Bepridil and Amiodarone Simultaneously Target the Alzheimer's Disease - and -Secretase via Distinct Mechanisms

German Center for Neurodegenerative Diseases Munich and Adolf Butenandt-Institute, Biochemistry, University of Munich, 80336 Munich, Germany.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 06/2010; 30(26):8974-83. DOI: 10.1523/JNEUROSCI.1199-10.2010
Source: PubMed


The two proteases beta-secretase and gamma-secretase generate the amyloid beta peptide and are drug targets for Alzheimer's disease. Here we tested the possibility of targeting the cellular environment of beta-secretase cleavage instead of the beta-secretase enzyme itself. beta-Secretase has an acidic pH optimum and cleaves the amyloid precursor protein in the acidic endosomes. We identified two drugs, bepridil and amiodarone, that are weak bases and are in clinical use as calcium antagonists. Independently of their calcium-blocking activity, both compounds mildly raised the membrane-proximal, endosomal pH and inhibited beta-secretase cleavage at therapeutically achievable concentrations in cultured cells, in primary neurons, and in vivo in guinea pigs. This shows that an alkalinization of the cellular environment could be a novel therapeutic strategy to inhibit beta-secretase. Surprisingly, bepridil and amiodarone also modulated gamma-secretase cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target beta- and gamma-secretase through distinct molecular mechanisms. In addition to Alzheimer's disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins.

Download full-text


Available from: Uli Zeitschel, Jul 09, 2014
52 Reads
  • Source
    • "The PS1 lines were then stably transfected with YC3.6/pcDNA3 construct (kindly provided by Dr. A. Miyawaki) and respectively isolated by G418 antibiotic resistance leading to generation of double stable lines [20]. The APP-, C99- and APPsw/PS1-M146L-overexpressing HEK293 lines were kindly provided by Dr. S. Lichtenthaler and Dr. H. Steiner and cultured as it has been previously described [21], [22]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Disrupted intracellular calcium homeostasis is believed to occur early in the cascade of events leading to Alzheimer's disease (AD) pathology. Particularly familial AD mutations linked to Presenilins result in exaggerated agonist-evoked calcium release from endoplasmic reticulum (ER). Here we report the development of a fully automated high-throughput calcium imaging assay utilizing a genetically-encoded FRET-based calcium indicator at single cell resolution for compound screening. The established high-throughput screening assay offers several advantages over conventional high-throughput calcium imaging technologies. We employed this assay for drug discovery in AD by screening compound libraries consisting of over 20,000 small molecules followed by structure-activity-relationship analysis. This led to the identification of Bepridil, a calcium channel antagonist drug in addition to four further lead structures capable of normalizing the potentiated FAD-PS1-induced calcium release from ER. Interestingly, it has recently been reported that Bepridil can reduce Aβ production by lowering BACE1 activity. Indeed, we also detected lowered Aβ, increased sAPPα and decreased sAPPβ fragment levels upon Bepridil treatment. The latter findings suggest that Bepridil may provide a multifactorial therapeutic modality for AD by simultaneously addressing multiple aspects of the disease.
    PLoS ONE 11/2013; 8(11):e80645. DOI:10.1371/journal.pone.0080645 · 3.23 Impact Factor
  • Source
    • "Primary neuronal cultures were obtained from the cerebral cortex of embryonic day (E16) C57/BL6 mouse embryos, incubated with 200 U of papain (Sigma Aldrich) (30 min at 34°C) and subsequently mechanically dissociated and transduced using lentiviral shRNA knock-down constructs as described [21], [64]. Breeding, maintenance and all experimental procedures on animals were approved and licensed by the Regierung von Oberbayern, Munich and were performed in accordance with German legislation and with the European Communities Council Directive (86/609/EEC). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Regulated intramembrane proteolysis of the amyloid precursor protein (APP) by the protease activities α-, β- and γ-secretase controls the generation of the neurotoxic amyloid β peptide. APLP2, the amyloid precursor-like protein 2, is a homolog of APP, which shows functional overlap with APP, but lacks an amyloid β domain. Compared to APP, less is known about the proteolytic processing of APLP2, in particular in neurons, and the cleavage sites have not yet been determined. APLP2 is cleaved by the β-secretase BACE1 and additionally by an α-secretase activity. The two metalloproteases ADAM10 and ADAM17 have been suggested as candidate APLP2 α-secretases in cell lines. Here, we used RNA interference and found that ADAM10, but not ADAM17, is required for the constitutive α-secretase cleavage of APLP2 in HEK293 and SH-SY5Y cells. Likewise, in primary murine neurons knock-down of ADAM10 suppressed APLP2 α-secretase cleavage. Using mass spectrometry we determined the proteolytic cleavage sites in the APLP2 sequence. ADAM10 was found to cleave APLP2 after arginine 670, whereas BACE1 cleaves after leucine 659. Both cleavage sites are located in close proximity to the membrane. γ-secretase cleavage was found to occur at different peptide bonds between alanine 694 and valine 700, which is close to the N-terminus of the predicted APLP2 transmembrane domain. Determination of the APLP2 cleavage sites enables functional studies of the different APLP2 ectodomain fragments and the production of cleavage-site specific antibodies for APLP2, which may be used for biomarker development.
    PLoS ONE 06/2011; 6(6):e21337. DOI:10.1371/journal.pone.0021337 · 3.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR-DNA binding protein 43-positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties, pharmacological stimulation of GRN production is a promising approach to rescue GRN haploinsufficiency and prevent disease progression. We therefore searched for compounds capable of selectively increasing GRN levels. Here, we demonstrate that four independent and highly selective inhibitors of vacuolar ATPase (bafilomycin A1, concanamycin A, archazolid B, and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, clinically used alkalizing drugs, including chloroquine, bepridil, and amiodarone, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagy, or endocytosis. Importantly, alkalizing reagents rescue GRN deficiency in organotypic cortical slice cultures from a mouse model for GRN deficiency and in primary cells derived from human patients with GRN loss-of-function mutations. Thus, alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically used to prevent GRN-dependent neurodegeneration.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 02/2011; 31(5):1885-94. DOI:10.1523/JNEUROSCI.5757-10.2011 · 6.34 Impact Factor
Show more