piperidines and sulindac-sulfide

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

γ-Secretase-mediated cleavage of amyloid precursor protein (APP) results in the production of Alzheimer disease-related amyloid-β (Aβ) peptides. The Aβ42 peptide in particular plays a pivotal role in Alzheimer disease pathogenesis and represents a major drug target. Several γ-secretase modulators (GSMs), such as the nonsteroidal anti-inflammatory drugs (R)-flurbiprofen and sulindac sulfide, have been suggested to modulate the Alzheimer-related Aβ production by targeting the APP. Here, we describe novel GSMs that are selective for Aβ modulation and do not impair processing of Notch, EphB2, or EphA4. The GSMs modulate Aβ both in cell and cell-free systems as well as lower amyloidogenic Aβ42 levels in the mouse brain. Both radioligand binding and cellular cross-competition experiments reveal a competitive relationship between the AstraZeneca (AZ) GSMs and the established second generation GSM, E2012, but a noncompetitive interaction between AZ GSMs and the first generation GSMs (R)-flurbiprofen and sulindac sulfide. The binding of a (3)H-labeled AZ GSM analog does not co-localize with APP but overlaps anatomically with a γ-secretase targeting inhibitor in rodent brains. Combined, these data provide compelling evidence of a growing class of in vivo active GSMs, which are selective for Aβ modulation and have a different mechanism of action compared with the original class of GSMs described.

Topics: Alanine; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Azepines; Binding, Competitive; Brain; Carbamates; Cell-Free System; Dibenzazepines; Dipeptides; Drug Interactions; Female; Flurbiprofen; Guinea Pigs; HEK293 Cells; Humans; Imidazoles; Mice; Mice, Inbred C57BL; Piperidines; Protein Binding; Protein Processing, Post-Translational; Pyrans; Pyridines; Pyrimidines; Rats; Receptor, EphA4; Receptor, EphB2; Receptors, Notch; Sulfonamides; Sulindac