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).
Table 1.
Kd values obtained by ITC experiments and the IC50 values in plasma for TBBPA, tafamidis, and diflunisal.
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.