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The quaternary structure stability of proteins is typically studied under conditions that accelerate their aggregation/unfolding processes on convenient laboratory time scales. Such conditions include high temperature or pressure, chaotrope-mediated unfolding, or low or high pH. These approaches have the limitation of being nonphysiological and that the concentration of the protein in solution is changing as the reactions proceed. We describe a methodology to define the quaternary structure stability of the amyloidogenic homotetrameric protein transthyretin (TTR) under physiological conditions. This methodology expands from a described approach based on the measurement of the rate of subunit exchange of TTR with a tandem flag-tagged (FT₂) TTR counterpart. We demonstrate that subunit exchange of TTR with FT₂·TTR can be analyzed and quantified using a semi-native polyacrylamide gel electrophoresis technique. In addition, we biophysically characterized two FT₂·TTR variants derived from wild-type and the amyloidogenic variant Val122Ile TTR, both of which are associated with cardiac amyloid deposition late in life. The FT₂·TTR variants have similar amyloidogenic potential and similar thermodynamic and kinetic stabilities compared to those of their nontagged counterparts. We utilized the methodology to study the potential of the small molecule SOM0226, a repurposed drug under clinical development for the prevention and treatment of the TTR amyloidoses, to stabilize TTR. The results enabled us to characterize the binding energetics of SOM0226 to TTR. The described technique is well-suited to study the quaternary structure of other human aggregation-prone proteins under physiological conditions.

13 pags, 6 figs, 1 tab. -- Supplementary information is available at the Publisher's web, 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., This work was funded by the Spanish Ministry of Economy and Competitiveness (BFU2013-44763 to S.V., BFU2012-37116 to D.R., RTC-2014-1931-1 to R.I. and BFU2013-47064-P to A.V.-C.), by SUDOE INTERREG IV B (SOE4/P1/E831 to S.V. and A.V.-C.), by the Spanish Ministry of Science and Innovation (BFU2010-19451 to A.V.-C.), by Diputacion General de Aragon (Protein Targets Group B89 to A. V.-C.) and by the National Institutes of Health (NIA, AG-032285 to N. Reixach). S.V. has been granted an ICREA ACADEMIA award. SOM Biotech was funded by the Spanish Ministry of Health DIN 10-13 grant and the Catalan Competitiveness Agency FINEBT10-2-0037 grant. N. Reig and R.I. were funded by a Torres Quevedo grant by the Spanish Ministry of Economy and Competitiveness. R.S. was supported by CAPES-Science without Borders and S.E. by Consejo Nacional de Promocion Cientifica y Tecnologica (CONICET). We also thank Dr. Debora Foguel for the WT-TTR plasmid used in this study to produce recombinant WT-TTR and Dr Antoni Planas and Dr Ellen Cotrina for technical work. The human cardiac AC16 cell line was developed by Mercy M Davidson at Columbia University46. X-ray experiments were performed at the BL-13 beamline at the ALBA synchrotron in collaboration with the ALBA staff.