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The FDA-approved natural product dihydroergocristine reduces the production of the Alzheimer's disease amyloid-β peptides.

Lei X, Yu J, Niu Q, Liu J, Fraering PC, Wu F - Sci Rep (2015)

Bottom Line: Micromolar concentrations of DHEC substantially reduced Aβ levels in different cell types, including a cell line derived from an AD patient.Structure-activity relationship studies implied that the key moiety for inhibiting γ-secretase is the cyclized tripeptide moiety of DHEC.A Surface Plasmon Resonance assay showed that DHEC binds directly to γ-secretase and Nicastrin, with equilibrium dissociation constants (Kd) of 25.7 nM and 9.8 μM, respectively.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
Known γ-secretase inhibitors or modulators display an undesirable pharmacokinetic profile and toxicity and have therefore not been successful in clinical trials for Alzheimer's disease (AD). So far, no compounds from natural products have been identified as direct inhibitors of γ-secretase. To search for bioactive molecules that can reduce the amount of amyloid-beta peptides (Aβ) and that have better pharmacokinetics and an improved safety profile, we completed a screen of ~400 natural products by using cell-based and cell-free γ-secretase activity assays. We identified dihydroergocristine (DHEC), a component of an FDA- (Food and Drug Administration)-approved drug, to be a direct inhibitor of γ-secretase. Micromolar concentrations of DHEC substantially reduced Aβ levels in different cell types, including a cell line derived from an AD patient. Structure-activity relationship studies implied that the key moiety for inhibiting γ-secretase is the cyclized tripeptide moiety of DHEC. A Surface Plasmon Resonance assay showed that DHEC binds directly to γ-secretase and Nicastrin, with equilibrium dissociation constants (Kd) of 25.7 nM and 9.8 μM, respectively. This study offers DHEC not only as a new chemical moiety for selectively modulating the activity of γ-secretase but also a candidate for drug repositioning in Alzheimer's disease.

No MeSH data available.


Related in: MedlinePlus

Dihydroergocristine inhibits the intracellular production of Aβ and the activity of γ-secretase, without affecting Notch processing.(a) Effects of DHEC on endogenous APP-CTF accumulation and Aβ generation in HEK293 cells. HEK293 cells were incubated with DMSO (control), the indicated concentrations of DHEC or 20 μM DAPT in 24-well plates for 24 h, before Western blot analysis of APP-FL and APP-CTF (left panel). The levels of β-actin were used as equal loading controls. The corresponding media from the DMSO-, DHEC- or DAPT- treated groups (n = 3) were collected, and the Aβ total level was measured by ELISA (right panel). The Aβ data are expressed as a percentage of the control value and presented as the means ± sd. Asterisks indicate significant differences (***P < 0.001; one-way ANOVA with Bonferroni’s multiple comparisons tests) in Aβ total production of the treated samples compared with the controls (DMSO). (b) Effects of DHEC on endogenous γ-secretase activity in fibroblast cells from an AD patient. Fibroblast cells from an AD patient carrying the PS1 missense mutation A246E were treated with various compounds, and the levels of APP-FL, APP-CTF and β-actin, as well as Aβ, were measured as described above. The Aβ data are expressed as a percentage of the control value and presented as the means ± sd. (n = 3). Asterisks indicate significant differences (**P < 0.01; ***P < 0.001; one-way ANOVA with Bonferroni’s multiple comparisons tests) in Aβ total production of the samples compared with the controls (DMSO). (c) Effects of DHEC on the cleavage of human Notch1 and APP in HEK293 cells overexpressing the human Notch1 extracellular truncation (NEXT; left panel) and APP (right panel), respectively. After 24  h transient transfection of HEK293 cells with Notch1 NEXT or hAPP plasmids, cells were incubated with DHEC or DAPT at the indicated concentrations for one additional day before Western Blot analysis of NICD (Ab1744) and APP-CTF (C-T15). The levels of β-actin served as equal loading controls. The densitometric quantifications for the Western Blots are shown in Supplementary Fig. S2. For full blots, please see Supplementary Fig. S9.
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f1: Dihydroergocristine inhibits the intracellular production of Aβ and the activity of γ-secretase, without affecting Notch processing.(a) Effects of DHEC on endogenous APP-CTF accumulation and Aβ generation in HEK293 cells. HEK293 cells were incubated with DMSO (control), the indicated concentrations of DHEC or 20 μM DAPT in 24-well plates for 24 h, before Western blot analysis of APP-FL and APP-CTF (left panel). The levels of β-actin were used as equal loading controls. The corresponding media from the DMSO-, DHEC- or DAPT- treated groups (n = 3) were collected, and the Aβ total level was measured by ELISA (right panel). The Aβ data are expressed as a percentage of the control value and presented as the means ± sd. Asterisks indicate significant differences (***P < 0.001; one-way ANOVA with Bonferroni’s multiple comparisons tests) in Aβ total production of the treated samples compared with the controls (DMSO). (b) Effects of DHEC on endogenous γ-secretase activity in fibroblast cells from an AD patient. Fibroblast cells from an AD patient carrying the PS1 missense mutation A246E were treated with various compounds, and the levels of APP-FL, APP-CTF and β-actin, as well as Aβ, were measured as described above. The Aβ data are expressed as a percentage of the control value and presented as the means ± sd. (n = 3). Asterisks indicate significant differences (**P < 0.01; ***P < 0.001; one-way ANOVA with Bonferroni’s multiple comparisons tests) in Aβ total production of the samples compared with the controls (DMSO). (c) Effects of DHEC on the cleavage of human Notch1 and APP in HEK293 cells overexpressing the human Notch1 extracellular truncation (NEXT; left panel) and APP (right panel), respectively. After 24  h transient transfection of HEK293 cells with Notch1 NEXT or hAPP plasmids, cells were incubated with DHEC or DAPT at the indicated concentrations for one additional day before Western Blot analysis of NICD (Ab1744) and APP-CTF (C-T15). The levels of β-actin served as equal loading controls. The densitometric quantifications for the Western Blots are shown in Supplementary Fig. S2. For full blots, please see Supplementary Fig. S9.

Mentions: After primary screening of the compounds in T100 cells, a total of 8 natural products were found to inhibit the cellular activity of γ-secretase, in a dose-dependent manner and with an IC50 < 30 μM. Of these, NSC409663 (DHEC), which was identified from the natural product library of the National Cancer Institute (NCI, Bethesda, USA), was the only compound that affected the activity of γ-secretase in both cell-based and cell-free assays (Figs 1 and 2). DHEC, which has been used for the treatment of glaucoma18, is also a component of the drug ergoloid mesylates. Ergoloid mesylates contains a mixture of four ergot alkaloids (DHEC, dihydroergocornine, α-dihydroergocryptine and β–dihydroergocryptine; refs 14,15). In our study, DHEC had an IC50 value of ~25 μM for inhibiting the activity of γ-secretase in T100 cells without affecting cell viability (Supplementary Fig. S1b). In HEK293 cells, DHEC also caused a significant dose-dependent accumulation of the carboxy-terminal fragments of APP (APP-CTFs, Fig. 1a; left panel; Supplementary Fig. S2a), and 10 μM DHEC resulted in a ~30% reduction in Aβ production (Fig. 1a; right panel), which did not influence the levels of full length APP (APP-FL) or cell viability at all tested doses (Fig. 1a, left panel; Supplementary Fig. S1c), as expected from a γ-secretase inhibitor19. Furthermore, 20 μM DHEC caused the accumulation of APP-CTFs and led to ~35% reduction in total Aβ (Fig. 1b; Supplementary Fig. S2b) in fibroblast cells from an Alzheimer’s disease patient carrying a missense mutation (A246E) in the presenilin 1 (PS1) gene. As predicted, DAPT caused a dose-dependent accumulation of APP-CTFs in HEK293 (Supplementary Fig. S3a) and fibroblast (Supplementary Fig. S3b) cells. Similarly, total Aβ levels were markedly reduced by treatment of both HEK293 and fibroblast cells with DAPT (Supplementary Fig. S3a,b and Fig. 1a,b, right panels).


The FDA-approved natural product dihydroergocristine reduces the production of the Alzheimer's disease amyloid-β peptides.

Lei X, Yu J, Niu Q, Liu J, Fraering PC, Wu F - Sci Rep (2015)

Dihydroergocristine inhibits the intracellular production of Aβ and the activity of γ-secretase, without affecting Notch processing.(a) Effects of DHEC on endogenous APP-CTF accumulation and Aβ generation in HEK293 cells. HEK293 cells were incubated with DMSO (control), the indicated concentrations of DHEC or 20 μM DAPT in 24-well plates for 24 h, before Western blot analysis of APP-FL and APP-CTF (left panel). The levels of β-actin were used as equal loading controls. The corresponding media from the DMSO-, DHEC- or DAPT- treated groups (n = 3) were collected, and the Aβ total level was measured by ELISA (right panel). The Aβ data are expressed as a percentage of the control value and presented as the means ± sd. Asterisks indicate significant differences (***P < 0.001; one-way ANOVA with Bonferroni’s multiple comparisons tests) in Aβ total production of the treated samples compared with the controls (DMSO). (b) Effects of DHEC on endogenous γ-secretase activity in fibroblast cells from an AD patient. Fibroblast cells from an AD patient carrying the PS1 missense mutation A246E were treated with various compounds, and the levels of APP-FL, APP-CTF and β-actin, as well as Aβ, were measured as described above. The Aβ data are expressed as a percentage of the control value and presented as the means ± sd. (n = 3). Asterisks indicate significant differences (**P < 0.01; ***P < 0.001; one-way ANOVA with Bonferroni’s multiple comparisons tests) in Aβ total production of the samples compared with the controls (DMSO). (c) Effects of DHEC on the cleavage of human Notch1 and APP in HEK293 cells overexpressing the human Notch1 extracellular truncation (NEXT; left panel) and APP (right panel), respectively. After 24  h transient transfection of HEK293 cells with Notch1 NEXT or hAPP plasmids, cells were incubated with DHEC or DAPT at the indicated concentrations for one additional day before Western Blot analysis of NICD (Ab1744) and APP-CTF (C-T15). The levels of β-actin served as equal loading controls. The densitometric quantifications for the Western Blots are shown in Supplementary Fig. S2. For full blots, please see Supplementary Fig. S9.
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Related In: Results  -  Collection

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f1: Dihydroergocristine inhibits the intracellular production of Aβ and the activity of γ-secretase, without affecting Notch processing.(a) Effects of DHEC on endogenous APP-CTF accumulation and Aβ generation in HEK293 cells. HEK293 cells were incubated with DMSO (control), the indicated concentrations of DHEC or 20 μM DAPT in 24-well plates for 24 h, before Western blot analysis of APP-FL and APP-CTF (left panel). The levels of β-actin were used as equal loading controls. The corresponding media from the DMSO-, DHEC- or DAPT- treated groups (n = 3) were collected, and the Aβ total level was measured by ELISA (right panel). The Aβ data are expressed as a percentage of the control value and presented as the means ± sd. Asterisks indicate significant differences (***P < 0.001; one-way ANOVA with Bonferroni’s multiple comparisons tests) in Aβ total production of the treated samples compared with the controls (DMSO). (b) Effects of DHEC on endogenous γ-secretase activity in fibroblast cells from an AD patient. Fibroblast cells from an AD patient carrying the PS1 missense mutation A246E were treated with various compounds, and the levels of APP-FL, APP-CTF and β-actin, as well as Aβ, were measured as described above. The Aβ data are expressed as a percentage of the control value and presented as the means ± sd. (n = 3). Asterisks indicate significant differences (**P < 0.01; ***P < 0.001; one-way ANOVA with Bonferroni’s multiple comparisons tests) in Aβ total production of the samples compared with the controls (DMSO). (c) Effects of DHEC on the cleavage of human Notch1 and APP in HEK293 cells overexpressing the human Notch1 extracellular truncation (NEXT; left panel) and APP (right panel), respectively. After 24  h transient transfection of HEK293 cells with Notch1 NEXT or hAPP plasmids, cells were incubated with DHEC or DAPT at the indicated concentrations for one additional day before Western Blot analysis of NICD (Ab1744) and APP-CTF (C-T15). The levels of β-actin served as equal loading controls. The densitometric quantifications for the Western Blots are shown in Supplementary Fig. S2. For full blots, please see Supplementary Fig. S9.
Mentions: After primary screening of the compounds in T100 cells, a total of 8 natural products were found to inhibit the cellular activity of γ-secretase, in a dose-dependent manner and with an IC50 < 30 μM. Of these, NSC409663 (DHEC), which was identified from the natural product library of the National Cancer Institute (NCI, Bethesda, USA), was the only compound that affected the activity of γ-secretase in both cell-based and cell-free assays (Figs 1 and 2). DHEC, which has been used for the treatment of glaucoma18, is also a component of the drug ergoloid mesylates. Ergoloid mesylates contains a mixture of four ergot alkaloids (DHEC, dihydroergocornine, α-dihydroergocryptine and β–dihydroergocryptine; refs 14,15). In our study, DHEC had an IC50 value of ~25 μM for inhibiting the activity of γ-secretase in T100 cells without affecting cell viability (Supplementary Fig. S1b). In HEK293 cells, DHEC also caused a significant dose-dependent accumulation of the carboxy-terminal fragments of APP (APP-CTFs, Fig. 1a; left panel; Supplementary Fig. S2a), and 10 μM DHEC resulted in a ~30% reduction in Aβ production (Fig. 1a; right panel), which did not influence the levels of full length APP (APP-FL) or cell viability at all tested doses (Fig. 1a, left panel; Supplementary Fig. S1c), as expected from a γ-secretase inhibitor19. Furthermore, 20 μM DHEC caused the accumulation of APP-CTFs and led to ~35% reduction in total Aβ (Fig. 1b; Supplementary Fig. S2b) in fibroblast cells from an Alzheimer’s disease patient carrying a missense mutation (A246E) in the presenilin 1 (PS1) gene. As predicted, DAPT caused a dose-dependent accumulation of APP-CTFs in HEK293 (Supplementary Fig. S3a) and fibroblast (Supplementary Fig. S3b) cells. Similarly, total Aβ levels were markedly reduced by treatment of both HEK293 and fibroblast cells with DAPT (Supplementary Fig. S3a,b and Fig. 1a,b, right panels).

Bottom Line: Micromolar concentrations of DHEC substantially reduced Aβ levels in different cell types, including a cell line derived from an AD patient.Structure-activity relationship studies implied that the key moiety for inhibiting γ-secretase is the cyclized tripeptide moiety of DHEC.A Surface Plasmon Resonance assay showed that DHEC binds directly to γ-secretase and Nicastrin, with equilibrium dissociation constants (Kd) of 25.7 nM and 9.8 μM, respectively.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China.

ABSTRACT
Known γ-secretase inhibitors or modulators display an undesirable pharmacokinetic profile and toxicity and have therefore not been successful in clinical trials for Alzheimer's disease (AD). So far, no compounds from natural products have been identified as direct inhibitors of γ-secretase. To search for bioactive molecules that can reduce the amount of amyloid-beta peptides (Aβ) and that have better pharmacokinetics and an improved safety profile, we completed a screen of ~400 natural products by using cell-based and cell-free γ-secretase activity assays. We identified dihydroergocristine (DHEC), a component of an FDA- (Food and Drug Administration)-approved drug, to be a direct inhibitor of γ-secretase. Micromolar concentrations of DHEC substantially reduced Aβ levels in different cell types, including a cell line derived from an AD patient. Structure-activity relationship studies implied that the key moiety for inhibiting γ-secretase is the cyclized tripeptide moiety of DHEC. A Surface Plasmon Resonance assay showed that DHEC binds directly to γ-secretase and Nicastrin, with equilibrium dissociation constants (Kd) of 25.7 nM and 9.8 μM, respectively. This study offers DHEC not only as a new chemical moiety for selectively modulating the activity of γ-secretase but also a candidate for drug repositioning in Alzheimer's disease.

No MeSH data available.


Related in: MedlinePlus