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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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Monitoring Aβ aggregation by ThT fluorescence assay and measuring Aβ-associated cytotoxicity using MTT reduction assay.(A) Time course of ThT fluorescence of Aβ samples. 50 µM of Aβ monomer was incubated at 37°C. 5 µL of Aβ sample was taken daily for 8 dyes for ThT fluorescence analysis. ThT fluorescence was measured in arbitrary units (a.u.). Values represent means ± standard deviation (n = 3). (B) Viability of neuroblastoma SH-SY5Y cells incubated with ERB analog controls and pre-formed Aβ samples in the absence or presence of ERB analog. Preformed Aβ aggregates were prepared by incubating 50 µM of Aβ monomer in the absence or presence of ERB analog (EOB, PHB, EOY, ERB, EOY, or ROB) at 37°C for 5 days. Aggregates were then administered to SH-SY5Y cells at a final concentration of 5 µM. After 48 hours, MTT reducing activity was measured. Values represent means ± standard deviation (n≥3). Values are normalized to the viability of cells administered with PBS only. Two-sided Student’s t-tests were applied to the MTT reduction data of Aβ aggregates in the presence of ERB analog at day 5 compared to that of the Aβ only control. (NS; Not significant, *; P<0.001, **; P<0.05).
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pone-0057288-g002: Monitoring Aβ aggregation by ThT fluorescence assay and measuring Aβ-associated cytotoxicity using MTT reduction assay.(A) Time course of ThT fluorescence of Aβ samples. 50 µM of Aβ monomer was incubated at 37°C. 5 µL of Aβ sample was taken daily for 8 dyes for ThT fluorescence analysis. ThT fluorescence was measured in arbitrary units (a.u.). Values represent means ± standard deviation (n = 3). (B) Viability of neuroblastoma SH-SY5Y cells incubated with ERB analog controls and pre-formed Aβ samples in the absence or presence of ERB analog. Preformed Aβ aggregates were prepared by incubating 50 µM of Aβ monomer in the absence or presence of ERB analog (EOB, PHB, EOY, ERB, EOY, or ROB) at 37°C for 5 days. Aggregates were then administered to SH-SY5Y cells at a final concentration of 5 µM. After 48 hours, MTT reducing activity was measured. Values represent means ± standard deviation (n≥3). Values are normalized to the viability of cells administered with PBS only. Two-sided Student’s t-tests were applied to the MTT reduction data of Aβ aggregates in the presence of ERB analog at day 5 compared to that of the Aβ only control. (NS; Not significant, *; P<0.001, **; P<0.05).

Mentions: In order to evaluate the modulation capability of ERB and its analogs (EOY, EOB, PHB, and ROB), we employed the widely-used MTT reduction assay [16], [29], [33], [36], [46], [47]. Aβ aggregates were prepared by incubating Aβ monomers with or without 3x ERB analog. In the absence of any ERB analog, Aβ aggregation was monitored by ThT fluorescence assay. The ThT fluorescence of Aβ aggregates started to increase at day 4 and reached the plateau at day 6 (Figure 2A), indicating that Aβ protofibrils and fibrils were primarily formed from day 4. In order to evaluate cytotoxicity of Aβ aggregates containing Aβ intermediates, we chose Aβ samples incubated for 5 days in the absence or presence of 3x ERB analog. The preformed Aβ aggregates were then administered to neuroblastoma SH-SY5Y cells, and cell viability was determined by MTT reduction (Figure 2B). We determined whether Aβ monomer or ERB analog is cytotoxic to neuroblastoma SH-SY5Y cells, and the results are shown in Figure 2B. Aβ monomers (5 µM) caused a mild reduction (11%) in the cell viability. All ERB analogs (15 µM) except ROB also caused only mild reduction in the cell viability ranging from 0 to 8%. However, 3x ROB substantially reduced the cell viability (34%). ROB has been tested to ablate certain types of cancer cells including melanoma [48], [49], and so it is not surprising that ROB is cytotoxic to SH-SY5Y cells.


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

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

Monitoring Aβ aggregation by ThT fluorescence assay and measuring Aβ-associated cytotoxicity using MTT reduction assay.(A) Time course of ThT fluorescence of Aβ samples. 50 µM of Aβ monomer was incubated at 37°C. 5 µL of Aβ sample was taken daily for 8 dyes for ThT fluorescence analysis. ThT fluorescence was measured in arbitrary units (a.u.). Values represent means ± standard deviation (n = 3). (B) Viability of neuroblastoma SH-SY5Y cells incubated with ERB analog controls and pre-formed Aβ samples in the absence or presence of ERB analog. Preformed Aβ aggregates were prepared by incubating 50 µM of Aβ monomer in the absence or presence of ERB analog (EOB, PHB, EOY, ERB, EOY, or ROB) at 37°C for 5 days. Aggregates were then administered to SH-SY5Y cells at a final concentration of 5 µM. After 48 hours, MTT reducing activity was measured. Values represent means ± standard deviation (n≥3). Values are normalized to the viability of cells administered with PBS only. Two-sided Student’s t-tests were applied to the MTT reduction data of Aβ aggregates in the presence of ERB analog at day 5 compared to that of the Aβ only control. (NS; Not significant, *; P<0.001, **; P<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585355&req=5

pone-0057288-g002: Monitoring Aβ aggregation by ThT fluorescence assay and measuring Aβ-associated cytotoxicity using MTT reduction assay.(A) Time course of ThT fluorescence of Aβ samples. 50 µM of Aβ monomer was incubated at 37°C. 5 µL of Aβ sample was taken daily for 8 dyes for ThT fluorescence analysis. ThT fluorescence was measured in arbitrary units (a.u.). Values represent means ± standard deviation (n = 3). (B) Viability of neuroblastoma SH-SY5Y cells incubated with ERB analog controls and pre-formed Aβ samples in the absence or presence of ERB analog. Preformed Aβ aggregates were prepared by incubating 50 µM of Aβ monomer in the absence or presence of ERB analog (EOB, PHB, EOY, ERB, EOY, or ROB) at 37°C for 5 days. Aggregates were then administered to SH-SY5Y cells at a final concentration of 5 µM. After 48 hours, MTT reducing activity was measured. Values represent means ± standard deviation (n≥3). Values are normalized to the viability of cells administered with PBS only. Two-sided Student’s t-tests were applied to the MTT reduction data of Aβ aggregates in the presence of ERB analog at day 5 compared to that of the Aβ only control. (NS; Not significant, *; P<0.001, **; P<0.05).
Mentions: In order to evaluate the modulation capability of ERB and its analogs (EOY, EOB, PHB, and ROB), we employed the widely-used MTT reduction assay [16], [29], [33], [36], [46], [47]. Aβ aggregates were prepared by incubating Aβ monomers with or without 3x ERB analog. In the absence of any ERB analog, Aβ aggregation was monitored by ThT fluorescence assay. The ThT fluorescence of Aβ aggregates started to increase at day 4 and reached the plateau at day 6 (Figure 2A), indicating that Aβ protofibrils and fibrils were primarily formed from day 4. In order to evaluate cytotoxicity of Aβ aggregates containing Aβ intermediates, we chose Aβ samples incubated for 5 days in the absence or presence of 3x ERB analog. The preformed Aβ aggregates were then administered to neuroblastoma SH-SY5Y cells, and cell viability was determined by MTT reduction (Figure 2B). We determined whether Aβ monomer or ERB analog is cytotoxic to neuroblastoma SH-SY5Y cells, and the results are shown in Figure 2B. Aβ monomers (5 µM) caused a mild reduction (11%) in the cell viability. All ERB analogs (15 µM) except ROB also caused only mild reduction in the cell viability ranging from 0 to 8%. However, 3x ROB substantially reduced the cell viability (34%). ROB has been tested to ablate certain types of cancer cells including melanoma [48], [49], and so it is not surprising that ROB is cytotoxic to SH-SY5Y cells.

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