Structure-based design of kinetic stabilizers that ameliorate the transthyretin amyloidoses

Abstract

Small molecules that bind to normally unoccupied thyroxine (T(4)) binding sites within transthyretin (TTR) in the blood stabilize the tetrameric ground state of TTR relative to the dissociative transition state and dramatically slow tetramer dissociation, the rate-limiting step for the process of amyloid fibril formation linked to neurodegeneration and cell death. These so-called TTR kinetic stabilizers have been designed using structure-based principles and one of these has recently been shown to halt the progression of a human TTR amyloid disease in a clinical trial, providing the first pharmacologic evidence that the process of amyloid fibril formation is causative. Structure-based design has now progressed to the point where highly selective, high affinity TTR kinetic stabilizers that lack undesirable off-target activities can be produced with high frequency.

Figure 1

The transthyretin (TTR) amyloidogenesis cascade. For amyloidogenesis to occur, the TTR tetramer must dissociate into four folded monomers and undergo partial denaturation in order to subsequently misassemble into a spectrum of aggregate structures including cross-β-sheet amyloid fibrils.

Figure 2

A table summarizing the transthyretin amyloidoses.

Figure 3

Structure of transthyretin•(T₄)₂ complex and the basis for TTR kinetic stabilization. (a) Crystal structure of TTR with T₄ bound to each T₄ binding site (PDB accession code 2ROX [16]). (b) Close-up view of one T₄ binding site with T₄ bound. Primed amino acids refer to those comprising symmetry-related halogen binding pockets (HBPs) of TTR monomers. (b) Kinetic stabilization of TTR by small molecule binding to the natively folded tetramer stabilizes the ground state and increases the tetramer dissociation barrier, preventing amyloidogenesis and pathology. (d) Schematic depiction of one of the two T₄ binding pockets within TTR occupied by a small molecule TTR kinetic stabilizer, where Y represents a linker of variable chemical structure (e.g. NH, O, CH=CH, C(O)NH, etc.) joining the two aryl rings, which typically bear a combination of alkyl, carboxyl, halide, trifluoromethyl, or hydroxyl substituents (X and Z). The inner and outer binding subsites are indicated.

Figure 4

Crystal structures of WT–TTR in complex with kinetic stabilizers from substructure X, Y and Z optimization studies. Ribbon diagram depiction of WT–TTR highlighting one of the two identical T₄ binding sites containing a schematic representation of a typical TTR kinetic stabilizer. (a) Aryl-X substructure optimization, PDB accession codes 2QGE, 2QGC and 2QGD, as indicated. (b) Linker-Y substructure optimization, PDB accession codes 3CN0, 3CN1 and 3CN4, as indicated. (c) Aryl-Z substructure optimization, PDB accession codes 3ESN, 3ESO and 3ESP, as indicated. All structures have ‘Connolly analytical’ surface representation of the pocket (green = hydrophobic, purple = polar), defined as all residues within 8 Å of ligand. Lysine 15′ and serines 117′ and 117 are shown with corresponding bonds with ligand. Figure is generated using the program MOE (2008.10), Chemical Computing Group, Montreal, Canada.

Figure 5

Overview of the substructure combination strategy. Potent and selective TTR kinetic stabilizers were created by combining highly ranked aryl-X and aryl-Z rings with appropriate linker-Y.