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Flavone derivatives as inhibitors of insulin amyloid-like fibril formation.

Malisauskas R, Botyriute A, Cannon JG, Smirnovas V - PLoS ONE (2015)

Bottom Line: Maximal values of ThT fluorescence varied two fold or more in one third of all cases, but this did not correlate with changes in t50.However, the main conclusion is that the positions are not additive.The structures and their effects must be thought of in the context of the whole molecule.

View Article: PubMed Central - PubMed

Affiliation: Department of Biothermodynamics and Drug Design, Vilnius University Institute of Biotechnology, Vilnius, Lithuania; Department of Chemistry and Bioengineering, Vilnius Gediminas Technical University, Vilnius, Lithuania.

ABSTRACT
Several natural and synthetic flavone derivatives have been reported to inhibit formation of amyloid fibrils or to remodel existing fibrils. These studies suggest that the numbers and positions of hydroxyl groups on the flavone rings determine their effectiveness as amyloid inhibitors. In many studies the primary method for determining the effectiveness of inhibition is measuring Thioflavin T (ThT) fluorescence. This method demonstrably results in a number of false positives for inhibition. We studied the effects of 265 commercially available flavone derivatives on insulin fibril formation. We enhanced the effectiveness of ThT fluorescence measurements by fitting kinetic curves to obtain halftime of aggregation (t50). Maximal values of ThT fluorescence varied two fold or more in one third of all cases, but this did not correlate with changes in t50. Changes in t50 values were more accurate measures of inhibition of amyloid formation. We showed that without a change in an assay, but just by observing complete kinetic curves it is possible to eliminate numbers of false positive and sometimes even false negative results. Examining the data from all 265 flavones we confirmed previous observations that identified the importance of hydroxyl groups for inhibition. Our evidence suggests the importance of hydroxyl groups at locations 5, 6, 7, and 4', and the absence of a hydroxyl group at location 3, for inhibiting amyloid formation. However, the main conclusion is that the positions are not additive. The structures and their effects must be thought of in the context of the whole molecule.

No MeSH data available.


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Distribution of inhibition potential by the number of side groups.
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pone.0121231.g002: Distribution of inhibition potential by the number of side groups.

Mentions: Beyond testing ThT fluorescence we looked for patterns in the effects of substituent groups on the flavone rings in inhibiting fibrillation. Aggregation time dependence on the number of substituents is shown in Fig. 2. Flavone without any side groups shows no impact on the rate of fibril formation. As noted above, the majority of flavones, no matter how many substituent groups, have little effect on fibrillation rates. We now note the outliers. No flavones with one or two side groups inhibit fibrillation. Most of the best fibrillation accelerators have two side groups, while one has three substituents. The first strong inhibitor, 7,8,2’-trihydroxyflavone, also has three side groups. The tetra-substituted flavones include the two strongest inhibitors, Scutellarein and Luteolin. Penta-substituted flavones include one medium and one strong inhibitor (3,6,2’,4’,5’-Pentahydroxyflavone). All hexahydroxyflavones tested show some inhibition, and one of these is a strong inhibitor (Gossypetin). However, we had only six such flavones available, so it is impossible to make strong generalizations. Overall we can state that flavones with two or fewer hydroxyl groups have no inhibition potential. The best inhibitors yet measured are tetra-substituted flavones, though there are also good inhibitors among tri-, penta-, and hexa-substituted flavones. Such distribution means that three residues around the flavone backbone are enough to have a potent inhibitor, however, the inhibition potential can be both increased and decreased by additional residues.


Flavone derivatives as inhibitors of insulin amyloid-like fibril formation.

Malisauskas R, Botyriute A, Cannon JG, Smirnovas V - PLoS ONE (2015)

Distribution of inhibition potential by the number of side groups.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4370379&req=5

pone.0121231.g002: Distribution of inhibition potential by the number of side groups.
Mentions: Beyond testing ThT fluorescence we looked for patterns in the effects of substituent groups on the flavone rings in inhibiting fibrillation. Aggregation time dependence on the number of substituents is shown in Fig. 2. Flavone without any side groups shows no impact on the rate of fibril formation. As noted above, the majority of flavones, no matter how many substituent groups, have little effect on fibrillation rates. We now note the outliers. No flavones with one or two side groups inhibit fibrillation. Most of the best fibrillation accelerators have two side groups, while one has three substituents. The first strong inhibitor, 7,8,2’-trihydroxyflavone, also has three side groups. The tetra-substituted flavones include the two strongest inhibitors, Scutellarein and Luteolin. Penta-substituted flavones include one medium and one strong inhibitor (3,6,2’,4’,5’-Pentahydroxyflavone). All hexahydroxyflavones tested show some inhibition, and one of these is a strong inhibitor (Gossypetin). However, we had only six such flavones available, so it is impossible to make strong generalizations. Overall we can state that flavones with two or fewer hydroxyl groups have no inhibition potential. The best inhibitors yet measured are tetra-substituted flavones, though there are also good inhibitors among tri-, penta-, and hexa-substituted flavones. Such distribution means that three residues around the flavone backbone are enough to have a potent inhibitor, however, the inhibition potential can be both increased and decreased by additional residues.

Bottom Line: Maximal values of ThT fluorescence varied two fold or more in one third of all cases, but this did not correlate with changes in t50.However, the main conclusion is that the positions are not additive.The structures and their effects must be thought of in the context of the whole molecule.

View Article: PubMed Central - PubMed

Affiliation: Department of Biothermodynamics and Drug Design, Vilnius University Institute of Biotechnology, Vilnius, Lithuania; Department of Chemistry and Bioengineering, Vilnius Gediminas Technical University, Vilnius, Lithuania.

ABSTRACT
Several natural and synthetic flavone derivatives have been reported to inhibit formation of amyloid fibrils or to remodel existing fibrils. These studies suggest that the numbers and positions of hydroxyl groups on the flavone rings determine their effectiveness as amyloid inhibitors. In many studies the primary method for determining the effectiveness of inhibition is measuring Thioflavin T (ThT) fluorescence. This method demonstrably results in a number of false positives for inhibition. We studied the effects of 265 commercially available flavone derivatives on insulin fibril formation. We enhanced the effectiveness of ThT fluorescence measurements by fitting kinetic curves to obtain halftime of aggregation (t50). Maximal values of ThT fluorescence varied two fold or more in one third of all cases, but this did not correlate with changes in t50. Changes in t50 values were more accurate measures of inhibition of amyloid formation. We showed that without a change in an assay, but just by observing complete kinetic curves it is possible to eliminate numbers of false positive and sometimes even false negative results. Examining the data from all 265 flavones we confirmed previous observations that identified the importance of hydroxyl groups for inhibition. Our evidence suggests the importance of hydroxyl groups at locations 5, 6, 7, and 4', and the absence of a hydroxyl group at location 3, for inhibiting amyloid formation. However, the main conclusion is that the positions are not additive. The structures and their effects must be thought of in the context of the whole molecule.

No MeSH data available.


Related in: MedlinePlus