Genistein, a natural product from soy, is a potent inhibitor of transthyretin amyloidosis

Abstract

The misfolding of transthyretin (TTR), including rate-limiting tetramer dissociation and partial monomer denaturation, is sufficient for TTR misassembly into amyloid and other abnormal quaternary structures associated with three amyloid diseases: senile systemic amyloidosis, familial amyloid polyneuropathy, and familial amyloid cardiomyopathy. Small molecules can bind to one or both of the unoccupied TTR thyroid hormone-binding sites, stabilizing the native tetramer more than the dissociative transition state, thereby raising the kinetic barrier for tetramer dissociation. Herein we demonstrate that genistein, the major isoflavone natural product in soy, works in this fashion and is an excellent inhibitor of transthyretin tetramer dissociation and amyloidogenesis, reducing acid-mediated fibril formation to <10% of that exhibited by TTR alone. Genistein also inhibits the amyloidogenesis of the most common familial amyloid polyneuropathy and familial amyloid cardiomyopathy mutations in TTR: V30M and V122I, respectively. Genistein additionally inhibits tetramer dissociation under physiological conditions thought to lead to slow amyloidogenesis in humans. Furthermore, this natural product exhibits highly selective binding to TTR in plasma over all of the other plasma proteins. Isothermal titration calorimetry shows that genistein binds to TTR with negative cooperativity (K(d1) = 40 nM, K(d2) = 1.4 microM). The benefits of using a nutraceutical such as genistein to treat orphan diseases such as the TTR amyloidoses include known oral bioavailability and safety data. It is conceivable that some patients could benefit from simply increasing their intake of soy products or supplements.

Fig. 1.

Line drawings depicting the structures of genistein (1), genistin (2), daidzein (3), daidzin (4), and apigenin (5) and the numbering of the isoflavone ring system.

Fig. 2.

Schematic representation of the tetrameric structure of TTR depicting the two thyroxine-binding sites. The two binding sites are interconverted by two C₂ axes perpendicular to the crystallographic twofold axis. Each binding site, filled with thyroxine, has an inner and outer binding pocket.

Fig. 3.

Partial acid denaturation-mediated aggregation of WT (A), V30M (B), and V122I (C) TTR. Blue bars represent data from an aggregate formation assay wherein tetrameric TTR (3.6 μM) is preincubated with inhibitor (3.6, 7.2, or 36 μM) for 30 min before lowering the pH to 4.4 (72 h). The y axis in each bar graph (optical density at 350 nm) represents aggregate formation relative to TTR (WT or variant, 3.6 μM) without inhibitor assigned as 100%. Hence 5% aggregate formation equates to 95% inhibition. The absolute turbidity OD₃₅₀ values for the uninhibited reactions are WT, 1.25; V30M, 1.36; and V122I, 1.10.

Fig. 4.

The rate of urea-mediated (6 M) tetramer dissociation for WT (A) (green circles), V122I (B), and V30M (C) TTR in the absence of small molecules. TTR dissociation is slowed dramatically when WT and the variants are preincubated with genistein. Far-UV CD ellipticity integrated over 214–218 nm at two concentrations of genistein (1.8 μM, ▴; 3.6 μM, ♦) was compared with that of TTR (WT or variant, 1.8 μM) without genistein to determine the fraction of TTR that dissociated and rapidly unfolded at each time point. As a reference, WT TTR tetramer dissociation occurs with a first-order rate constant of 0.033 h^-1 under these conditions.

Fig. 5.

Rate of WT TTR (1.8 μM tetramer) homotetramer subunit exchange with subunits from homotetramers of FT (1.8 μM tetramer) can be used to follow the rate of tetramer dissociation, because dissociation is rate-limiting for subunit exchange under physiological conditions. Subunit exchange kinetics decrease substantially at an equimolar concentration of genistein (3.6 μM, ▴), whereas, at twice the TTR concentration, genistein completely eliminates tetramer dissociation (7.2 μM, ♦). Percentage exchange is calculated by dividing the concentration of (TTR)₂(FT)₂ at each time point by its equilibrium concentration in the absence of inhibitor.