Tetrabromobisphenol A Is an Efficient Stabilizer of the Transthyretin Tetramer

Abstract

Amyloid formation of the human plasma protein transthyretin (TTR) is associated with several human disorders, including familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis. Dissociation of TTR's native tetrameric assembly is the rate-limiting step in the conversion into amyloid, and this feature presents an avenue for intervention because binding of an appropriate ligand to the thyroxin hormone binding sites of TTR stabilizes the native tetrameric assembly and impairs conversion into amyloid. The desired features for an effective TTR stabilizer include high affinity for TTR, high selectivity in the presence of other proteins, no adverse side effects at the effective concentrations, and a long half-life in the body. In this study we show that the commonly used flame retardant tetrabromobisphenol A (TBBPA) efficiently stabilizes the tetrameric structure of TTR. The X-ray crystal structure shows TBBPA binding in the thyroxine binding pocket with bromines occupying two of the three halogen binding sites. Interestingly, TBBPA binds TTR with an extremely high selectivity in human plasma, and the effect is equal to the recently approved drug tafamidis and better than diflunisal, both of which have shown therapeutic effects against FAP. TBBPA consequently present an interesting scaffold for drug design. Its absorption, metabolism, and potential side-effects are discussed.

Fig 1. TBBPA prevents tetramer dissociation under acidic conditions.

WT-TTR (A) and V30M-TTR (B) dissolved in PBS at a tetrameric concentration of 15 μM were pre-incubated alone or in the presence of TBBPA, diflunisal, or tafamidis (each at 15 μM) for 2 h. Aggregation of WT-TTR and V30M-TTR was initiated by lowering the pH to 4.5 using a sodium-acetate/acetic acid buffer followed by incubation for 72 h. The percentage of aggregation relative to the control was monitored by measuring the turbidity at 400 nm. Statistical analysis was performed using one-way ANOVA, and data are presented as the mean ± standard deviation of the percent fibril formation (n = 2, *** p < 0.001).

Fig 2. Probing the ability of the compounds to prevent TTR-mediated cytotoxicity in a human neuronal cell line.

TTR at a final tetrameric concentration of 15 μM was pre-incubated with TBBPA, diflunisal, or tafamidis (15 μM each) for 2 h. TTR or the TTR with inhibitor complexes were added to SH-SY5Y cells and incubated for 72 h. Cell viability was measured with a resazurin assay [46]. Data are reported as the mean cell viability ± the standard deviation (n = 3, ** p < 0.01; *** p < 0.001). The addition of TTR tetramer stabilizers leads to a statistically significant increase in the number of viable cells compared to cells exposed to TTR alone. The statistical significance was assessed using one-way ANOVA.

Fig 3. Determining the selectivity of TBBPA and tafamidis for TTR in human plasma.

TBBPA and tafamidis were titrated at different concentrations in human plasma from a single healthy donor and incubated for 2 h. The dissociation of plasma TTR was initiated by the addition of urea at a final concentration of 4.0 M. The dissociation to monomeric TTR was monitored by western blot and quantified with the ImageJ 2.0 software. The inhibitory concentration at 50% in plasma (IC₅₀) was estimated from three independent experiments. (A) TBBPA. (B) Tafamidis.

Fig 4

(A) The TTR-TBBPA structure shows the orientation of the ligand within the T4 binding sites. (B) Close-up view of the dimer-dimer interface of monomers B and B'. The σA-weighted (m|Fo|−D|Fc|) electron density is contoured at 3 times the root-mean-square value of the map and is shown in orange. To reduce model bias, the TBBPA molecules were excluded from the coordinate file that was subjected to one round of simulated annealing before calculation of the electron density map. The anomalous log-likelihood-gradient (LLG) map shown in dark blue shows the positions of the eight symmetry-related bromine atoms and verifies the modeled orientation of the TBBPA compound in the binding site. HBP1–3 and HBP1'–3' are indicated with numbers.

Fig 5. Molecular interaction between TBBPA and TTR at the B—B' hormone-binding site.

Carbon atoms from the B monomer and TBBPA are colored in blue, and carbon atoms from the B' monomer are colored in yellow. Lys15 and Leu17 have two rotamer conformations (conf1 and conf2), of which only the ones interacting with the bound TBBPA ligand are shown. The Nz atom of the B'-Lys15 side chain forms a hydrogen bond with a water molecule and the O5 atom of the TBBPA ligand, whereas the Nz atom of B-Lys15 adopts a different orientation to allow for a hydrophobic interaction with the TBBPA benzene ring. The side chain of Ser117 has three rotamer conformations of which one (occupancy 25%) makes a hydrogen bond to the same residue in the A monomer. The HBP1–3 and HBP1'–3' binding sites are indicated with black numbers, and bromine atoms are colored in purple.