Stilbene Boronic Acids Form a Covalent Bond with Human Transthyretin and Inhibit Its Aggregation

Abstract

Transthyretin (TTR) is a homotetrameric protein. Its dissociation into monomers leads to the formation of fibrils that underlie human amyloidogenic diseases. The binding of small molecules to the thyroxin-binding sites in TTR stabilizes the homotetramer and attenuates TTR amyloidosis. Herein, we report on boronic acid-substituted stilbenes that limit TTR amyloidosis in vitro. Assays of affinity for TTR and inhibition of its tendency to form fibrils were coupled with X-ray crystallographic analysis of nine TTR·ligand complexes. The ensuing structure-function data led to a symmetrical diboronic acid that forms a boronic ester reversibly with serine 117. This diboronic acid inhibits fibril formation by both wild-type TTR and a common disease-related variant, V30M TTR, as effectively as does tafamidis, a small-molecule drug used to treat TTR-related amyloidosis in the clinic. These findings establish a new modality for covalent inhibition of fibril formation and illuminate a path for future optimization.

Figure 1

Three-dimensional structure of the TTR·resveratrol complex. TTR monomers (tan, red, green, and purple ribbons) have a β-sandwich fold and assemble into a tetramer, which binds to two molecules of resveratrol (ball-and-stick). The rings of resveratrol (1) occupy inner and outer pockets of the two T₄-binding sites at the dimer–dimer interfaces. The image was created with the program PyMOL and PDB entry 1dvs.

Figure 2

Three-dimensional structures of TTR·ligand complexes that contain a boronic acid group. One monomer (chain B) of the TTR tetramer is shown, and is in the same orientation in each panel. The main chain of TTR is rendered as a ribbon, and the side chains of Lys15 and Ser117 are shown explicitly. Ligands are depicted in a ball-and-stick rendition with CPK coloring and boron atoms labeled explicitly. Alternative conformations of Ser117 or the ligand are shown in some panels. Arrows indicate the O₁₁₇^γ–B bond in the TTR·10 and TTR·11 complexes. Images were created with the program PyMOL. (A) TTR·2 (PDB entry 5u48). (B) TTR·4 (5u4a). (C) TTR·6 (5u4c). (D) TTR·8 (5u4e). (E) TTR·10 (5u4f). (F) TTR·11 (5u4g).

Figure 3

Three-dimensional structures of TTR·ligand complexes that do not contain a boronic acid group. One monomer of the TTR tetramer is shown, and is in the same orientation in each panel. The main chain of TTR is rendered as a ribbon, and the side chains of Lys15 and Ser117 are shown explicitly. Ligands are depicted in a ball-and-stick rendition with CPK coloring. Alternative conformations of Ser117 or the ligand are shown in panels B and C. Images were created with the program PyMOL. (A) TTR·3 (PDB entry 5u49, chain A). (B) TTR·5 (5u4b, chain B). (C) TTR·7 (5u4d, chain B).

Figure 4

Halogen-bonding interactions in the TTR·10 and TTR·11 complexes. One monomer (chain B) of the TTR tetramer is shown. Chloro groups in the two ester-forming boronates exhibit C–Cl···O₁₀₈′ angles that are nearly linear and Cl··· O₁₀₈′ distances (dashed yellow lines) that are 3.6–3.8 Å (Table S12). Images were created with the program PyMOL. (A) TTR·10 (PDB entry 5u4f). (B) TTR·11 (5u4g).

Scheme 1

Routes for the Synthesis of Stilbenes 2–6

Scheme 2

Routes for the Synthesis of Stilbenes 7–11

Chart 1

Diphenol Ligands

Chart 2

Carboxylic Acid Ligands

Chart 3

Diboronic Acid and Related Ligands