Transthyretin - TTR (OMIM 176300) is a well characterized molecule that consists of a tetramer of identical subunits of 127 amino acids each; the molecular structure has been determined by X-ray analysis (1). TTR is a plasma transport protein for thyroxine - T4 - and for retinol, through the association with retinol binding protein (RBP).

Over 80 different mutations in transthyretin (TTR) have been reported. The vast majority is inherited in an autosomal dominant manner and is related to amyloid deposition, affecting predominantly peripheral nerve and/or the heart. A small portion of TTR mutations is apparently non-amyloidogenic. Among these are mutations responsible for hyperthyroxinemia, presenting high affinity for T4 (a TTR ligand). Compound heterozygotic individuals for TTR mutants have been described; noteworthy is the clinically protective effect exerted by a non-pathogenic over a pathogenic mutation explained by stabilization of TTR tetrameric native structure. This fact lead to possible avenues of therapeutic intervention including stabilization via the central hydrophobic channel that runs through the molecule where thyroid hormones bind.

Amyloidogenic TTR mutations

Familial amyloidotic polyneuropathy - FAP - was first described by Andrade (2) in 1952 in the Northern area of Portugal; kindreds had an age of onset of clinical symptoms in the third to fourth decade of life. Early impairment of temperature and pain sensation in the feet and autonomic dysfunction leading to paresis, malabsorption, sphincter dysfunction, electrocardiographic abnormalities, emaciation and death were typical clinical features. The genetic defect in these Portuguese FAP kindreds was ascribed to a valine to methionine substitution at position 30 (3) – TTR Val30Met - resulting from a single A to G nucleotide change (4). Over 500 kindreds have been identified in Portugal, constituting the largest focus of FAP worldwide; the patient prevalence rate in the area where FAP is common in Portugal has been estimated as 105x10 -5 (5), and the gene carrier frequency as 1 in 625 (6). The second largest known Val30Met focus is Northern Sweden where more that 350 families have been diagnosed (7); other relevant foci include Japan and the Island of Maiorca (8,9). A few cases of homozygosity for the Met 30 gene occur but do not lead to a more severe form of the disease (10).

Many other TTR mutations associated with FAP clinically not differing from the original description by Andrade have been described; others, give rise to variable phenotypes such as the presence of both neuropathy and cardiomyopathy, presentation of carpal tunnel syndrome, predominant vitreous TTR deposition and leptomeningeal involvement. A few TTR mutations are related to cardiomyopathy without neurological symptoms. The most common TTR mutation associated with cardiac amyloidosis is Val122Ile, described in the Black population; after the age of 60, isolated cardiac amyloidosis is four times more common among blacks than whites in the United States and 3.9 percent of blacks are heterozygous for Val122Ile; a few cases of homozygosity for this mutant have been found (11). A list of TTR mutations associated with amyloidosis is presented in Table 1 (bibliographic references can be found at: http://www.ibmc.up.pt/~mjsaraiv/ttrmut.html

Table 1 - Transthyretin mutations in amyloidoses



Table 2 - Non-amyloid TTR mutations and compound heterozygotes

Non-amyloidogenic TTR mutations

Several TTR mutations without pathogenic consequences and compound heterozygotes carriers of TTR mutations have been described and are presented in Table 2 (bibliographic references can be found at: http://www.ibmc.up.pt/~mjsaraiv2ttrmut.html

The allele frequency has been estimated in screening studies in different populations; this is the case of Gly6Ser present in about 12% of the Caucasian population (12) and the Thr119Met mutation found in about 0.8% of Portuguese and German populations investigated (5). Of particular importance is compound heterozygosity of non-amyloid and amyloid mutations usually occurring in different alleles. Thus, the polymorphic Gly6Ser mutation has been described in association with different amyloid mutants as documented in Table 2; this mutation does not influence the clinical outcome of Val30Met carriers (13), whereas the Thr119Met and the Arg104His mutations do. Thus, differences in clinical presentation and severity of symptoms among Portuguese and Japanese Val30Met patients carrying respectively the Met119 and the His104 mutations are observed with a clear protective effect exerted by the nonpathogenic mutation (14,15), which confer more stability to the molecule (see below). Substitutions in position 109 have been found in individuals with euthyroid hyperthyroxinemia and lead to an increase in the affinity for T4 (16).

Aggregation pathway

The three dimensional structure of TTR revealed an extensive ?-sheet structure. Each monomer contains two ?-sheets, composed of strands DAGH and CBEF, which interact face-to-face through hydrogen bonds between strands HH´ and FF´ to form a dimer (represented in Fig. 1). In the tetramer, hydrogen bonds between main chain atoms belonging to loop AB of one monomer and strand H´ from the other monomer as well as hydrophobic contacts are important.



Fig. 1 Title

The effects introduced by amyloidogenic mutations have been the subject of intensive study mainly by X-ray crystallography but, with the exception of the Leu55Pro mutation, did not reveal drastic changes; so far, the solved structures point to a clear destabilization of the tetrameric structure of the protein. The structural studies by X-ray diffraction on the particularly aggressive mutant TTR - Leu55Pro revealed aggregation of monomeric TTR, consistent with data from synchrotron analyses of "ex vivo" fibrils (17); important changes in secondary structure by the disruption of strand D which becomes part of a long loop that connects strands C and E were observed. Disruption of the D strand affects the hydrogen bonding with the A strand, exposing new surfaces involved in aggregation; in particular, the contacts of the ?-helix and the AB loop are different, suggesting these regions are important in amyloidogenesis. In fact, deletion or multiple substitutions in the D strand lead to highly amyloidogenic mutants (18); when monoclonal antibodies were generated against these mutants, they recognize circulating TTR form carriers of FAP mutations, pointing out that modified tetrameric species circulate in FAP patients (19). These species have a higher tendency to dissociate into monomers, which then polymerize into amyloid fibrils.

The Leu55Pro structure also pointed out for an important bond between Tyr78 and the AB loop, in stabilizing the quaternary structure. Based on this information a putative TTR mutant Tyr78Phe was constructed, leading to a highly amyloidogenic TTR recognized in its soluble tetrameric form by monoclonal antibodies specific for the amyloid fold (see above) and forming amyloid fibrils readily at neutral pH (20). In summary, mutations that destabilize the D strand, or that loose the AB loops of the tetramer and dimer-dimer interactions increase the susceptibility of amyloid formation.

Possible therapeutic strategies in FAP: TTR stabilizers and fibril disrupters

Based on our knowledge of the aggregation pathway, possible therapeutic strategies in FAP encompass either preventing dissociation of the native tetrameric structure into monomers, the building blocks of fibrils, through tetrameric stabilizers or disrupting the fibril structure through fibril disrupters.

Development of TTR stabilizers derives for most part from early work on the protective Thr119Met TTR mutation. X-ray analysis of the TTR Thr119Met:T4 complex demonstrated that this variant presents alterations in the T4 binding channel including dimer-dimer contacts. When compared to the wild type protein or to the amyloidogenic TTR Val30Met, the TTR Thr119Met variant shows new atomic interactions involving hydrogen bonds occurring within monomers, dimers and the tetramer (21). These additional interactions explain the higher stability of the variant and its protective effect over the Val30Met mutation in compound heterozygotic carriers of TTR Val30Met / TTR Thr119Met. Thus, tetrameric stabilizers can act either by binding TTR in the central hydrophobic channel that runs through the molecule, where the hydrophobic hormone T4 binds, to prevent dissociation into monomers (as documented in figure 2) or by affecting interactions within the monomer.




Fig. 2 – Complex of TTR tetramer and retinol binding protein with
stabilizer in TTR central hydrophobic channel

Several compounds have been reported as amyloid fibril formation inhibitors, «in vitro», as is the case of some NSAIDs including flufenamic acid, diflunisal and diclofenac (22,23). For TTR amyloidoses it has been proposed that they would exert their effect through binding to T4 binding sites in TTR as demonstrated by the crystal structure of some of the complexes, in particular the TTR-diclofenac complex (23). However, all the assays reported were performed using isolated recombinant TTR «in vitro», in the absence of physiological modulating factors, namely other plasma binding proteins. Therefore, it is of most importance to characterize the interaction of the different compounds with TTR concerning binding specificity and selectivity to assure their preferential binding to TTR in plasma over other thyroxine binding proteins, in particular the highly abundant albumin and the most potent T4 binding protein of human serum, thyroxine.binding globubin, TBG. Binding by proteins other than TTR leads to decreased drug availability in plasma and compromise their use as therapeutic agents for TTR amyloidosis; this fact led to the development of novel derivatives that show TTR binding in «ex vivo» assays for T4 and for tetrameric stability. For instance, an iodinated diflunisal derivative – IDIF – has been shown to present high specificity and high binding affinity to TTR as shown in the T4 binding protein profile obtained after electrophoresis of plasma. Unlike diclofenac, diflunisal and flufenamic acid, which bound to albumin and to TBG, IDIF and BrDIF, displaced T4 preferentially from TTR (24).

IDIF disclosed not only selective TTR binding affinity but also efficiently stabilized TTR from dissociation into monomers in plasma from heterozygotic TTR Val30Met carriers and from controls. This was demonstrated by plasma TTR resistance to dissociation, after incubation with the compounds, in isoelectric focusing experiments. Similar «ex vivo» results were obtained using TTR Val30Met transgenic mouse plasma. In this "ex vivo" assay diclofenac and flufenamic acid, previously reported as inhibitors of TTR fibril formation did not seem to stabilize the TTR tetramer. Taken together, "ex vivo" and "in vitro" studies present evidence for the selectivity and efficiency of novel diflunisal derivates as TTR stabilizers and inhibitors of fibril formation vis a vis reported TTR fibril inhibitors. The criteria of: (i) "ex vivo" TTR binding selectivity in T4 binding sites; (ii) "ex vivo" TTR tetrameric stabilization; and (iii) definition of the inhibitory step of fibrillogenesis, must be taken into consideration in further testing of drugs with therapeutic interest in TTR amyloidosis.

As for TTR fibril disrupters, the effect of the drug 4’-iodo-4’-deoxydoxorubicin (I-DOX) on the in vitro assembly of TTR Leu55Pro fibrils has been investigated by following the fibril growth over a 15 day period in the presence and absence of this drug. I-DOX did not inhibit fibril formation up to 10 days since fibrils of approximately 7- 8 nm width, identical to the fibrils present in the non-treated sample were observed. However, after 15 days of I-DOX treatment, only round particles approximately 5- 6 nm wide, resembling soluble native TTR were observed. A series of other compounds including tetracyclines, and nitrophenols have also been studied for their effects on amyloid fibril formation/disagregation (25). Among these, doxycycline resulted in the complete disaggregation of fibrils. In addition, the species generated upon I-DOX or tetracyclines were non-toxic, as revealed by the lack of significant caspase-3 activation on a Schwanoma cell line, making this class of compounds, in particular doxycycline, of potential use in the treatment of TTR amyloidoses.