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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

Effects of dihydroergocristine on the processing of human APP C100-Flag.(a) Dose-dependent effects of DHEC on the cleavage of C100-Flag by purified γ-secretase. Purified γ-secretase solubilized in 0.2% CHAPSO-HEPES was incubated at 37 °C for 4 h with 1 μM C100-Flag substrate, 0.1% PC, and the indicated concentrations of dihydroergocristine (DHEC) or DMSO (control, 100%). Reactions were stopped by adding 0.5% SDS, and the resultant products were separated in 16% Tricine-SDS-PAGE gels, transferred to a membrane and detected with anti-AICD-Flag antibody (C-T15) and anti-Aβ antibody (6E10; lower panel). The density of the AICD-Flag and Aβ total bands was quantified by Odyssey software (Supplementary data Fig. S5). (b) Dose-dependent effects of DHEC on Aβ40 and Aβ42 production in the cell-free assay performed with purified C100-Flag and γ-secretase. Reactions in the in vitro assay upon treatment with DHEC, using purified γ-secretase and C100-Flag, were tested as described above. The residue was then separated by Bicine/urea SDS-PAGE, together with an Aβ standard of synthetic human Aβ38, Aβ40 and Aβ42, followed by Western blot detection with anti-Aβ antibody (6E10, left panel), or quantification by ELISA (right panel). Black bars, Aβ [1–40]; gray bars, Aβ[1–42]. Data were presented as the means ± sd. (n = 3) Asterisks indicate significant differences (**P < 0.01; ***P < 0.001; two-way ANOVA with Bonferroni’s multiple comparisons tests) in Aβ40 or Aβ42 production of DHEC-treated samples compared with the control (DMSO). (c) Surface plasmon resonance assay analysis of the binding of DHEC to γ-secretase or NCT. Solutions of various concentrations of DHEC were injected into the chamber with a γ-secretase (left panel) or NCT (right panel)-coated sensor chip. The change in response units over time is shown.
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f2: Effects of dihydroergocristine on the processing of human APP C100-Flag.(a) Dose-dependent effects of DHEC on the cleavage of C100-Flag by purified γ-secretase. Purified γ-secretase solubilized in 0.2% CHAPSO-HEPES was incubated at 37 °C for 4 h with 1 μM C100-Flag substrate, 0.1% PC, and the indicated concentrations of dihydroergocristine (DHEC) or DMSO (control, 100%). Reactions were stopped by adding 0.5% SDS, and the resultant products were separated in 16% Tricine-SDS-PAGE gels, transferred to a membrane and detected with anti-AICD-Flag antibody (C-T15) and anti-Aβ antibody (6E10; lower panel). The density of the AICD-Flag and Aβ total bands was quantified by Odyssey software (Supplementary data Fig. S5). (b) Dose-dependent effects of DHEC on Aβ40 and Aβ42 production in the cell-free assay performed with purified C100-Flag and γ-secretase. Reactions in the in vitro assay upon treatment with DHEC, using purified γ-secretase and C100-Flag, were tested as described above. The residue was then separated by Bicine/urea SDS-PAGE, together with an Aβ standard of synthetic human Aβ38, Aβ40 and Aβ42, followed by Western blot detection with anti-Aβ antibody (6E10, left panel), or quantification by ELISA (right panel). Black bars, Aβ [1–40]; gray bars, Aβ[1–42]. Data were presented as the means ± sd. (n = 3) Asterisks indicate significant differences (**P < 0.01; ***P < 0.001; two-way ANOVA with Bonferroni’s multiple comparisons tests) in Aβ40 or Aβ42 production of DHEC-treated samples compared with the control (DMSO). (c) Surface plasmon resonance assay analysis of the binding of DHEC to γ-secretase or NCT. Solutions of various concentrations of DHEC were injected into the chamber with a γ-secretase (left panel) or NCT (right panel)-coated sensor chip. The change in response units over time is shown.

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)

Effects of dihydroergocristine on the processing of human APP C100-Flag.(a) Dose-dependent effects of DHEC on the cleavage of C100-Flag by purified γ-secretase. Purified γ-secretase solubilized in 0.2% CHAPSO-HEPES was incubated at 37 °C for 4 h with 1 μM C100-Flag substrate, 0.1% PC, and the indicated concentrations of dihydroergocristine (DHEC) or DMSO (control, 100%). Reactions were stopped by adding 0.5% SDS, and the resultant products were separated in 16% Tricine-SDS-PAGE gels, transferred to a membrane and detected with anti-AICD-Flag antibody (C-T15) and anti-Aβ antibody (6E10; lower panel). The density of the AICD-Flag and Aβ total bands was quantified by Odyssey software (Supplementary data Fig. S5). (b) Dose-dependent effects of DHEC on Aβ40 and Aβ42 production in the cell-free assay performed with purified C100-Flag and γ-secretase. Reactions in the in vitro assay upon treatment with DHEC, using purified γ-secretase and C100-Flag, were tested as described above. The residue was then separated by Bicine/urea SDS-PAGE, together with an Aβ standard of synthetic human Aβ38, Aβ40 and Aβ42, followed by Western blot detection with anti-Aβ antibody (6E10, left panel), or quantification by ELISA (right panel). Black bars, Aβ [1–40]; gray bars, Aβ[1–42]. Data were presented as the means ± sd. (n = 3) Asterisks indicate significant differences (**P < 0.01; ***P < 0.001; two-way ANOVA with Bonferroni’s multiple comparisons tests) in Aβ40 or Aβ42 production of DHEC-treated samples compared with the control (DMSO). (c) Surface plasmon resonance assay analysis of the binding of DHEC to γ-secretase or NCT. Solutions of various concentrations of DHEC were injected into the chamber with a γ-secretase (left panel) or NCT (right panel)-coated sensor chip. The change in response units over time is shown.
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Related In: Results  -  Collection

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f2: Effects of dihydroergocristine on the processing of human APP C100-Flag.(a) Dose-dependent effects of DHEC on the cleavage of C100-Flag by purified γ-secretase. Purified γ-secretase solubilized in 0.2% CHAPSO-HEPES was incubated at 37 °C for 4 h with 1 μM C100-Flag substrate, 0.1% PC, and the indicated concentrations of dihydroergocristine (DHEC) or DMSO (control, 100%). Reactions were stopped by adding 0.5% SDS, and the resultant products were separated in 16% Tricine-SDS-PAGE gels, transferred to a membrane and detected with anti-AICD-Flag antibody (C-T15) and anti-Aβ antibody (6E10; lower panel). The density of the AICD-Flag and Aβ total bands was quantified by Odyssey software (Supplementary data Fig. S5). (b) Dose-dependent effects of DHEC on Aβ40 and Aβ42 production in the cell-free assay performed with purified C100-Flag and γ-secretase. Reactions in the in vitro assay upon treatment with DHEC, using purified γ-secretase and C100-Flag, were tested as described above. The residue was then separated by Bicine/urea SDS-PAGE, together with an Aβ standard of synthetic human Aβ38, Aβ40 and Aβ42, followed by Western blot detection with anti-Aβ antibody (6E10, left panel), or quantification by ELISA (right panel). Black bars, Aβ [1–40]; gray bars, Aβ[1–42]. Data were presented as the means ± sd. (n = 3) Asterisks indicate significant differences (**P < 0.01; ***P < 0.001; two-way ANOVA with Bonferroni’s multiple comparisons tests) in Aβ40 or Aβ42 production of DHEC-treated samples compared with the control (DMSO). (c) Surface plasmon resonance assay analysis of the binding of DHEC to γ-secretase or NCT. Solutions of various concentrations of DHEC were injected into the chamber with a γ-secretase (left panel) or NCT (right panel)-coated sensor chip. The change in response units over time is shown.
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