Amyloidosis is a systemic disease caused by structural alteration of proteins that tend to precipitate in the extracellular space as insoluble fibrils. FAC is a hereditary TTR-related systemic amyloidosis with predominant cardiac involvement resulting from myocardial infiltration of amyloid protein. There are currently no FDA-approved drugs for prevention or treatment of TTR-induced amyloid cardiomyopathy and the therapy for most patients is confined to symptomatic relief. Liver transplantation, which removes the source of the pathologic protein, has been the treatment of choice for hereditary TTR amyloidoses. Heart transplantation is performed as a palliative measure for a subset of FAC patients who display predominately cardiac pathology and only mild systemic involvement (44
Small molecules targeting the formation, clearance, or assembly of toxic aggregates provide a promising strategy to treat amyloidoses (3
). A number of disease causing mutations (e.g., L55P, V30M, and V122I) influence the thermodynamic stability and the kinetics of dissociation of the TTR tetramer in vitro, and these properties of the variant tetramers appear to be correlated to the severity of the resulting disease (47
). Moreover, suppressor mutations in TTR (e.g., T119M) that kinetically stabilize the native tetramer and protect against the development of pathology in compound heterozygotes carrying a disease-associated mutation, have been identified (49
Binding of small molecule ligands to the unoccupied thyroxine (T4
) binding sites of TTR stabilizes the TTR tetramer and slows tetramer dissociation and amyloidogenesis in vitro. These kinetic stabilizers could potentially be used as small molecule therapeutics for the treatment of TTR amyloidoses (for review see: (16
Recently clinical trials of TTR kinetic stabilizers have been initiated in FAP patients. The two compounds, which are being tested are the NSAID, diflunisal (19
), and the benzoxazole, tafamidis (51
). Due to the lack of animal models that faithfully reproduce the pathology of human TTR-mediated familial amyloidoses, the efficacy of TTR kinetic stabilizers was assessed in human FAP patients. Several transgenic mouse models that overexpress human mutant TTR (V30M) do not reproduce the tissue distribution of TTR deposits observed in peripheral nerves of human FAP patients carrying the same mutation (52
). No mouse models that express transgenic human V122I TTR have been generated. In addition, the level of TTR overexpression required to observe TTR amyloid deposits in mice is very high. Because of the stoichiometric requirements of small molecule to TTR tetramer the concentrations of drug required to stabilize transgenic human TTR are difficult to achieve in these mouse models (2
). Almost all existing TTR kinetic stabilizers were identified by structure-based drug designs and time consuming assays, performed under non-physiological binding conditions. One such assay uses the displacement of radioactively-labeled T4
from TTR (28
). However, the use of radioactivity makes it unsuitable for HTS. Another assay is based on the ability of TTR binders to stabilize TTR during acid-mediated aggregation (29
). This is a simple but cumbersome assay (72 hours), which requires long incubation times, large amounts of protein and little kinetic information about ligand binding. In contrast, the FP-based assay we developed provided a simple, sensitive, and robust method that detected the binding of small molecules to the T4
binding pocket of TTR under physiological conditions.
Preliminary results indicate that probe 1
bound to TTR in human serum (fig. S10
). The measurement of TTR concentrations in blood samples is clinically used as a sensitive indicator of the nutritional status of patients. The half-life of TTR in humans in vivo
is about 2–4 days, which is much shorter than that of other nutritional markers such as albumin (approximately 20 days). Therefore, TTR concentration is more sensitive to changes in protein-energy status than albumin as it reflects recent dietary intake rather than overall nutritional status. In addition to being an excellent small molecule screening tool, this new FP assay has the potential to be developed as an alternative to the more error-prone immunoprecipitation-turbidity assay that is used by many clinical laboratories to determine plasma TTR concentrations (54
The FP assay was adapted for HTS and enabled us to identify a variety of highly potent and structurally diverse TTR kinetic stabilizers. Most TTR ligands up to date were identified by rational design. Consequently their chemical diversity is limited and many have COX-inhibitory activity. Genomic variations in the human population are known to influence the response to pharmacotherapy. Hence, in the current era of pharmacogenomics it seems desirable not to limit disease therapy to a “one drug fits all” system. Unlike many previously reported halogenated TTR ligands, most of the new chemical TTR binding scaffolds we discovered using this HTS, had minimal COX-1 or THR activity. Many of the known kinetic stabilizers of TTR, including diflunisal, which has been in clinical trials for FAP since 2006 (19
), show inhibitory activity towards COX. While there is never a simple way to predict whether a compound will be effective in all patients or whether individuals will suffer from adverse side effects, it is well known that COX inhibitors are associated with a number of adverse side effects, especially in patients with already compromised cardiac function, which makes their use in patients suffering from cardiomyopathy problematic. These adverse reactions include renal dysfunction and elevated blood pressure and they may precipitate heart failure in vulnerable individuals (20
Our newly identified TTR kinetic stabilizers were able to rescue cardiomyocytes from the proteotoxicity of TTR aggregates thought to cause FAC and SSA in patients. Thus these compounds offer additional classes of highly effective TTR tetramer stabilizers. Several of the new TTR ligands we identified appeared to be very selective for TTR in human serum, where they stabilized human TTR even in the presence of all other serum proteins such as albumin. Eight of the twelve ligands we tested were more potent TTR stabilizers (at 50 μM) than diflunisal, which is currently in clinical trials for the treatment of FAP. Binding to serum proteins is an important factor in determining the overall distribution, metabolism, activity, and toxicity of a drug. When ligands exhibit excellent TTR amyloid fibril inhibition data in vitro, yet display poor serum selectivity, it is assumed that they prefer to bind to the drug-binding sites in albumin and/or similar sites in other proteins found in plasma. It is unlikely that such promiscuous inhibitors will prevent TTR misfolding and amyloidosis in a complex environment like human blood or CSF.
The ligands we identified should be effective against SSA and FAC, because they bound both WT and V122I mutant TTR to kinetically stabilize tetramers. In conclusion, we discovered a number of new chemical scaffolds that were able to stabilize wt and mutant TTR and our data provide the incentive to further evaluate the efficacy of the most selective and potent ligands against FAC and SSA. No FDA-approved drugs or accepted therapeutic strategies are at this time available for treating SSA and FAC. Hence, we envision that several of the new TTR kinetic stabilizers described in this manuscript might be promising leads to be developed as therapeutics for FAC and SSA.