Novel -Secretase Enzyme Modulators Directly Target Presenilin Protein

Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, 80336 Munich, Germany.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2011; 286(43):37181-6. DOI: 10.1074/jbc.C111.276972
Source: PubMed


γ-Secretase is essential for the generation of the neurotoxic 42-amino acid amyloid β-peptide (Aβ(42)). The aggregation-prone hydrophobic peptide, which is deposited in Alzheimer disease (AD) patient brain, is generated from a C-terminal fragment of the β-amyloid precursor protein by an intramembrane cleavage of γ-secretase. Because Aβ(42) is widely believed to trigger AD pathogenesis, γ-secretase is a key AD drug target. Unlike inhibitors of the enzyme, γ-secretase modulators (GSMs) selectively lower Aβ(42) without interfering with the physiological function of γ-secretase. The molecular target(s) of GSMs and hence the mechanism of GSM action are not established. Here we demonstrate by using a biotinylated photocross-linkable derivative of highly potent novel second generation GSMs that γ-secretase is a direct target of GSMs. The GSM photoprobe specifically bound to the N-terminal fragment of presenilin, the catalytic subunit of γ-secretase, but not to other γ-secretase subunits. Binding was differentially competed by GSMs of diverse structural classes, indicating the existence of overlapping/multiple GSM binding sites or allosteric alteration of the photoprobe binding site. The β-amyloid precursor protein C-terminal fragment previously implicated as the GSM binding site was not targeted by the compound. The identification of presenilin as the molecular target of GSMs directly establishes allosteric modulation of enzyme activity as a mechanism of GSM action and may contribute to the development of therapeutically active GSMs for the treatment of AD.

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    • "We hope that our observations will provide a motivation for undertaking such challenging studies. Second, particularly intriguing and potentially useful is our discovery that compounds known to modulate g-secretase activity (De Strooper and Annaert, 2010; Ebke et al., 2011; Eriksen et al., 2003; Fraering et al., 2004; Golde et al., 2013; Jumpertz et al., 2012; Kukar et al., 2008; Ohki et al., 2011) also affect rhomboid proteolysis. This observation raises the possibility that the holistic effect of g-secretase modulation may also involve simultaneous changes in the cell membrane. "
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    ABSTRACT: Rhomboid proteases are integral membrane enzymes that regulate cell signaling, adhesion, and organelle homeostasis pathways, making substrate specificity a key feature of their function. Interestingly, we found that perturbing the membrane pharmacologically in living cells had little effect on substrate processing but induced inappropriate cleavage of nonsubstrates by rhomboid proteases. A subclass of drugs known to modulate γ-secretase activity acted on the membrane directly and induced nonsubstrate cleavage by rhomboid proteases but left true substrate cleavage sites unaltered. These observations highlight an active role for the membrane in guiding rhomboid selectivity and caution that membrane-targeted drugs should be evaluated for cross-activity against membrane-resident enzymes that are otherwise unrelated to the intended drug target. Furthermore, some γ-secretase-modulating activity or toxicity could partly result from global membrane effects.
    Full-text · Article · Aug 2014 · Cell Reports
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    • "Certain non-steroidal anti-inflammatory drugs (NSAIDs), such as Sulindac sulfide and Flurbiprofen, apart from inhibiting the COX enzyme also act as potent GSMs. These GSMs have been reported to bind directly to the APP's C-terminal trans-membrane domain (APP TMD) and interfere with the dimerization of APP [4] there by increasing the accessibility of c38 cleavage site of APP compared to the c42 cleavage site. This results in the production of the shorter Ab38 peptide rather than Ab42 [8]. "
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    ABSTRACT: Recently, γ-secretase modulators (GSM) have been shown to interact directly with the amyloid precursor protein (APP) and simultaneously inhibit the activity of the Presenilin domain of γ-secretase. A clear understanding of the molecular recognition pathways by which GSM can target both γ-secretase and Aβ precursor protein can lead to the development of more effective inhibitors. To examine whether this direct interaction with APP affects the downstream Aβ fibril formation, we chose to investigate three different molecules in this study: Sulindac sulfide, Segmacestat and E2012 from the class of generation I GSMs, γ-secretase inhibitors (GSI), and generation II GSM molecules, respectively. Firstly, through NMR based ligand titration, we identified that Sulindac Sulfide and Segmacestat interact strongly with Aβ40 monomers, whereas E2012 does not. Secondly, using saturation transfer difference (STD) NMR experiments, we found that all three molecules bind equally well with Aβ40 fibrils. To determine if these interactions with the monomer/fibril lead to a viable inhibition of the fibrillation process, we designed an NMR based time-dependent assay and accurately distinguished the inhibitors from the non-inhibitors within a short period of 12 hours. Based on this pre-seeded fibril assay, we conclude that none of these molecules inhibit the ongoing fibrillation, rather ligands such as Semagacestat and E2012 accelerated the rate of aggregation.
    Full-text · Article · Apr 2014 · Biochemical and Biophysical Research Communications
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    • "Using immunoprecipitation/mass spectrometry (IP/MS) analysis of conditioned media from Satori compound-treated versus control 2B7 cells, it was observed that the total Aβ levels are maintained with concomitant lowering of Aβ38 and Aβ42 and increases in Aβ37 and Aβ39 [34]. Furthermore, since increasing substrate levels do not result in an IC50 shift, it is likely that SPI-1865 binds to the gamma-secretase complex as do other GSMs [35-37] instead of the APP substrate [33,34]. In the studies described here, the effects of SPI-1865 on Aβ38, Aβ40 and Aβ42 in both wild-type and transgenic animals were examined. "
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    ABSTRACT: Introduction Modulation of the gamma-secretase enzyme, which reduces the production of the amyloidogenic Aβ42 peptide while sparing the production of other Aβ species, is a promising therapeutic approach for the treatment of Alzheimer's disease. Satori has identified a unique class of small molecule gamma-secretase modulators (GSMs) capable of decreasing Aβ42 levels in cellular and rodent model systems. The compound class exhibits potency in the nM range in vitro and is selective for lowering Aβ42 and Aβ38 while sparing Aβ40 and total Aβ levels. In vivo, a compound from the series, SPI-1865, demonstrates similar pharmacology in wild-type CD1 mice, Tg2576 mice and Sprague Dawley rats. Methods Animals were orally administered either a single dose of SPI-1865 or dosed for multiple days. Aβ levels were measured using a sensitive plate-based ELISA system (MSD) and brain and plasma exposure of drug were assessed by LC/MS/MS. Results In wild-type mice using either dosing regimen, brain Aβ42 and Aβ38 levels were decreased upon treatment with SPI-1865 and little to no statistically meaningful effect on Aβ40 was observed, reflecting the changes observed in vitro. In rats, brain Aβ levels were examined and similar to the mouse studies, brain Aβ42 and Aβ38 were lowered. Comparable changes were also observed in the Tg2576 mice, where Aβ levels were measured in brain as well as plasma and CSF. Conclusions Taken together, these data indicate that SPI-1865 is orally bioavailable, brain penetrant, and effective at lowering Aβ42 in a dose responsive manner. With this unique profile, the class of compounds represented by SPI-1865 may be a promising new therapy for Alzheimer's disease.
    Full-text · Article · Apr 2013 · Alzheimer's Research and Therapy
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