The transthyretin amyloidoses: from delineating the molecular mechanism of aggregation linked to pathology to a regulatory-agency-approved drug

Abstract

Transthyretin (TTR) is one of the many proteins that are known to misfold and aggregate (i.e., undergo amyloidogenesis) in vivo. The process of TTR amyloidogenesis causes nervous system and/or heart pathology. While several of these maladies are associated with mutations that destabilize the native TTR quaternary and/or tertiary structure, wild-type TTR amyloidogenesis also leads to the degeneration of postmitotic tissue. Over the past 20 years, much has been learned about the factors that influence the propensity of TTR to aggregate. This biophysical information led to the development of a therapeutic strategy, termed "kinetic stabilization," to prevent TTR amyloidogenesis. This strategy afforded the drug tafamidis which was recently approved by the European Medicines Agency for the treatment of TTR familial amyloid polyneuropathy, the most common familial TTR amyloid disease. Tafamidis is the first and currently the only medication approved to treat TTR familial amyloid polyneuropathy. Here we review the biophysical basis for the kinetic stabilization strategy and the structure-based drug design effort that led to this first-in-class pharmacologic agent.

Figure 1

Structure of transthyretin (TTR; PDB code: 2ROX). (A) Ribbon diagram depiction of TTR with the crystallographic two-fold axis (Z-axis) bisecting the T₄ binding channel comprising the weaker of TTR’s two dimer-dimer interfaces(B)B. Close-up view of onethyroid hormone binding site with T₄(shown as a ball-and-stick representation) bound, showing the iodide substituents occupying symmetry-related halogen binding pockets. Primed amino acids refer to those comprising symmetry-related subunits. Hydrogen bonds are shown as light blue, dashed lines. Figure adapted from Connelly et al.

Figure 2

Folding free energy landscape of an amyloidogenic protein that normally forms a well folded 3D structure, but can also aggregate as a consequence of a conformational change, e.g., TTR or lysozyme. Three energy wells are shown: the native state, a partially unfolded amyloidogenic intermediate, and an aggregated state. Conformational excursions convert the native state to the partially unfolded state, which can then aggregate. The stability of the aggregated state depends on the protein concentration. At low protein concentrations, it would be less stable than the native state, and therefore not substantially populated. As the protein concentration increases, it becomes increasingly stable, and will eventually become the most stable state.

Figure 3

Energy diagrams associated with three distinct mechanisms of protein aggregation. In a nucleated polymerization (top), the initial association events are unfavorable until a critical sizeis reached. The oligomer of this size is referred to as the nucleus. Subsequent steps are favorable, making further growth favorable for oligomers larger than the nucleus. In a nucleated conformational conversion (middle), facile initial association steps form amorphous oligomers. Oligomers of a certain size can undergo a rate-limiting conversion step, in which they change from an amorphous structure to a cross-β-sheet fibrillar state. Subsequent steps are favorable, as in the nucleated polymerization. In a downhill polymerization (bottom), the mechanism by which TTR aggregates, all of the association steps are favorable after formation of the amyloidogenic intermediate, and there is no kinetic barrier to oligomerization. The aggregates shown are ordered, but they need not be; TTR forms a collection of aggregate structures.

Figure 4

TTR amyloid cascade. In order for TTR to form amyloid, the tetramer must first dissociate (the rate-limiting step) and then the natively folded monomer must undergo partial denaturation to become competent to misassemble into a variety of aggregate morphologies, including oligomers and amyloid fibrils. Ligands (such as thyroxine, shown in gray and red) stabilize the tetramer and thus prevent amyloidogenesis.

Figure 5

Kinetic stabilization through T119M TTR subunit incorporation into TTR tetramers.(A) Urea-mediated tetramer dissociation time courses of the T119M TTR homotetramer, wild type TTR homotetramer, or mixed tetramers produced by co-expression of the two different subunits, the stoichiometry being indicated on the right. (B) Free energy diagram illustrating that the increase in activation energy required for tetramer dissociation is proportional to the number of T119M subunits comprising the tetramer. (C) Ribbon diagram depiction of T119M TTR, where in the 119M side chains shown in CPK representation stabilize the weaker of TTR’s two dimer-dimer interfaces (PDB code: 1BZE). Figure adapted from Hammarstrom et al. .

Figure 6

The structural diversity of TTR kinetic stabilizer core structures. (A) Line drawings of the structural cores underpinning the 1000+ TTR kinetic stabilizers synthesized to date. Adapted from Johnson et al. (B) Schematic depiction of the substructure combination strategy to create potent and highly selective TTR kinetic stabilizers. Individual elements of candidate TTR kinetic stabilizers are varied and the most potent and selective substructures of the candidates are combined to create potent, highly selective TTR kinetic stabilizers. Adapted from Choi et al.

Figure 7

(A) Line drawing of tafamidis. (B) Structural model of how tafamidis is envisioned to bind to and kinetically stabilize TTR.

Figure 8

(A) Top: Close-up view of a bivalent TTR kinetic stabilizer bound to the thyroid hormone binding sites (PDB code:2FLM). Bottom: Schematic representation of a bivalent TTR kinetic stabilizer bound simultaneously to both T₄ binding sites of tetrameric TTR. Figure adapted from Green et al. (B) Close-up view of a covalent kinetic stabilizer attached via an amide bond to Lys 15 in one thyroid hormone binding site of TTR (PDB code: 3HJ0).