Trapping of palindromic ligands within native transthyretin prevents amyloid formation

Abstract

Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, 2,2'-(4,4'-(heptane-1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2'-(4,4'-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one stoichiometry, demonstrable in solution and by MS, was confirmed by X-ray crystallographic analysis showing simultaneous occupation of both T4 binding sites in each tetrameric TTR molecule by the pair of ligand head groups. Ligand binding by native TTR was irreversible under physiological conditions, and it stabilized the tetrameric assembly and inhibited amyloidogenic aggregation more potently than other known ligands. These superstabilizers are orally bioavailable and exhibit low inhibitory activity against cyclooxygenase (COX). They offer a promising platform for development of drugs to treat and prevent TTR amyloidosis.

Fig. 1.

Structures of TTR binding palindromic ligands mds84 and 4ajm15 together with compound Ia, 2-(3,5-dichlorophenylamino)benzoic acid, originally reported as compound 9 by Purkey et al. (16), compound Ib, 2-(3,5-dichloro-4-hydroxyphenylamino)benzoic acid, and compound II, Fx-1006A.

Fig. 2.

Ligand binding by TTR. (A) Displacement of ¹²⁵I-T4 from TTR in whole serum by 4ajm15 (●), mds84 (▲), and Fx-1006A (▼). Each point is the mean (SD) of three replicates. (B) Emission at 450 nm of 4ajm15 on addition to 1 μM TTR (●) or PBS (■) monitored after excitation at 340 nm. (Inset: spectra of 4ajm15 in PBS at three different concentrations.) Nanoflow electrospray mass spectra of WT TTR (C) and WT TTR complexed at 1:1 and 1:2 molar ratios with holo RBP (D) in absence (apo WT TTR; solid line) or presence of 1 molar equivalent of 4ajm15 (holo WT TTR; dashed line) recorded under nonactivating instrument conditions. The measured mass of apo and holo TTR is consistent with the binding of 1 mole of 4ajm15 per mole of TTR, even when TTR is complexed with one and two holo RBP molecules.

Fig. 3.

(A) Omit map (contoured at 2σ and 1.9 Å resolution) showing the complex electron density for the contents of one hormone binding site of TTR cocrystallized with 4ajm15 and fitted with two copies [one blue in halogen binding site 1 (HBS1) and one yellow (HBS3)] of the dichorophenyl constituent of the ligand generated by an 8-Å translation and ∼90° rotation. The outer density (yellow model; HBS3) is considerably weaker, and the refined atoms have higher B factors. (B) Rendered surface (slate) of the TTR dimer with superposed stick models (blue and yellow) of two complete molecules of 4ajm15 showing the sliding-fit model (Movie S1). (C) Electron density (Omit map contoured at 2σ and 1.2 Å resolution) for four molecules of compound Ib (Fig. 1) (one pair of symmetry-related molecules in each binding site) corresponding to the head group of 4ajm15, bound in the forward mode with chlorinated rings toward the center of the TTR tetramer. (D) Omit map (contoured at 2σ and 1.4 Å resolution) showing two symmetry-related mds84 molecules bound within the two hormone-binding sites of the TTR tetramer.

Fig. 4.

Kinetics of ligand binding by TTR. Fluorescence emission at 450 nm after excitation at 340 nm of TTR at 2 μM with T4 or mds84 at a threefold molar excess. Results are normalized to 100% fluorescence of the protein alone. The time constant was 11 s for mds84 and 3 s for T4.

Fig. 5.

(A) Displacement from TTR of mds84 and Fx-1006A by T4. (Left) Mass spectrum of a solution of holo WT TTR bound to mds84 before (Lower) and 6 d after (Upper) the addition of a 10-fold molar excess of T4. In both spectra, the measured mass indicates binding of 1 mole of mds84 per mole of tetramer. (Right) Mass spectrum of a solution of holo WT TTR with 2 moles of Fx-1006A bound per mole of protein before (Lower) and immediately after (Upper) addition of a 10-fold molar excess of T4. F designates holo TTR bound to two Fx-1006A molecules. Peaks corresponding to the displacement of one and two Fx-1006A molecules by T4 are designated FT and T, respectively. (B) Comparison of the subunit exchange in variant Leu55Pro TTR in the presence or absence of mds84. (Left) Mass spectra of an equimolar solution containing ¹²C¹⁵N, and ¹³C¹⁵N Leu55Pro variant TTR recorded at time 0 and after 48 h at room temperature; 48 h after mixing, additional peaks in the mass spectrum corresponding to 3:1, 2:2, and 1:3 (¹²C¹⁵N:¹³C¹⁵N) heterotetramers were observed. (Right) Mass spectra of the same equimolar solution of labeled and unlabeled proteins in the presence of a twofold molar excess of mds84. The binding of the ligand completely prevented any subunit exchange occurring in solution.

Fig. 6.

Inhibition by various ligands of TTR aggregation at low pH. Inhibition of aggregation of WT TTR at pH 4.4 (A) and amyloidogenic Leu55Pro variant TTR at pH 5.0 (B) by equimolar (solid bars) or threefold molar excess (open bars) of T4, Fx-1006A, and mds84 in solution in DMSO. Each bar shows the mean (SD) of three replicates.