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:The intramembrane-cleaving protease γ-secretase catalyzes the last step in the generation of toxic amyloid-β (Aβ) peptides and is a principal therapeutic target in Alzheimer's disease. Both preclinical and clinical studies have demonstrated that inhibition of γ-secretase is associated with prohibitive side effects due to suppression of Notch processing and signaling. Potentially safer are γ-secretase modulators (GSMs), which are small molecules that selectively lower generation of the highly amyloidogenic Aβ42 peptides but spare Notch processing. GSMs with nanomolar potency and favorable pharmacological properties have been described, but the molecular mechanism of GSMs remains uncertain and both the substrate amyloid precursor protein (APP) and subunits of the γ-secretase complex have been proposed as the molecular target of GSMs. We have generated a potent photo-probe based on an acidic GSM that lowers Aβ42 generation with an IC(50) of 290 nM in cellular assays. By combining in vivo photo-crosslinking with affinity purification, we demonstrated that this probe binds the N-terminal fragment of presenilin (PSEN), the catalytic subunit of the γ-secretase complex, in living cells. Labeling was not observed for APP or any of the other γ-secretase subunits. Binding was readily competed by structurally divergent acidic and non-acidic GSMs suggesting a shared mode of action. These findings indicate that potent acidic GSMs target presenilin to modulate the enzymatic activity of the γ-secretase complex.
| S-EPMC3253113 | 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:Neurexins are a large family of neuronal plasma membrane proteins, which function as trans-synaptic receptors during synaptic differentiation. The binding of presynaptic neurexins to postsynaptic partners, such as neuroligins, has been proposed to participate in a signaling pathway that regulates synapse formation/stabilization. The identification of mutations in neurexin genes associated with autism and mental retardation suggests that dysfunction of neurexins may underlie synaptic defects associated with brain disorders. However, the mechanisms that regulate neurexin function at synapses are still unclear. Here, we show that neurexins are proteolytically processed by presenilins (PS), the catalytic components of the γ-secretase complex that mediates the intramembraneous cleavage of several type I membrane proteins. Inhibition of PS/γ-secretase by using pharmacological and genetic approaches induces a drastic accumulation of neurexin C-terminal fragments (CTFs) in cultured rat hippocampal neurons and mouse brain. Neurexin-CTFs accumulate mainly at the presynaptic terminals of PS conditional double knockout (PS cDKO) mice lacking both PS genes in glutamatergic neurons of the forebrain. The fact that loss of PS function enhances neurexin accumulation at glutamatergic terminals mediated by neuroligin-1 suggests that PS regulate the processing of neurexins at glutamatergic synapses. Interestingly, presenilin 1 (PS1) is recruited to glutamatergic terminals mediated by neuroligin-1, thus concentrating PS1 at terminals containing β-neurexins. Furthermore, familial Alzheimer's disease (FAD)-linked PS1 mutations differentially affect β-neurexin-1 processing. Expression of PS1 M146L and PS1 H163R mutants in PS-/- cells rescues the processing of β-neurexin-1, whereas PS1 C410Y and PS1 ΔE9 fail to rescue the processing defect. These results suggest that PS regulate the synaptic function and processing of neurexins at glutamatergic synapses, and that impaired neurexin processing by PS may play a role in FAD.
| S-EPMC3084856 | 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:Severe early-onset familial Alzheimer's disease (FAD) is caused by more than 200 different mutations in the genes coding for presenilin, the catalytic subunit of the 4-subunit protease complex γ-secretase, which cleaves the C99 fragment of the amyloid precursor protein (APP) to produce Aβ peptides. γ-Secretase exists with either of two homologues, PS1 and PS2. All cryo-electron microscopic structures and computational work has so far focused on γ-secretase with PS1, yet PS2 mutations also cause FAD. A central question is thus whether there are structural and dynamic differences between PS1 and PS2. To address this question, we use the cryo-electron microscopic data for PS1 to develop the first structural and dynamic model of PS2-γ-secretase in the catalytically relevant mature membrane-bound state at ambient temperature, equilibrated by three independent 500 ns molecular dynamics simulations. We find that the characteristic nicastrin extra-cellular domain breathing mode and major movements in the cytosolic loop between TM6 and TM7 occur in both PS2- and PS1-γ-secretase. The overall structures and conformational states are similar, suggesting similar catalytic activities. However, at the sequence level, charge-controlled membrane-anchoring is extracellular for PS1 and intracellular for PS2, which suggests different subcellular locations. The tilt angles of the TM2, TM6, TM7 and TM9 helices differ in the two forms of γ-secretase, suggesting that the two proteins have somewhat different substrate processing and channel sizes. Our MD simulations consistently indicated that PS2 retains several water molecules near the catalytic site at the bilayer, as required for catalysis. The possible reasons for the differences of PS1 and PS2 are discussed in relation to their location and function.
| S-EPMC9065803 | biostudies-literature