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Halogenation generates effective modulators of amyloid-Beta aggregation and neurotoxicity.

Wong HE, Irwin JA, Kwon I - PLoS ONE (2013)

Bottom Line: Halogenation of organic compounds plays diverse roles in biochemistry, including selective chemical modification of proteins and improved oral absorption/blood-brain barrier permeability of drug candidates.However, halogenation of either the xanthenes or benzoate ring of fluorescein substantially enhanced the inhibitory capacity on Aβ aggregation.To our knowledge, this is the first report demonstrating that halogenation of aromatic rings substantially enhance inhibitory capacities of small molecules on Aβ-associated neurotoxicity via Aβ aggregation modulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA, USA.

ABSTRACT
Halogenation of organic compounds plays diverse roles in biochemistry, including selective chemical modification of proteins and improved oral absorption/blood-brain barrier permeability of drug candidates. Moreover, halogenation of aromatic molecules greatly affects aromatic interaction-mediated self-assembly processes, including amyloid fibril formation. Perturbation of the aromatic interaction caused by halogenation of peptide building blocks is known to affect the morphology and other physical properties of the fibrillar structure. Consequently, in this article, we investigated the ability of halogenated ligands to modulate the self-assembly of amyloidogenic peptide/protein. As a model system, we chose amyloid-beta peptide (Aβ), which is implicated in Alzheimer's disease, and a novel modulator of Aβ aggregation, erythrosine B (ERB). Considering that four halogen atoms are attached to the xanthene benzoate group in ERB, we hypothesized that halogenation of the xanthene benzoate plays a critical role in modulating Aβ aggregation and cytotoxicity. Therefore, we evaluated the modulating capacities of four ERB analogs containing different types and numbers of halogen atoms as well as fluorescein as a negative control. We found that fluorescein is not an effective modulator of Aβ aggregation and cytotoxicity. However, halogenation of either the xanthenes or benzoate ring of fluorescein substantially enhanced the inhibitory capacity on Aβ aggregation. Such Aβ aggregation inhibition by ERB analogs except rose bengal correlated well to the inhibition of Aβ cytotoxicity. To our knowledge, this is the first report demonstrating that halogenation of aromatic rings substantially enhance inhibitory capacities of small molecules on Aβ-associated neurotoxicity via Aβ aggregation modulation.

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Modulation of Aβ aggregation and cytotoxicity by FLN.(A) CD spectra of Aβ monomer incubated for 7 days at 37°C in the absence (Aβ aggregate) or presence of 10x FLN (FLN). (B) TEM images of 50 µM of Aβ incubated for seven days at 37°C in the absence of any dye (Aβ only), or in the presence of 3x FLN. Scale bar is 100 nm. (C) Dot blot images of Aβ aggregates formed without (Aβ only) or with 3x and 10x FLN using OC and 4G8 antibodies. For each antibody, all samples were spotted onto one nitrocellulose membrane. Each membrane was immuno-stained with the OC or 4G8 antibody. For clearer presentation of the data, the sections of each membrane were cut and re-arranged. (D) Viability of neuroblastoma SH-SY5Y cells. Three controls (PBS buffer, Aβ monomer, and FLN) and two Aβ aggregates formed in the absence or presence of 3x FLN at 37°C for 5 days. Values represent means ± standard deviation (n≥3). Values are normalized to the viability of cells administered with PBS buffer only. Two-sided Student’s t-tests were applied to the MTT reduction data. (*; P = 0.013).
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pone-0057288-g006: Modulation of Aβ aggregation and cytotoxicity by FLN.(A) CD spectra of Aβ monomer incubated for 7 days at 37°C in the absence (Aβ aggregate) or presence of 10x FLN (FLN). (B) TEM images of 50 µM of Aβ incubated for seven days at 37°C in the absence of any dye (Aβ only), or in the presence of 3x FLN. Scale bar is 100 nm. (C) Dot blot images of Aβ aggregates formed without (Aβ only) or with 3x and 10x FLN using OC and 4G8 antibodies. For each antibody, all samples were spotted onto one nitrocellulose membrane. Each membrane was immuno-stained with the OC or 4G8 antibody. For clearer presentation of the data, the sections of each membrane were cut and re-arranged. (D) Viability of neuroblastoma SH-SY5Y cells. Three controls (PBS buffer, Aβ monomer, and FLN) and two Aβ aggregates formed in the absence or presence of 3x FLN at 37°C for 5 days. Values represent means ± standard deviation (n≥3). Values are normalized to the viability of cells administered with PBS buffer only. Two-sided Student’s t-tests were applied to the MTT reduction data. (*; P = 0.013).

Mentions: Investigating the modulatory capacities of ERB congeners on Aβ cytotoxicity and aggregation reveals that even a subtle change in their chemical structure from the ERB structural template can affect their modulatory capacities. In order to further validate our hypothesis that the modulatory capacities of the ERB congeners are related with the presence of halogen atoms, we also evaluated the modulatory capacities of FLN as a negative control without any halogen atoms (Figure 1). The CD spectrum of the FLN-induced Aβ aggregates clearly exhibits the typical features of β-sheet rich structure (Figure 6A). The TEM image of the FLN-induced Aβ aggregates also indicates that protofibrils and fibrils are dominant species similar to the Aβ control (Figure 6B). Furthermore, the OC-reactivity of the Aβ aggregates formed with FLN at days 5 and 6 are very comparable to those of the Aβ control (Figure 6C), indicating that the FLN-induced Aβ aggregates had fibrillar aggregates as much as the Aβ control. The 4G8-reactivity of the FLN-induced Aβ aggregates with FLN remained unchanged up to day 7, whereas the 4G8-reactivity of the Aβ control dropped at day 5. Such a slightly higher 4G8-reactivity of the FLN-induced Aβ aggregates at day 5 is likely because the FLN-induced fibrils are not laterally stacked and so allow the 4G8 binding to its epitope better than the Aβ control. The CD, TEM, and dot-blot assay results conclusively demonstrate that FLN does not modulate the Aβ aggregation as much as EOY, ERB, or ROB.


Halogenation generates effective modulators of amyloid-Beta aggregation and neurotoxicity.

Wong HE, Irwin JA, Kwon I - PLoS ONE (2013)

Modulation of Aβ aggregation and cytotoxicity by FLN.(A) CD spectra of Aβ monomer incubated for 7 days at 37°C in the absence (Aβ aggregate) or presence of 10x FLN (FLN). (B) TEM images of 50 µM of Aβ incubated for seven days at 37°C in the absence of any dye (Aβ only), or in the presence of 3x FLN. Scale bar is 100 nm. (C) Dot blot images of Aβ aggregates formed without (Aβ only) or with 3x and 10x FLN using OC and 4G8 antibodies. For each antibody, all samples were spotted onto one nitrocellulose membrane. Each membrane was immuno-stained with the OC or 4G8 antibody. For clearer presentation of the data, the sections of each membrane were cut and re-arranged. (D) Viability of neuroblastoma SH-SY5Y cells. Three controls (PBS buffer, Aβ monomer, and FLN) and two Aβ aggregates formed in the absence or presence of 3x FLN at 37°C for 5 days. Values represent means ± standard deviation (n≥3). Values are normalized to the viability of cells administered with PBS buffer only. Two-sided Student’s t-tests were applied to the MTT reduction data. (*; P = 0.013).
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Related In: Results  -  Collection

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pone-0057288-g006: Modulation of Aβ aggregation and cytotoxicity by FLN.(A) CD spectra of Aβ monomer incubated for 7 days at 37°C in the absence (Aβ aggregate) or presence of 10x FLN (FLN). (B) TEM images of 50 µM of Aβ incubated for seven days at 37°C in the absence of any dye (Aβ only), or in the presence of 3x FLN. Scale bar is 100 nm. (C) Dot blot images of Aβ aggregates formed without (Aβ only) or with 3x and 10x FLN using OC and 4G8 antibodies. For each antibody, all samples were spotted onto one nitrocellulose membrane. Each membrane was immuno-stained with the OC or 4G8 antibody. For clearer presentation of the data, the sections of each membrane were cut and re-arranged. (D) Viability of neuroblastoma SH-SY5Y cells. Three controls (PBS buffer, Aβ monomer, and FLN) and two Aβ aggregates formed in the absence or presence of 3x FLN at 37°C for 5 days. Values represent means ± standard deviation (n≥3). Values are normalized to the viability of cells administered with PBS buffer only. Two-sided Student’s t-tests were applied to the MTT reduction data. (*; P = 0.013).
Mentions: Investigating the modulatory capacities of ERB congeners on Aβ cytotoxicity and aggregation reveals that even a subtle change in their chemical structure from the ERB structural template can affect their modulatory capacities. In order to further validate our hypothesis that the modulatory capacities of the ERB congeners are related with the presence of halogen atoms, we also evaluated the modulatory capacities of FLN as a negative control without any halogen atoms (Figure 1). The CD spectrum of the FLN-induced Aβ aggregates clearly exhibits the typical features of β-sheet rich structure (Figure 6A). The TEM image of the FLN-induced Aβ aggregates also indicates that protofibrils and fibrils are dominant species similar to the Aβ control (Figure 6B). Furthermore, the OC-reactivity of the Aβ aggregates formed with FLN at days 5 and 6 are very comparable to those of the Aβ control (Figure 6C), indicating that the FLN-induced Aβ aggregates had fibrillar aggregates as much as the Aβ control. The 4G8-reactivity of the FLN-induced Aβ aggregates with FLN remained unchanged up to day 7, whereas the 4G8-reactivity of the Aβ control dropped at day 5. Such a slightly higher 4G8-reactivity of the FLN-induced Aβ aggregates at day 5 is likely because the FLN-induced fibrils are not laterally stacked and so allow the 4G8 binding to its epitope better than the Aβ control. The CD, TEM, and dot-blot assay results conclusively demonstrate that FLN does not modulate the Aβ aggregation as much as EOY, ERB, or ROB.

Bottom Line: Halogenation of organic compounds plays diverse roles in biochemistry, including selective chemical modification of proteins and improved oral absorption/blood-brain barrier permeability of drug candidates.However, halogenation of either the xanthenes or benzoate ring of fluorescein substantially enhanced the inhibitory capacity on Aβ aggregation.To our knowledge, this is the first report demonstrating that halogenation of aromatic rings substantially enhance inhibitory capacities of small molecules on Aβ-associated neurotoxicity via Aβ aggregation modulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, University of Virginia, Charlottesville, VA, USA.

ABSTRACT
Halogenation of organic compounds plays diverse roles in biochemistry, including selective chemical modification of proteins and improved oral absorption/blood-brain barrier permeability of drug candidates. Moreover, halogenation of aromatic molecules greatly affects aromatic interaction-mediated self-assembly processes, including amyloid fibril formation. Perturbation of the aromatic interaction caused by halogenation of peptide building blocks is known to affect the morphology and other physical properties of the fibrillar structure. Consequently, in this article, we investigated the ability of halogenated ligands to modulate the self-assembly of amyloidogenic peptide/protein. As a model system, we chose amyloid-beta peptide (Aβ), which is implicated in Alzheimer's disease, and a novel modulator of Aβ aggregation, erythrosine B (ERB). Considering that four halogen atoms are attached to the xanthene benzoate group in ERB, we hypothesized that halogenation of the xanthene benzoate plays a critical role in modulating Aβ aggregation and cytotoxicity. Therefore, we evaluated the modulating capacities of four ERB analogs containing different types and numbers of halogen atoms as well as fluorescein as a negative control. We found that fluorescein is not an effective modulator of Aβ aggregation and cytotoxicity. However, halogenation of either the xanthenes or benzoate ring of fluorescein substantially enhanced the inhibitory capacity on Aβ aggregation. Such Aβ aggregation inhibition by ERB analogs except rose bengal correlated well to the inhibition of Aβ cytotoxicity. To our knowledge, this is the first report demonstrating that halogenation of aromatic rings substantially enhance inhibitory capacities of small molecules on Aβ-associated neurotoxicity via Aβ aggregation modulation.

Show MeSH
Related in: MedlinePlus