Novel γ-secretase enzyme modulators directly target presenilin protein.
ABSTRACT 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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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.Cell Reports 08/2014; 8(5). DOI:10.1016/j.celrep.2014.07.039 · 7.21 Impact Factor
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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.Biochemical and Biophysical Research Communications 04/2014; 447(4). DOI:10.1016/j.bbrc.2014.04.041 · 2.28 Impact Factor
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ABSTRACT: Presenilins (PSs) are catalytic components of the γ-secretase proteolytic complexes that produce Aβ and cell signaling peptides. γ-Secretase substrates are mostly membrane-bound peptides derived following proteolytic cleavage of the extracellular domain of type I transmembrane proteins. Recent work reveals that γ-secretase substrate processing is regulated by proteins termed γ-secretase substrate recruiting factors (γSSRFs) that bridge substrates to γ-secretase complexes. These factors constitute novel targets for pharmacological control of specific γ-secretase products, such as Aβ and signaling peptides. PS familial Alzheimer's disease (FAD) mutants cause a loss of γ-secretase cleavage function at epsilon sites of substrates thus inhibiting production of cell signaling peptides while promoting accumulation of uncleaved toxic substrates. Importantly, γ-secretase inhibitors may cause toxicity in vivo by similar mechanisms. Here we review novel mechanisms that control γ-secretase substrate selection and cleavage and examine their relevance to AD.Progress in Neurobiology 05/2012; 98(2):166-75. DOI:10.1016/j.pneurobio.2012.05.006 · 10.30 Impact Factor