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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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TEM images of 50µM of Aβ incubated for five days at 37°C in the absence of any dye (Aβ only), or in the presence of 3x EOB, EOY, PHB, ERB, or ROB.Scale bar is 100 nm.
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pone-0057288-g004: TEM images of 50µM of Aβ incubated for five days at 37°C in the absence of any dye (Aβ only), or in the presence of 3x EOB, EOY, PHB, ERB, or ROB.Scale bar is 100 nm.

Mentions: In order to determine whether Aβ cytotoxicity inhibition by ERB analogs is associated with Aβ aggregation modulation, we characterized the Aβ aggregates formed in the absence or presence of each ERB analog using CD, TEM, and dot-blot assays. CD analysis has been widely used to monitor secondary structure changes of proteins [53], [54], [55], [56]. The CD spectrum of Aβ monomer did not exhibit any spectral feature of α-helix and β-sheet, but showed typical features of dominantly disordered structure (Figure 3A). The CD spectrum of Aβ aggregates at day 5 exhibited the typical signatures of β-sheet structure, including a minimum at 217 nm (Figure 3A), which indicate that disordered Aβ monomers aggregated into β-sheet rich fibrillar aggregates. The TEM image of Aβ monomers incubated for 5 days also clearly show the existence of the Aβ aggregates consisting of protofibrils and short fibrils (Figure 4; Panel Aβ only). Recently, dot-blotting with Aβ-specific antibodies was widely used to detect the spectrum of Aβ aggregates with different conformations [16], [27], [37], [57], [58], [59], [60]. OC is a polyclonal antibody that reacts with neurotoxic fibrillar oligomers, protofibrils and fibrils [16], [58]. It was shown that Aβ-associated toxicity could be eliminated by reducing the OC-reactive species [16]. Dot-blot assay using the OC antibody confirmed the existence of fibrillar structure at day 5 (Figure 5; Panel OC). 4G8 is an Aβ-sequence-specific monoclonal antibody [61], [62], [63], [64] of which epitope is known to be residues 17 to 24 of Aβ. During transition from monomers to fibrils, β-sheet stacking buries the 4G8 epitope and ultimately limits 4G8 antibody access to the epitope leading to a significant reduction in the 4G8 reactivity [33], [36], [65]. Therefore, the reduction in 4G8 reactivity of Aβ aggregates at days 5 and 6 can be attributed to the formation of fibrils and the lateral fibril stacking (Figure 5; Panel 4G8). A11 is a polyclonal antibody that reacts with disordered prefibrillar aggregates [16]. The weak A11-reactivity of the Aβ aggregates at day 5 indicate that content of disordered prefibrillar Aβ aggregates was low (Figure S2). Therefore, the CD, TEM, and dot-blot results using Aβ-specific antibodies clearly show that the Aβ aggregates at day 5 mainly consist of fibrillar aggregates including protofibrils and short fibrils.


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

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

TEM images of 50µM of Aβ incubated for five days at 37°C in the absence of any dye (Aβ only), or in the presence of 3x EOB, EOY, PHB, ERB, or ROB.Scale bar is 100 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585355&req=5

pone-0057288-g004: TEM images of 50µM of Aβ incubated for five days at 37°C in the absence of any dye (Aβ only), or in the presence of 3x EOB, EOY, PHB, ERB, or ROB.Scale bar is 100 nm.
Mentions: In order to determine whether Aβ cytotoxicity inhibition by ERB analogs is associated with Aβ aggregation modulation, we characterized the Aβ aggregates formed in the absence or presence of each ERB analog using CD, TEM, and dot-blot assays. CD analysis has been widely used to monitor secondary structure changes of proteins [53], [54], [55], [56]. The CD spectrum of Aβ monomer did not exhibit any spectral feature of α-helix and β-sheet, but showed typical features of dominantly disordered structure (Figure 3A). The CD spectrum of Aβ aggregates at day 5 exhibited the typical signatures of β-sheet structure, including a minimum at 217 nm (Figure 3A), which indicate that disordered Aβ monomers aggregated into β-sheet rich fibrillar aggregates. The TEM image of Aβ monomers incubated for 5 days also clearly show the existence of the Aβ aggregates consisting of protofibrils and short fibrils (Figure 4; Panel Aβ only). Recently, dot-blotting with Aβ-specific antibodies was widely used to detect the spectrum of Aβ aggregates with different conformations [16], [27], [37], [57], [58], [59], [60]. OC is a polyclonal antibody that reacts with neurotoxic fibrillar oligomers, protofibrils and fibrils [16], [58]. It was shown that Aβ-associated toxicity could be eliminated by reducing the OC-reactive species [16]. Dot-blot assay using the OC antibody confirmed the existence of fibrillar structure at day 5 (Figure 5; Panel OC). 4G8 is an Aβ-sequence-specific monoclonal antibody [61], [62], [63], [64] of which epitope is known to be residues 17 to 24 of Aβ. During transition from monomers to fibrils, β-sheet stacking buries the 4G8 epitope and ultimately limits 4G8 antibody access to the epitope leading to a significant reduction in the 4G8 reactivity [33], [36], [65]. Therefore, the reduction in 4G8 reactivity of Aβ aggregates at days 5 and 6 can be attributed to the formation of fibrils and the lateral fibril stacking (Figure 5; Panel 4G8). A11 is a polyclonal antibody that reacts with disordered prefibrillar aggregates [16]. The weak A11-reactivity of the Aβ aggregates at day 5 indicate that content of disordered prefibrillar Aβ aggregates was low (Figure S2). Therefore, the CD, TEM, and dot-blot results using Aβ-specific antibodies clearly show that the Aβ aggregates at day 5 mainly consist of fibrillar aggregates including protofibrils and short fibrils.

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