The flavonoid luteolin, but not luteolin-7-o-glucoside, prevents a transthyretin mediated toxic response

Transthyretin (TTR) is a homotetrameric plasma protein with amyloidogenic properties that has been linked to the development of familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The in vivo role of TTR is associated with transport of thyroxine hormone T4 and retinol-binding protein. Loss of the tetrameric integrity of TTR is a rate-limiting step in the process of TTR amyloid formation, and ligands with the ability to bind within the thyroxin binding site (TBS) can stabilize the tetramer, a feature that is currently used as a therapeutic approach for FAP. Several different flavonoids have recently been identified that impair amyloid formation. The flavonoid luteolin shows therapeutic potential with low incidence of unwanted side effects. In this work, we show that luteolin effectively attenuates the cytotoxic response to TTR in cultured neuronal cells and rescues the phenotype of a Drosophila melanogaster model of FAP. The plant-derived luteolin analogue cynaroside has a glucoside group in position 7 of the flavone A-ring and as opposed to luteolin is unable to stabilize TTR tetramers and thus prevents a cytotoxic effect. We generated high-resolution crystal-structures of both TTR wild type and the amyloidogenic mutant V30M in complex with luteolin. The results show that the A-ring of luteolin, in contrast to what was previously suggested, is buried within the TBS, consequently explaining the lack of activity from cynaroside. The flavonoids represent an interesting group of drug candidates for TTR amyloidosis. The present investigation shows the potential of luteolin as a stabilizer of TTR in vivo. We also show an alternative orientation of luteolin within the TBS which could represent a general mode of binding of flavonoids to TTR and is of importance concerning the future design of tetramer stabilizing drugs

Influence of inhibitors on TTR aggregation under acidic conditions.

<p>TTR (final tetrameric concentration 15 μM) was pre-incubated with luteolin, diflunisal, diclofenac, or luteolin-7-<i>O</i>-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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128222#pone.0128222.ref017" target="_blank">17</a>]. 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-<i>O</i>-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).</p

Inhibition of TTR-induced cytotoxicity.

<p>TTR at a final tetrameric concentration of 18 μM was pre-incubated with luteolin, diclofenac, diflunisal, or luteolin-7-<i>O</i>-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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128222#pone.0128222.ref044" target="_blank">44</a>]. 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-<i>O</i>-glucoside had no effect on TTR toxicity. The statistical significance was assessed using one-way ANOVA.</p

Luteolin reverses the phenotype of a <i>D</i>. <i>melanogaster</i> model of FAP.

<p>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. *, <i>p</i> < 0.05 by Mann Whitney <i>U</i> test TTRV30M 3.0 mM LUT. **, <i>p</i> < 0.05 by Mann Whitney <i>U</i> test for TTRV30M vehicle.</p

Flavonoids.

<p>(A) luteolin, (B) general formula of the flavonoid scaffold, (C) luteolin-7-O-glucoside, and (D) luteolin-7-O-glucuronide.</p

The crystal structure of TTR-luteolin complex.

<p>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.</p

Detailed view of the luteolin-binding site in TTR.

<p>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 σ^A-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.</p

Enthalpic Forces Correlate with the Selectivity of Transthyretin-Stabilizing Ligands in Human Plasma

The plasma protein transthyretin (TTR) is linked to human amyloidosis. Dissociation of its native tetrameric assembly is a rate-limiting step in the conversion from a native structure into a pathological amyloidogenic fold. Binding of small molecule ligands within the thyroxine binding site of TTR can stabilize the tetrameric integrity and is a potential therapeutic approach. However, through the characterization of nine different tetramer-stabilizing ligands we found that unspecific binding to plasma components might significantly compromise ligand efficacy. Surprisingly the binding strength between a particular ligand and TTR does not correlate well with its selectivity in plasma. However, through analysis of the thermodynamic signature using isothermal titration calorimetry we discovered a better correlation between selectivity and the enthalpic component of the interaction. This is of specific interest in the quest for more efficient TTR stabilizers, but a high selectivity is an almost universally desired feature within drug design and the finding might have wide-ranging implications for drug design