Amyloid precursor protein (APP) and iron both play pivotal roles in the central nervous system, but whether and how iron influences the processing of endogenous APP in neurons remain unclear. Here, we investigated the regulatory effects and underlying mechanisms of iron on non-amyloidogenic and amyloidogenic processing of APP in rat primary cortical neurons. Treatment of the neurons with ferric ammonium citrate (FAC, 100 μmol/L) markedly facilitated the non-amyloidogenic processing of APP, as evidenced by a robust increase in α-secretase-derived carboxy-terminal fragment α (CTFα). Furthermore, the distribution of sAPPα was altered after iron treatment, and sAPPα remained in the cellular lysates instead of being secreted into the extracellular milieu. Moreover, the levels of APP amyloidogenic products, including sAPPβ and Aβ were both decreased. We further revealed that FAC did not alter the expression of β-secretase, but significantly suppressed its enzymatic activity in iron-treated neurons. In a cell-free β-secretase activity assay, FAC dose-dependently inhibited the activity of purified β-secretase with an IC value of 21.67 μmol/L. Our data provide the first evidence that iron overload alters the neuronal sAPPα distribution and directly inhibits β-secretase activity. These findings shed light on the regulatory mechanism of bio-metals on APP processing.
Aim: To study the conformational changes of Aβ 42 and discover novel inhibitors of both Aβ 42 aggregation and β-secretase (BACE1). Methods: A molecular dynamics (MD) simulation at a microsecond level was performed to explore stable conformations of Aβ 42 monomer in aqueous solution. Subsequently, structure-based virtual screening was used to search for inhibitors of both Aβ 42 aggregation and BACE1. Protein purification and in vitro activity assays were performed to validate the inhibition of the compounds identified via virtual screening. Results: The initial α-helical conformation of Aβ 42 , which was unstable in aqueous solution, turned into a β-sheet mixed with a coil structure through a transient and fully random coil. The conformation of Aβ 42 mainly comprising β-sheets and coils structure was used for further virtual screening. Five compounds were identified as inhibitors for Aβ 42 aggregation, and one of them, AE-848, was discovered to be a dual inhibitor of both Aβ 42 aggregation and BACE1, with IC 50 values of 36.95 μmol/L and 22.70 μmol/L, respectively. Conclusion: A helical to β-sheet conformational change in Aβ 42 occurred in a 1.8 microsecond MD simulation. The resulting β-sheet structure of the peptide is an appropriate conformation for the virtual screening of inhibitors against Aβ 42 aggregation. Five compounds were identified as inhibitors of Aβ 42 aggregation by in vitro activity assays. It was particularly interesting to discover a dual inhibitor that targets both Aβ 42 aggregation and BACE1, the two crucial players in the pathogenesis of Alzheimer's disease.
Aim: To identify a small molecule L655,240 as a novel β-secretase (BACE1) inhibitor and to investigate its effects on β-amyloid (Aβ) generation in vitro. Methods: Fluorescence resonance energy transfer (FRET) was used to characterize the inhibitory effect of L655,240 on BACE1. Surface plasmon resonance (SPR) technology-based assay was performed to study the binding affinity of L655,240 for BACE1. The selectivity of L655,240 toward BACE1 over other aspartic proteases was determined with enzymatic assay. The effects of L655,240 on Aβ40, Aβ42, and sAPPβ production were studied in HEK293 cells stably expressing APP695 Swedish mutant K595N/M596L (HEK293-APPswe cells). The activities of BACE1, γ-secretase and α-secretase were assayed, and both the mRNA and protein levels of APP and BACE1 were evaluated using real-time PCR (RT-PCR) and Western blot analysis. Results: L655,240 was determined to be a competitive, selective BACE1 inhibitor (IC 50 =4.47±1.37 μmol/L), which bound to BACE1 directly (K D =17.9±0.72 μmol/L). L655,240 effectively reduced Aβ40, Aβ42, and sAPPβ production by inhibiting BACE1 without affecting the activities of γ-secretase and α-secretase in HEK293-APPswe cells. L655,240 has no effect on APP and BACE1 mRNA or protein levels in HEK293-APPswe cells. Conclusion:The small molecule L655,240 is a novel BACE1 inhibitor that can effectively decreases Aβ production in vitro, thereby highlighting its therapeutic potential for the treatment of Alzheimer's disease.
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