Piperidine acetic acid based γ-secretase modulators directly bind to Presenilin-1.
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ABSTRACT:
SUBMITTER: Crump CJ
PROVIDER: S-EPMC3249837 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:?-Secretase is essential for the generation of the neurotoxic 42-amino acid amyloid ?-peptide (A?(42)). The aggregation-prone hydrophobic peptide, which is deposited in Alzheimer disease (AD) patient brain, is generated from a C-terminal fragment of the ?-amyloid precursor protein by an intramembrane cleavage of ?-secretase. Because A?(42) is widely believed to trigger AD pathogenesis, ?-secretase is a key AD drug target. Unlike inhibitors of the enzyme, ?-secretase modulators (GSMs) selectively lower A?(42) without interfering with the physiological function of ?-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 ?-secretase is a direct target of GSMs. The GSM photoprobe specifically bound to the N-terminal fragment of presenilin, the catalytic subunit of ?-secretase, but not to other ?-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 ?-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.
| S-EPMC3199465 | biostudies-literature
Project description:A central pathogenic event of Alzheimer's disease (AD) is the accumulation of the A?42 peptide, which is generated from amyloid-? precursor protein (APP) via cleavages by ?- and ?-secretase. We have developed a class of soluble 2-aminothiazole ?-secretase modulators (SGSMs) that preferentially decreases A?42 levels. However, the effects of SGSMs in AD animals and cells expressing familial AD mutations, as well as the mechanism of ?-secretase modulation remain largely unknown. Here, a representative of this SGSM scaffold, SGSM-36, was investigated using animals and cells expressing FAD mutations. SGSM-36 preferentially reduced A?42 levels without affecting either ?- and ?-secretase processing of APP nor Notch processing. Furthermore, an allosteric site was identified within the ?-secretase complex that allowed access of SGSM-36 using cell-based, fluorescence lifetime imaging microscopy analysis. Collectively, these studies provide mechanistic insights regarding SGSMs of this class and reinforce their therapeutic potential in AD.
| S-EPMC5652037 | biostudies-literature
Project description:Presenilin is the catalytic component of the γ-secretase complex, a membrane-embedded aspartyl protease that plays a central role in biology and in the pathogenesis of Alzheimer's disease. Upon assembly with its three protein cofactors (nicastrin, Aph-1 and Pen-2), presenilin undergoes autoproteolysis into two subunits, each of which contributes one of the catalytic aspartates to the active site. A family of presenilin homologs, including signal peptide peptidase, possess proteolytic activity without the need for other protein factors, and these simpler intramembrane aspartyl proteases have given insight into the action of presenilin within the γ-secretase complex. Cellular and molecular studies support a nine-transmembrane topology for presenilins and their homologs, and small-molecule inhibitors and cysteine scanning with crosslinking have suggested certain presenilin residues and regions that contribute to substrate recognition and handling. Identification of partial complexes has also offered clues to protein-protein interactions within the γ-secretase complex. Biophysical methods have allowed 3D views of the γ-secretase complex and presenilins. Most recently, the crystal structure of a microbial presenilin homolog has confirmed a nine-transmembrane topology and intramembranous location and proximity of the two conserved and essential aspartates. The crystal structure also provides a platform for the formulation of specific hypotheses regarding substrate interaction and catalysis as well as the pathogenic mechanism of Alzheimer-causing presenilin mutations. This article is part of a Special Issue entitled: Intramembrane Proteases.
| S-EPMC3801419 | biostudies-literature
Project description:?-Secretase modulators (GSMs) have received much attention as potential therapeutic agents for Alzheimer's disease (AD). GSMs increase the ratio between short and long forms of the amyloid-? (A?) polypeptides produced by ?-secretase and thereby decrease the amount of the toxic amyloid species. However, the mechanism of action of these agents is still poorly understood. One recent paper [Richter et al. (2010) Proc. Natl. Acad. Sci. U. S. A.107, 14597-14602] presented data that were interpreted to support direct binding of the GSM sulindac sulfide to A?(42), supporting the notion that GSM action is linked to direct binding of these compounds to the A? domain of its immediate precursor, the 99-residue C-terminal domain of the amyloid precursor protein (C99, also known as the ?-CTF). Here, contrasting results are presented that indicate there is no interaction between monomeric sulindac sulfide and monomeric forms of A?42. Instead, it was observed that sulindac sulfide is itself prone to form aggregates that can bind nonspecifically to A?42 and trigger its aggregation. This observation, combined with data from previous work [Beel et al. (2009) Biochemistry48, 11837-11839], suggests both that the poor behavior of some NSAID-based GSMs in solution may obscure results of binding assays and that NSAID-based GSMs do not function by directly targeting C99. It was also observed that another GSM, flurbiprofen, fails to bind to monomeric A?42 or to C99 reconstituted into bilayered lipid vesicles. These results disfavor the hypothesis that these NSAID-based GSMs exert their modulatory effect by directly targeting a site located in the A?42 domain of free C99.
| S-EPMC3272834 | biostudies-literature
Project description:Selective lowering of Abeta42 levels (the 42-residue isoform of the amyloid-beta peptide) with small-molecule gamma-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer's disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the gamma-secretase complex, but instead labelled the beta-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-beta peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP gamma-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28-36 of amyloid-beta, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-beta act as GSMs, and some GSMs alter the production of cell-derived amyloid-beta oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Abeta42 production and inhibition of amyloid-beta aggregation, which may synergistically reduce amyloid-beta deposition in Alzheimer's disease. These data also demonstrate the existence and feasibility of 'substrate targeting' by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of 'druggable' targets.
| S-EPMC2678541 | biostudies-literature
Project description:In neuronal cells, presenilin-dependent gamma-secretase activity cleaves amyloid precursor proteins to release Abeta peptides, and also catalyzes the release of the intracellular domain of the transmembrane receptor Notch. Accumulation of aberrant Abeta peptides appears to be causally related to Alzheimer's disease. Inhibition of Abeta peptide production is therefore a potential target for therapeutic intervention. Notch proteins play an important role in cell fate determination in many different organisms and at different stages of development, for example in mammalian T cell development. We therefore addressed whether structurally diverse gamma-secretase inhibitors impair Notch function by studying thymocyte development in murine fetal thymic organ cultures. Here we show that high concentrations of the most potent inhibitors blocked thymocyte development at the most immature stage. In contrast, lower concentrations or less potent inhibitors impaired differentiation at a later stage, most notably suppressing the development of CD8 single-positive T cells. These phenotypes are consistent with an impairment of Notch signaling by gamma-secretase inhibitors and define a strict Notch dose dependence of consecutive stages during thymocyte development.
| S-EPMC55417 | biostudies-literature
Project description:BACKGROUND:γ-Secretase is a multiprotein protease that cleaves amyloid protein precursor (APP) and other type I transmembrane proteins. It has two catalytic subunits, presenilins 1 and 2 (PS1 and 2). In our previous report, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates and slightly different potencies of PS1 versus PS2 inhibition for select γ-secretase inhibitors (GSIs) on various substrates. In this study, we investigated whether γ-secretase modulators (GSMs) and inverse γ-secretase modulators (iGSMs) modulate γ-secretase processivity using multiple different substrates. We next used HEK 293T cell lines in which PSEN1 or PSEN2 was selectively knocked out to investigate processivity and response to GSMs and iGSMs. METHODS:For cell-free γ-secretase cleavage assay, recombinant substrates were incubated with CHAPSO-solubilized CHO or HEK 293T cell membrane with GSMs or iGSMs in suitable buffer. For cell-based assay, cDNA encoding substrates were transfected into HEK 293T cells. Cells were then treated with GSMs or iGSMs, and conditioned media were collected. Aβ and Aβ-like peptide production from cell-free and cell-based assay were measured by ELISA and mass spectrometry. RESULT:These studies demonstrated that GSMs are highly selective for effects on APP, whereas iGSMs have a more promiscuous effect on many substrates. Surprisingly, iGSMs actually appear to act as like GSIs on select substrates. The data with PSEN1 or PSEN2 knocked out HEK 293T reveal that PS1 has higher processivity and response to GSMs than PS2, but PS2 has higher response to iGSM. CONCLUSION:Collectively, these data indicate that GSMs are likely to have limited target-based toxicity. In addition, they show that iGSMs may act as substrate-selective GSIs providing a potential new route to identify leads for substrate-selective inhibitors of certain γ-secretase-mediated signaling events. With growing concerns that long-term β-secretase inhibitor is limited by target-based toxicities, such data supports continued development of GSMs as AD prophylactics.
| S-EPMC7236921 | biostudies-literature
Project description:A complex composed of presenilin (PS), nicastrin, PEN-2, and APH-1 is absolutely required for ?-secretase activity in vivo. Evidence has emerged to suggest a role for PS as the catalytic subunit of ?-secretase, but it has not been established that PS is catalytically active in the absence of associated subunits. We now report that bacterially synthesized, recombinant PS (rPS) reconstituted into liposomes exhibits ?-secretase activity. Moreover, an rPS mutant that lacks a catalytic aspartate residue neither exhibits reconstituted ?-secretase activity nor interacts with a transition-state ?-secretase inhibitor. Importantly, we demonstrate that rPS harboring mutations that cause early onset familial Alzheimer's disease (FAD) lead to elevations in the ratio of A?42 to A?40 peptides produced from a wild-type APP substrate and that rPS enhances the A?42/A?40 peptide ratio from FAD-linked mutant APP substrates, findings that are entirely consistent with the results obtained in in vivo settings. Thus, ?-secretase cleavage specificity is an inherent property of the polypeptide. Finally, we demonstrate that PEN2 is sufficient to promote the endoproteolysis of PS1 to generate the active form of ?-secretase. Thus, we conclusively establish that activated PS is catalytically competent and the bimolecular interaction of PS1 and PEN2 can convert the PS1 zymogen to an active protease.
| S-EPMC3003001 | biostudies-literature
Project description:gamma-Secretase is known to play a pivotal role in the pathogenesis of Alzheimer disease through production of amyloidogenic Abeta42 peptides. Early onset familial Alzheimer disease mutations in presenilin (PS), the catalytic core of gamma-secretase, invariably increase the Abeta42:Abeta40 ratio. However, the mechanism by which these mutations affect gamma-secretase complex formation and cleavage specificity is poorly understood. We show that our in vitro assay system recapitulates the effect of PS1 mutations on the Abeta42:Abeta40 ratio observed in cell and animal models. We have developed a series of small molecule affinity probes that allow us to characterize active gamma-secretase complexes. Furthermore we reveal that the equilibrium of PS1- and PS2-containing active complexes is dynamic and altered by overexpression of Pen2 or PS1 mutants and that formation of PS2 complexes is positively correlated with increased Abeta42:Abeta40 ratios. These data suggest that perturbations to gamma-secretase complex equilibrium can have a profound effect on enzyme activity and that increased PS2 complexes along with mutated PS1 complexes contribute to an increased Abeta42:Abeta40 ratio.
| S-EPMC2631949 | biostudies-literature
Project description:Presenilin (PS), a causative molecule of familial Alzheimer disease, acts as a crucial component of the ?-secretase complex, which is required to cleave type I transmembrane proteins such as amyloid precursor protein and Notch. However, it also functions through ?-secretase-independent pathways. Recent reports suggested that PS could regulate the expression level of cell surface receptors, including the PDGF and EGF receptors, followed by modulating their downstream pathways via ?-secretase-independent mechanisms. The main purpose of this study was to clarify the effect of PS on expression of the insulin receptor (IR) as well as on insulin signaling. Here, we demonstrate that PS inhibited IR transcription and reduced IR expression, and this was followed by down-regulation of insulin signaling. Moreover, we suggest that neither ?-secretase activity nor Wnt/?-catenin signaling can reduce the expression of IR, but a PS-mediated increase in the intracellular Ca(2+) level can be associated with it. These results clearly indicate that PS can functionally regulate insulin signaling by controlling IR expression.
| S-EPMC3137102 | biostudies-literature