et al found that some -secretase modulators altered Aβ levels in the media even in the absence of cells.…”