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.

Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis

“…The subunits of recombinant wild-type and Ser52Pro variant TTR proteins had molecular masses of 13,892.2 ± 0.9 Da and 13,902.1 ± 1.5 Da, respectively, corresponding to the expected theoretical mass values of 13,892.6 Da and 13,902.7 Da, including the additional N-terminal methionine residue, Met0. Correct assembly of the subunits into native homotetrameric wild-type and variant TTR was confirmed by both size-exclusion chromatography and native mass spectrometry, and these intact proteins bound the natural ligand, thyroxine, normally, showing the same IC 50 with mds84 and Tafamidis (13,14) as native wild-type TTR isolated from normal human serum (Table S1). However, the guanidine thiocyanate (Gdn-SCN)-mediated transition from folded tetramer to unfolded monomers ( Fig.…”

“…Binding of Tafamidis and mds84 (13,14), which both enter the TTR inner channel, had no significant effect on the products (Fig. 5A) or kinetics (Fig.…”

Stabilization of protein–protein interactions by small molecules

“…In this context, TTR amyloidosis has received widespread attention and several chemical classes of TTR kinetic stabilizers have been reported to date [72]. Among these, a benzoxazole series [73] was further optimized to 2-(3,5-dichlorophenyl)-benzoxazole-6-carboxylic acid (tafamidis; Table 1) [74], a potent TTR stabilizer that has been approved in Europe and Japan for the treatment of transthyretin-related hereditary amyloidosis. Tafamidis binds selectively to TTR variants and stabilizes the TTR dimer interface [74].…”

“…Among these, a benzoxazole series [73] was further optimized to 2-(3,5-dichlorophenyl)-benzoxazole-6-carboxylic acid (tafamidis; Table 1) [74], a potent TTR stabilizer that has been approved in Europe and Japan for the treatment of transthyretin-related hereditary amyloidosis. Tafamidis binds selectively to TTR variants and stabilizes the TTR dimer interface [74]. As can be seen from the crystal structure, tafamidis bound to TTR in a symmetric pocket established at the interface of the TTR.…”

Prion-Like Propagation of Protein Aggregation and Related Therapeutic Strategies

“…Thus, based on knowledge of tetramer structure, an alternative strategy was adopted to stabilize this form with a small molecule, Tafamidis. [90]. In a randomized, controlled trial it preserved nerve fiber function and slowed neurological deterioration in patients with amyloidosis [91].…”