Nonspecificity of Binding of γ-Secretase Modulators to the Amyloid Precursor Protein

Abstract

Evidence has been presented (Kukar et al. 2008 Nature 453, 925-929) that certain γ-secretase modulators (GSMs) target the 99 residue C-terminal domain (C99) of the amyloid precursor protein, a substrate of γ-secretase, but not the protease complex itself. Here, NMR results demonstrate a lack of specific binding of these GSMs to monodisperse C99 in LMPG micelles. In addition, results indicate that C99 was likely to have been aggregated in some of the key experiments of the previous work, and that binding of GSMs to these C99 aggregates is also of a non-specific nature.Among the therapeutic targets for Alzheimer's disease (AD), the amyloid pathway has long been paramount (1). Familial early-onset AD (FAD) is associated with autosomal dominant mutations in the amyloid precursor protein (APP) and in the catalytic subunits (presenilin 1 and presenilin 2) of the intramembrane protease that processes it, γ-secretase (2). According to the amyloid hypothesis, oligomeric forms of Aβ are the principal agents underlying disease pathogenesis (1). The Aβ peptide is generated by proteolysis of APP. Cleavage of APP by β-secretase yields C99 2 , which is then heterogeneously processed by γ-secretase to generate Aβ species with a variety of lengths, principally Aβ40 (3). Familial AD is associated with an increase in the Aβ42/Aβ40 ratio, with Aβ42 being the primary species deposited in the brain parenchyma of most individuals with AD (4). Because Aβ is thought to be central to the pathogenesis of AD, inhibiting its production is a potential therapeutic strategy (1). Although significant progress has been made in the identification and development of potent γ-secretase inhibitors, their clinical application has been limited by significant toxicities resulting from interference with processing of other γ-secretase substrates, particularly Notch (5). Indeed, γ-secretase is a highly promiscuous protease with more than sixty identified targets (6).The discovery that a subset of non-steroidal anti-inflammatory drugs could selectively reduce Aβ42 production without abrogating Notch cleavage suggested an alternative therapeutic strategy for AD (7). The Aβ42-lowering activity of these γ-secretase modulators (GSMs) was recapitulated in cell-free assays of γ-secretase activity. Several groups have produced data suggesting that GSMs interact allosterically with presenilin, thereby modifying the enzyme's conformation (8-10). Moreover, GSMs were observed to influence the cleavage of an unrelated substrate by signal peptide peptidase -an enzyme with homology to the presenilin subunit of γ-secretase, suggesting that the modulators interact with the enzyme rather than substrate † This work was supported by grants from the US NIH (PO1 GM080513, to CRS) and from the Alzheimer's Association (IIRG-07-59379, to CRS). A recent report from the Golde laboratory, however, postulates that GSMs specifically target APP and its C-terminal derivatives, providing an alternative explanation to the apparent specificity that GSMs exert on cleavage of C99 (1...

Amyloid beta 42 peptide (Aβ42)-lowering compounds directly bind to Aβ and interfere with amyloid precursor protein (APP) transmembrane dimerization

“…In this respect, the effects of 20 μM indomethacin and 2.5 μM sulindac sulfide can be directly compared to GxxxG mutants G29A and G33A (5,7). From previous NMR measurements with purified and monodisperse C99 in lyso-myristoylphosphatidylglycerol micelles, the authors have concluded that interactions with sulindac sulfide are of nonspecific nature (15), which is seemingly in contradiction with our results obtained from the ToxR assay. Here, in a natural lipid bilayer, Aβ42-lowering compounds affected APP-TMS dimerization, although we cannot specify whether existing dimers are disrupted or if binding to monomers inhibits the reassociation and the dimer formation.…”

“…As sulindac sulfide was the most potent compound to bind to Aβ42, we further analyzed this interaction by NMR spectroscopy. We recorded a solution-state two-dimensional 1 H, 15 N correlation spectrum of Aβ42 in the presence and absence of sulindac sulfide (Fig. 3).…”

“…Evidence was also provided that GSMs target the enzyme's substrate when GSM photoprobes labeled APP (14). However, recent NMR results have questioned specific APP binding sites of GSMs (15). Thus, the precise molecular mechanism by which GSMs modulate Aβ formation is still unclear and the targets will have to be characterized.…”

β-Amyloid Precursor Protein Mutants Respond to γ-Secretase Modulators

“…In addition, the lack of a consistent response to GSMs regarding an inversely correlated production of A␤ 42 and A␤ 38 by PS FAD mutants observed earlier argued against a strict precursor-product relationship between A␤ 42 and A␤ 38 (24,25). Moreover, while this manuscript was in preparation, a biophysical study failed to demonstrate GSMbinding to the APP substrate, however, and suggested that the reported GSM-APP interaction (18) was unspecific (26).…”

Novel γ-Secretase Enzyme Modulators Directly Target Presenilin Protein

“…Certain low potency GSMs such as the NSAIDs flurbiprofen and sulindac sulfide have been reported to bind to the APP CTF as well as to A␤ in the N-terminal third of the APP transmembrane domain (8 -10), although this interaction was questioned by others (11). Another study identified the ␥-secretase subunit PEN-2 as the principal target of non-NSAID-type second generation GSMs (12).…”