Transthyretin (TTR) is a soluble human plasma protein that can be converted into amyloid by acid-mediated dissociation of the homotetramer into monomers. The pH required for disassembly also results in tertiary structural changes within the monomeric subunits. To understand whether these tertiary structural changes are required for amyloidogenicity, we created the Phe87Met/Leu110Met TTR variant (M-TTR) that is monomeric according to analytical ultracentrifugation and gel filtration analyses and nonamyloidogenic at neutral pH. Results from far-and near-UV circular dichroism spectroscopy, onedimensional proton NMR spectroscopy, and X-ray crystallography, as well as the ability of M-TTR to form a complex with retinol binding protein, indicate that M-TTR forms a tertiary structure at pH 7 that is very similar if not identical to that found within the tetramer. Reducing the pH results in tertiary structural changes within the M-TTR monomer, rendering it amyloidogenic, demonstrating the requirement for partial denaturation. M-TTR exhibits stability toward acid and urea denaturation that is nearly identical to that characterizing wild-type (WT) TTR at low concentrations (0.01-0.1 mg/mL), where monomeric WT TTR is significantly populated at intermediate urea concentrations prior to the tertiary structural transition. However, the kinetics of denaturation and fibril formation are much faster for M-TTR than for tetrameric WT TTR, particularly at near-physiological concentrations, because of the barrier associated with the tetramer to folded monomer preequilibrium. These results demonstrate that the tetramer to folded monomer transition is insufficient for fibril formation; further tertiary structural changes within the monomer are required.

Twelve analogues of diclofenac (1), a nonsteroidal antiinflammatory drug and known inhibitor of transthyretin (TTR) amyloid formation, were prepared and evaluated as TTR amyloid formation inhibitors. High activity was exhibited by five of the compounds. Structure-activity relationships reveal that a carboxylic acid is required for activity, but changes in its position as well as the positions of other substituents are tolerated. High-resolution X-ray crystal structures of four of the active compounds bound to TTR were obtained. These demonstrate the significant flexibility with which TTR can accommodate ligands within its two binding sites.

Polychlorinated biphenyls (PCBs) and their hydroxylated metabolites (OH-PCBs) are known to bind to transthyretin (TTR) in vitro, possibly explaining their bioaccumulation, rodent toxicity, and presumed human toxicity. Herein, we show that several OH-PCBs bind selectively to TTR in blood plasma; however, only one of the PCBs tested binds TTR in plasma. Some of the OH-PCBs displace thyroid hormone (T4) from TTR, rationalizing the toxicity observed in rodents, where TTR is the major T4 transporter. Thyroid binding globulin and albumin are the major T4 carriers in humans, making it unlikely that enough T4 could be displaced from TTR to be toxic. OH-PCBs are excellent TTR amyloidogenesis inhibitors in vitro because they bind to the TTR tetramer, imparting kinetic stability under amyloidogenic denaturing conditions. Four OH-PCB/TTR cocrystal structures provide further insight into inhibitor binding interactions.