Repositioning tolcapone as a potent inhibitor of transthyretin amyloidogenesis and associated cellular toxicity
Abstract
Transthyretin (TTR) is a plasma homotetrameric protein implicated in fatal systemic amyloidoses. TTR tetramer dissociation precedes pathological TTR aggregation. Native state stabilizers are promising drugs to treat TTR amyloidoses. Here we repurpose tolcapone, an FDA-approved molecule for Parkinson's disease, as a potent TTR aggregation inhibitor. Tolcapone binds specifically to TTR in human plasma, stabilizes the native tetramer in vivo in mice and humans and inhibits TTR cytotoxicity. Crystal structures of tolcapone bound to wild-type TTR and to the V122I cardiomyopathy-associated variant show that it docks better into the TTR T4 pocket than tafamidis, so far the only drug on the market to treat TTR amyloidoses. These data indicate that tolcapone, already in clinical trials for familial amyloid polyneuropathy, is a strong candidate for therapeutic intervention in these diseases, including those affecting the central nervous system, for which no small-molecule therapy exists.
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2 a b Supplementary Figure 1. Tolcapone prevents WT, V122I-TTR and A25T-TTR aggregation as measured by total protein precipitation. a) WT and V122I-TTR were incubated with different concentrations of tolcapone under aggregation conditions (pH 4.4 and 37°C) for 72 hours. The samples were then centrifuged at 20,000 x g for 1 h at 4 °C and the supernatants (soluble protein) were carefully removed. The precipitated TTR from each sample was resolubilized by addition of 8 M guanidine solution followed by 1 h incubation at RT. TTR concentration was measured by spectroscopy at 280 nm and total amount of resolubilized TTR was calculated. The data is presented as % insoluble TTR with respect to samples in which no tolcapone were added (n = 1). b) The same experiment was performed for A25T-TTR at pH 5.0 and 37°C for 22 hours.
3 a b Supplementary Figure 2. Fluorescence and absorbance spectra of WT-TTR in the presence and absence of Tolcapone. a) Fluorescence spectra of apo-WT-TTR, WT-TTR:tolcapone (2 µM:10 µM) and tolcapone alone (1 µM and 10 µM). Excitation wavelength 280 nm; emission collected from 300 to 400 nm. b) Absorbance spectra of tolcapone and of WT-TTR (2 µM) in the presence or absence of tolcapone.
4 Supplementary Figure 3. Interaction of TTR with Tafamidis assessed by ITC. WT (left) and V122I-TTR (right); upper panels: thermograms (thermal power versus time) after baseline correction; bottom panels: binding isotherm (normalized heat versus molar ratio of reactants). 0 1 2 3 4 5 6 7 8 9 -10 -5 0 -0.3 -0.2 -0.1 0.0 0.1 0.2 010 20 30 40 50 time (min) dQ/dt (µcal/s) [Tafamidis]T/[TTR WT]T Q (kcal/mol of injectant) [Tafamidis]T[TTR+WT]T+ 0 1 2 3 4 5 6 7 8 9 -10 -5 0 -0.3 -0.2 -0.1 0.0 0.1 0.2 010 20 30 40 50 time (min) dQ/dt (µcal/s) Molar Ratio Q (kcal/mol of injectant) [Tafamidis]T[TTR+WT]T+ Kd1 +29+nM+ Kd2 +1100+nM+ + + Kd1+5.7+nM+ Kd2+260+nM+ + +
5 Supplementary Figure 4. TTR dimer interfaces. a) Surface representation of the dimer in the WT TTR crystal structure (PDB: 1DVQ). b) Surface representation of the dimer interface in the tolcapone bound TTR crystal structure (PDB: 4D7B). c) Surface representation of the dimer interface in the tafamidis bound TTR crystal structure (PDB 3TCT). Dimer interface ordered water molecules in the crystal structures are represented by solid balls. a b c
6 a b Supplementary Figure 5. Stereo images of the electron density maps. Stereo views of the TTR wild type (a) and of the TTR-V122I (b) in complex with tolcapone with a 2Fo-Fc map (blue mesh) contoured at 1.5 σ.
7 Supplementary Figure 6. Selected images of the film used in main figure 4a.
8 Supplementary Figure 7. Selected images of the gel used in main figure 4b.
9 Supplementary Figure 8. Selected images of the dot blot used in main figure 5c.
10 Supplementary Table 1. Y78F-TTR aggregation inhibitory activity of selected compounds. Compound( n( Model( EC50a(( µ M)( RAb((%)( SOM01( 2" Linear" >100" -" SOM02( 2" Linear" 56" -" SOM03( 2" Linear" >100" -" SOM04( 2" Exponential" 59" 35.5" SOM05( 2" Linear" >100" -" SOM06( 2" Exponential" 58" 37.2" SOM07( 4" Exponential" >100" 26" SOM08( 2" Linear" >100" -" SOM09( 2" Linear" >100" -" SOM10( 2" Linear" >100" -" tolcapone( 4" Exponential" 3.92" 89.13" SOM12( 2" Linear" >100" -" SOM13( 2" Linear" 70" -" SOM14( 2" Linear" >100" -" SOM15( 2" Linear" >100" -" SOM16( 2" Exponential" >100" 38.3" SOM17( 2" Linear" >100" -" SOM18( 2" Linear" 84" -" SOM19( 2" Linear" 87.38" -" SOM20( 2" Exponential" 58" 46.7" SOM21( 2" Linear" 78.61" -" SOM22( 2" Linear" >100" -" SOM23( 2" Linear" >100" -" SOM24( 4" Exponential" >100" 31.6" SOM25( 2" Linear" >100" -" SOM26( 2" Linear" >100" -" SOM27( 2" Exponential" >100" 34.9" SOM28( 2" Exponential" >100" 25.8" SOM29( 2" Exponential" >100" 24.6" tafamidis( 6" Exponential" 5.36" 59.57" a EC50: concentration of inhibitor at which the initial rate of TTR aggregation is one-half that of Y78F-TTR without inhibitor. b RA (%): percent reduction of TTR aggregation rate at high inhibitor concentration relative to the rate in its absence.