Tafamidis, a potent and selective transthyretin kinetic stabilizer that inhibits the amyloid cascade

Abstract

The transthyretin amyloidoses (ATTR) are invariably fatal diseases characterized by progressive neuropathy and/or cardiomyopathy. ATTR are caused by aggregation of transthyretin (TTR), a natively tetrameric protein involved in the transport of thyroxine and the vitamin A-retinol-binding protein complex. Mutations within TTR that cause autosomal dominant forms of disease facilitate tetramer dissociation, monomer misfolding, and aggregation, although wild-type TTR can also form amyloid fibrils in elderly patients. Because tetramer dissociation is the rate-limiting step in TTR amyloidogenesis, targeted therapies have focused on small molecules that kinetically stabilize the tetramer, inhibiting TTR amyloid fibril formation. One such compound, tafamidis meglumine (Fx-1006A), has recently completed Phase II/III trials for the treatment of Transthyretin Type Familial Amyloid Polyneuropathy (TTR-FAP) and demonstrated a slowing of disease progression in patients heterozygous for the V30M TTR mutation. Herein we describe the molecular and structural basis of TTR tetramer stabilization by tafamidis. Tafamidis binds selectively and with negative cooperativity (K(d)s ~2 nM and ~200 nM) to the two normally unoccupied thyroxine-binding sites of the tetramer, and kinetically stabilizes TTR. Patient-derived amyloidogenic variants of TTR, including kinetically and thermodynamically less stable mutants, are also stabilized by tafamidis binding. The crystal structure of tafamidis-bound TTR suggests that binding stabilizes the weaker dimer-dimer interface against dissociation, the rate-limiting step of amyloidogenesis.

Fig. 1.

The TTR amyloid cascade. Amyloid formation by TTR requires rate-limiting tetramer dissociation to a pair of folded dimers, which then quickly dissociate into folded monomers. Partial unfolding of the monomers yields the aggregation-prone amyloidogenic intermediate. The amyloidogenic intermediate of TTR (Lower Right) retains much of its native structure (shown in purple), probably with some β-strand dissociation (shown in turquoise). The amyloidogenic intermediate can misassemble to form a variety of aggregate morphologies, including spherical oligomers, amorphous aggregates, and fibrils. Tafamidis binding to the TTR tetramer (Upper Left) dramatically slows dissociation, thereby efficiently inhibiting aggregation.

Fig. 2.

Inhibition of WT, V30M, and V122I TTR fibril formation under acidic conditions after 72 h. Purified TTR homotetramers (WT, V30M, or V122I at 3.6 μM) were mixed with tafamidis at final concentrations of 0, 0.9, 1.8, 2.7, 3.6, 4.5, 5.4, 6.3, and 7.2 μM and then incubated for 30 min at 25 °C. The pH was then adjusted to 4.4 (WT and V30M) or 4.5 (V122I) to allow for stabilization by tafamidis to be tested under conditions of comparable kinetics of fibril formation for the three alleles. The samples were incubated at 37 °C for 72 h and turbidity was then measured at 350 and 400 nm using a UV-visible (vis) spectrometer. For each allele, the endpoint turbidity in the absence of tafamidis (which varies slightly by allele) was defined as 100% fibril formation. Therefore, 5% fibril formation corresponds to a compound inhibiting 95% of TTR fibril formation after 72 h (60). Error bars represent the minimum and maximum values from three technical replicates.

Fig. 3.

Tafamidis stabilizes WT-TTR to urea-mediated denaturation. WT-TTR (1.8 μM) was incubated with 0, 1.8, and 3.6 μM tafamidis (corresponding to TTR:tafamidis molar ratios of 0, 1, and 2). Denaturation was initiated by adding urea to a final concentration of 6.5 M. After 72 h, circular dichroism spectra were collected between 220 and 213 nm, with scanning every 0.5 nm and an averaging time of 10 s. Each wavelength was scanned once. The values for the amplitude were averaged between 220 and 213 nm to determine the extent of β-sheet loss throughout the experiment. Results were expressed as a percentage of fraction unfolded relative to the amount observed in the absence of tafamidis (17).

Fig. 4.

Subunit exchange between WT-TTR and FLAG-tagged WT-TTR under physiologic conditions, monitored by anion exchange chromatography. Black square, WT-TTR; white square, FLAG-tagged WT-TTR. Homotetramers of WT-TTR (0 FLAG; 1.8 μM) and WT-TTR tagged with an N-terminal acidic FLAG tag (4 FLAG; 1.8 μM) were mixed with tafamidis at different concentrations (tafamidis:TTR tetramer molar ratios: 0.25, 0.50, 0.75, 1.00, 1.25, and 1.50) and incubated at 25 °C at a pH of 7. Samples were analyzed by anion exchange chromatography at the indicated times. (A) FPLC trace after 96-h incubation of 0 Flag TTR with 4 Flag TTR in the absence (gray) or presence (black) of tafamidis at a tafamidis:TTR tetramer molar ratio of 1.5. (B) Dose-dependent stabilization of TTR by tafamidis. At each time point, the extent of exchange was calculated by dividing the peak area of each tetramer by the sum of the peak areas for all of the tetramers. The fraction exchange was calculated by dividing the extent of exchange of 2 FLAG at each data point by 0.375 multiplied by 100. The predicted complete extent of exchange for 2 FLAG is 0.375, based on the statistical distribution of 1:4:6:4:1 for tetramer 0 FLAG–4 FLAG, respectively. Values for samples of tafamidis:TTR tetramer molar ratio = 1.5 at 220 and 264 h (not shown) were 9.5% and 9.8%, respectively.

Fig. 5.

Crystal structure of tafamidis bound to TTR. The coordinates are available in the Protein Data Bank under accession code 3TCT. (A) 3D ribbon diagram depiction of the TTR tetramer with tafamidis bound. The four TTR monomers are individually colored. (B) Magnified image of tafamidis bound in one of the T₄-binding sites. Connolly analytical surface representation (translucent gray, hydrophobic; translucent purple, polar) depicts the hydrophobicity of the binding site. The 3,5-chloro groups are placed in the HBPs 3 and 3′ making hydrophobic interactions, whereas the carboxylate of tafamidis engages in water-mediated H-bonds with the Lys15/15′ and Glu54/54′ residues of TTR represented by dotted lines (Lys15′-water H-bond not shown owing to tafamidis orientation). Fig. 5 was generated using the software Molecular Operating Environment, MOE (Chemical Computing Group).

Fig. 6.

Tafamidis stabilizes TTR in plasma from patients with WT, V30M, and V122I alleles under conditions of urea denaturation. (A) Dose-dependent stabilization of WT-TTR. Human plasma (pool, n = 11 healthy volunteers; Golden West Biologicals); premeasured TTR level is 3.6 μM. Under urea-mediated denaturation stress, TTR tetramer disappears over the course of 4 d. The addition of tafamidis stabilized TTR tetramer in a dose-dependent manner. Human plasma from patients with V30M TTR-FAP (B, n = 4) and V122I TTR-CM (C, n = 4) was obtained and pooled by genotype. Stabilization by tafamidis (7.2 μM) was compared with vehicle (DMSO). Tafamidis stabilized both V30M/WT and V122I/WT heterotetramers.