Fig 1.
(A) luteolin, (B) general formula of the flavonoid scaffold, (C) luteolin-7-O-glucoside, and (D) luteolin-7-O-glucuronide.
Fig 2.
Inhibition of TTR-induced cytotoxicity.
TTR at a final tetrameric concentration of 18 μM was pre-incubated with luteolin, diclofenac, diflunisal, or luteolin-7-O-glucoside (10 μM each) for 2 h. TTR or the TTR + inhibitor mixtures were added to SH-SY5Y cells and incubated for 72 h. Cell viability was measured with a resazurin assay [44]. Data are presented as mean ± standard deviation (n = 3, **P < 0.01; ***P < 0.001; ns P > 0.05). The addition of luteolin, diflunisal and diclofenac leads to a statistically significant difference in the number of viable cells compared to cells exposed to TTR alone, but the addition of luteolin-7-O-glucoside had no effect on TTR toxicity. The statistical significance was assessed using one-way ANOVA.
Fig 3.
Luteolin reverses the phenotype of a D. melanogaster model of FAP.
Climbing activity using a DAMS5 System is shown for wild-type flies and TTRV30M expressing flies without drug (vehicle) and with drug treatment at 6 days after eclosion. TTRV30M flies with vehicle alone showed reduced climbing activity compared to wild-type control flies. TTRV30M flies showed improved activity after drug treatment and a sufficient rescue effect was observed in flies treated with 3.0 mM luteolin. Values in graph represent mean ± S.E. *, p < 0.05 by Mann Whitney U test TTRV30M 3.0 mM LUT. **, p < 0.05 by Mann Whitney U test for TTRV30M vehicle.
Fig 4.
Influence of inhibitors on TTR aggregation under acidic conditions.
TTR (final tetrameric concentration 15 μM) was pre-incubated with luteolin, diflunisal, diclofenac, or luteolin-7-O-glucoside (each at 15 μM) for 2 h. The TTR complexes were subjected to pH 4.6 for 72 h, and fibril formation was evaluated by turbidity measurements at 400 nm [17]. The amount of aggregated TTR is reported as a fraction of fibril formation relative to the amount of aggregates produced in the absence of inhibitors. There was a significant decrease in TTR fibril formation after the addition of luteolin, diflunisal, and diclofenac compared with TTR alone, but no inhibitory effect was observed after treatment with luteolin-7-O-glucoside. The statistical significance of the obtained results was assessed using one-way ANOVA. Data are presented as mean ± standard deviation of the % fibril formation (n = 2 ***P < 0.001; ns P > 0.05).
Fig 5.
The crystal structure of TTR-luteolin complex.
A) The TTR monomers in the dimer structure are shown as ribbons and are labeled A and B. The symmetry-related monomers are labeled A´, and B´. Two luteolin molecules bind at the thyroxin-binding channels, and are shown as sticks. For clarity, only one of the symmetry-related luteolin orientations is shown. B) The quality of the electron density map at the BB´ dimer-dimer interface. The σA-weighted (m|Fo|-D|Fc|) electron density contoured at 3 times the root-mean-square value of the map is shown in orange. To reduce model bias the luteolin molecule was excluded from the coordinate file that was subjected to one round of simulated annealing refinement before calculation of the map.
Fig 6.
Detailed view of the luteolin-binding site in TTR.
A and B) Two orientations of the TTR-luteolin complex that show interactions made between the luteolin A-ring and TTR. Residues and the luteolin molecule from the B and B’ monomers are shown in blue and yellow, respectively. Hydrogen bonds are shown as dotted lines. The O5 and O7 oxygen form hydrogen bonds to the Oγ1 atoms of B-Ser117 and B-Thr119 (and B´-Ser117 and B’-Thr119 over the tetramer interface). Both Ser117 and Thr119 are refined in two conformations. C) The σA-weighted (2m|Fo|-D|Fc|) electron density calculated from the refined coordinates is contoured at the root-mean-square value of the mapover residues B- and B’-Lys15 (blue). The density shows that no direct hydrogen bond is formed between the Nz atom of Lys15 to luteolin.