amyloid-beta-peptides and sulindac-sulfide

Alzheimer's disease is characterized by deposition of the amyloid β-peptide (Aβ) in brain tissue of affected individuals. In recent years, many potential lead structures have been suggested that can potentially be used for diagnosis and therapy. However, the mode of action of these compounds is so far not understood. Among these small molecules, the nonsteroidal anti-inflammatory drug (NSAID) sulindac sulfide received a lot of attention. In this manuscript, we characterize the interaction between the monomeric Aβ peptide and the NSAID sulindac sulfide. We find that sulindac sulfide efficiently depletes the pool of toxic oligomers by enhancing the rate of fibril formation. In vitro, sulindac sulfide forms colloidal particles which catalyze the formation of fibrils. Aggregation is immediate, presumably by perturbing the supersaturated Aβ solution. We find that sulindac sulfide induced Aβ aggregates are structurally homogeneous. The C-terminal part of the peptide adopts a β-sheet structure, whereas the N-terminus is disordered. The salt bridge between D23 and K28 is present, similar as in wild type fibril structures. (13)C-(19)F transferred echo double resonance experiments suggest that sulindac sulfide colocalizes with the Aβ peptide in the aggregate.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Anti-Inflammatory Agents, Non-Steroidal; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Molecular Sequence Data; Peptide Fragments; Protein Aggregates; Sulindac

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, Semagacestat 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 Semagacestat 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 12h. 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.

Topics: Alanine; Amyloid; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Azepines; Binding Sites; Humans; Imidazoles; Kinetics; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments; Piperidines; Protease Inhibitors; Protein Conformation; Protein Multimerization; Sulindac

The pathogenesis of Alzheimer's disease (AD) is correlated with the toxicity of amyloid β-peptide (Aβ), especially Aβ42. γ-Secretase modulators (GSMs) are compounds that alter production of Aβ42 without interfering with the physiological function of γ-secretase. Aβ42-lowering GSMs have been studied with the hope of using them as therapeutic or prophylactic drugs for AD. However, the mechanism of action of GSMs is not well defined. We examined the effect of Aβ42-lowering GSMs on model cells producing large amounts of Aβ42: CHO cells expressing CTF1-51, a precursor peptide of Aβ that is mainly cleaved into Aβ42. Our results indicate that the effect of GSM in the model was weak. Thus, we conclude that CTF1-51 cleavage mainly yields Aβ42 and suppresses the effects of some GSMs, a phenomenon that may be related to their mechanism of action.

Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; CHO Cells; Cricetinae; Cricetulus; Ibuprofen; Peptide Fragments; Sulindac

Gamma-secretase is a member of an unusual class of proteases with intramembrane catalytic sites. This enzyme cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Biochemical and genetic studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin (PS) composed of its N- and C-terminal fragments (PS-NTF/CTF), a mature glycosylated form of nicastrin (NCT), Aph-1, and Pen-2. Recent data from studies in Drosophila, mammalian, and yeast cells suggest that PS, NCT, Aph-1, and Pen-2 are necessary and sufficient to reconstitute gamma-secretase activity. However, many unresolved issues, in particular the possibility of other structural or regulatory components, would be resolved by actually purifying the enzyme. Here, we report a detailed, multistep purification procedure for active gamma-secretase and an initial characterization of the purified protease. Extensive mass spectrometry of the purified proteins strongly suggests that PS-NTF/CTF, mNCT, Aph-1, and Pen-2 are the components of active gamma-secretase. Using the purified gamma-secretase, we describe factors that modulate the production of specific Abeta species: (1) phosphatidylcholine and sphingomyelin dramatically improve activity without changing cleavage specificity within an APP substrate; (2) increasing CHAPSO concentrations from 0.1 to 0.25% yields a approximately 100% increase in Abeta42 production; (3) exposure of an APP-based recombinant substrate to 0.5% SDS modulates cleavage specificity from a disease-mimicking pattern (high Abeta42/43) to a physiological pattern (high Abeta40); and (4) sulindac sulfide directly and preferentially decreases Abeta42 cleavage within the purified complex. Taken together, our results define a procedure for purifying active gamma-secretase and suggest that the lipid-mediated conformation of both enzyme and substrate regulate the production of the potentially neurotoxic Abeta42 and Abeta43 peptides.

Topics: Amino Acid Sequence; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; CHO Cells; Cricetinae; Endopeptidases; gamma-Aminobutyric Acid; Humans; Hydrolysis; Kinetics; Lipids; Macromolecular Substances; Mass Spectrometry; Membrane Glycoproteins; Membrane Proteins; Mice; Molecular Sequence Data; Oligopeptides; Peptide Fragments; Peptide Hydrolases; Peptides; Presenilin-1; Protease Inhibitors; Receptors, Notch; Sodium Dodecyl Sulfate; Substrate Specificity; Sulindac; Triglycerides

Epidemiological studies have documented a reduced prevalence of Alzheimer's disease among users of nonsteroidal anti-inflammatory drugs (NSAIDs). It has been proposed that NSAIDs exert their beneficial effects in part by reducing neurotoxic inflammatory responses in the brain, although this mechanism has not been proved. Here we report that the NSAIDs ibuprofen, indomethacin and sulindac sulphide preferentially decrease the highly amyloidogenic Abeta42 peptide (the 42-residue isoform of the amyloid-beta peptide) produced from a variety of cultured cells by as much as 80%. This effect was not seen in all NSAIDs and seems not to be mediated by inhibition of cyclooxygenase (COX) activity, the principal pharmacological target of NSAIDs. Furthermore, short-term administration of ibuprofen to mice that produce mutant beta-amyloid precursor protein (APP) lowered their brain levels of Abeta42. In cultured cells, the decrease in Abeta42 secretion was accompanied by an increase in the Abeta(1-38) isoform, indicating that NSAIDs subtly alter gamma-secretase activity without significantly perturbing other APP processing pathways or Notch cleavage. Our findings suggest that NSAIDs directly affect amyloid pathology in the brain by reducing Abeta42 peptide levels independently of COX activity and that this Abeta42-lowering activity could be optimized to selectively target the pathogenic Abeta42 species.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspartic Acid Endopeptidases; Brain; CHO Cells; Cricetinae; Disease Models, Animal; Endopeptidases; Enzyme-Linked Immunosorbent Assay; Humans; Ibuprofen; Indomethacin; Mass Spectrometry; Mice; Mice, Transgenic; Peptide Fragments; Prostaglandin-Endoperoxide Synthases; Sulindac; Tumor Cells, Cultured