Deuterium-proton exchange on the native wild-type transthyretin tetramer identifies the stable core of the individual subunits and indicates mobility at the subunit interface

Transthyretin is a human protein capable of amyloid formation that is believed to cause several types of amyloid disease, depending on the sequence deposited. Previous studies have demonstrated that wild-type transthyretin (TTR), although quite stable, forms amyloid upon dissociation from its native tetrameric form into monomers with an altered conformation. Many naturally occurring single-site variants of TTR display decreased stability in vitro, manifested by the early onset familial amyloid diseases in vivo. Only subtle structural changes were observed in X-ray crystallographic structures of these disease associated variants. In this study, the stability of the wild-type TTR tetramer was investigated at the residue-resolution level by monitoring (2)H-H exchange via NMR spectroscopy. The measured protection factors for slowly-exchanging amide hydrogen atoms reveal a stable core consisting of strands A, B, E, F, and interestingly, the loop between strands A and B. In addition, the faster exchange of amide groups from residues at the subunit interfaces suggests unexpected mobility in these regions. This information is crucial for future comparisons between disease-associated and wild-type tetramers. Such studies can directly address the regions of TTR that become destabilized as a consequence of single amino acid substitutions, providing clues to aspects of TTR amyloidogenesis.     

Quantification of the thermodynamically linked quaternary and tertiary structural stabilities of transthyretin and its disease-associated variants: the relationship between stability and amyloidosis

Urea denaturation studies were carried out as a function of transthyretin (TTR) concentration to quantify the thermodynamically linked quaternary and tertiary structural stability and to improve our understanding of the relationship between mutant folding energetics and amyloid disease phenotype. Urea denaturation of TTR involves at least two equilibria: dissociation of tetramers into folded monomers and monomer unfolding. To deal with the thermodynamic linkage of these equilibria, we analyzed concentration-dependent denaturation data by globally fitting them to an equation that simultaneously accounts for the two-step denaturation process. Using this method, the quaternary and tertiary structural stabilities of well-behaved TTR sequences, wild-type (WT) TTR and the disease-associated variant V122I, were scrutinized. The V122I variant is linked to late onset familial amyloid cardiomyopathy, the most common familial TTR amyloid disease. V122I TTR exhibits a destabilized quaternary structure and a stable tertiary structure relative to those of WT TTR. Three other variants of TTR were also examined, L55P, V30M, and A25T TTR. The L55P mutation is associated with the most aggressive familial TTR amyloid disease. L55P TTR has a complicated denaturation pathway that includes dimers and trimers, so globally fitting its concentration-dependent urea denaturation data yielded error-laden estimates of stability parameters. Nevertheless, it is clear that L55P TTR is substantially less stable than WT TTR, primarily because its tertiary structure is unstable, although its quaternary structure is destabilized as well. V30M is the most common mutation associated with neuropathic forms of TTR amyloid disease. V30M TTR is certainly destabilized relative to WT TTR, but like L55P TTR, it has a complex denaturation pathway that cannot be fit to the aforementioned two-step denaturation model. Literature data suggest that V30M TTR has stable quaternary structure but unstable tertiary structure. The A25T mutant, associated with central nervous system amyloidosis, is highly aggregation-prone and exhibits drastically reduced quaternary and tertiary structural stabilities. The observed differences in stability among the disease-associated TTR variants highlight the complexity and heterogeneity of TTR amyloid disease, an observation that has important implications for the treatment of these maladies.     

The beta-strand D of transthyretin trapped in two discrete conformations

Conformational changes in native and variant forms of the human plasma protein transthyretin (TTR) induce several types of amyloid diseases. Biochemical and structural studies have mapped the initiation site of amyloid formation onto residues at the outer C and D beta-strands and their connecting loop. In this study, we characterise an engineered variant of transthyretin, Ala108Tyr/Leu110Glu, which is kinetically and thermodynamically more stable than wild-type transthyretin, and as a consequence less amyloidogenic. Crystal structures of the mutant were determined in two space groups, P2(1)2(1)2 and C2, from crystals grown in the same crystallisation set-up. The structures are identical with the exception for residues Leu55-Leu58, situated at beta-strand D and the following DE loop. In particular, residues Leu55-His56 display large shifts in the C2 structure. There the direct hydrogen bonding between beta-strands D and A has been disrupted and is absent, whereas the beta-strand D is present in the P2(1)2(1)2 structure. This difference shows that from a mixture of metastable TTR molecules, only the molecules with an intact beta-strand D are selected for crystal growth in space group P2(1)2(1)2. The packing of TTR molecules in the C2 crystal form and in the previously determined amyloid TTR (ATTR) Leu55Pro crystal structure is close-to-identical. This packing arrangement is therefore not unique in amyloidogenic mutants of TTR.     

Why is Leu55-->Pro55 transthyretin variant the most amyloidogenic: insights from molecular dynamics simulations of transthyretin monomers

Transthyretin (TTR) is one of the known human amyloidogenic proteins. Its native state is a homotetramer with each monomer having a beta-sandwich structure. Strong experimental evidence suggests that TTR dissociates into monomeric intermediates and that the monomers subsequently self-assemble to form amyloid deposits and insoluble fibrils. However, details on the early steps along the pathway of TTR amyloid formation are unclear, although various experimental approaches with resolutions at the molecular or residue level have provided some clues. It is highly likely that the stability and flexibility of monomeric TTR play crucial roles in the early steps of amyloid formation; thereby, it is essential to characterize initial conformational changes of TTR monomers. In this article we probe the possibility that the differences in the monomeric forms of wild-type (WT) TTR and its variants are responsible for differential amyloidogenesis. We begin with the simulations of WT, Val30-->Met (V30M), and Leu55-->Pro (L55P) TTR monomers. Nanosecond time scale molecular dynamics simulations at 300 K were performed using AMBER. The results indicate that the L55P-TTR monomer undergoes substantial structural changes relative to fluctuations observed in the WT and V30M TTR monomers. The observation supports earlier speculation that the L55P mutation may lead to disruption of the beta-sheet structure through the disorder of the "edge strands" that might facilitate amyloidogenesis.     

Native state hydrogen exchange study of suppressor and pathogenic variants of transthyretin

Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for numerous familial amyloid diseases, the exact phenotype being dependent on the sequence deposited. Many familial disease variants display decreased stability in vitro, and early onset pathology in vivo. Only subtle structural differences were observed upon crystallographic comparison of the disease-associated variants to the T119M interallelic trans-suppressor. Herein three human TTR single amino acid variant homotetramers including two familial amyloidotic polyneuropathy (FAP) causing variants (V30M and L55P), and a suppressor variant T119M (known to protect V30M carriers from disease by trans-suppression) were investigated in a residue-specific fashion by monitoring (2)H-(1)H exchange employing NMR spectroscopy. The measured protection factors for slowly exchanging amide hydrogen atoms reveal destabilization of the protein core in the FAP variants, the core consisting of strands A, B, E and G and the loop between strands A and B. The same core exhibits much slower exchange in the suppressor variant. Accelerated exchange rates were observed for residues at the subunit interfaces in L55P, but not in the T119M or V30M TTR. The correlation between destabilization of the TTR core strands and the tendency for amyloid formation supports the view that these strands are involved in amyloidogenicity, consistent with previous (2)H-(1)H exchange analysis of the WT-TTR amyloidogenic intermediate.     

Transthyretin amyloidosis: a tale of weak interactions.

Over 70 transthyretin (TTR) mutations have been associated with hereditary amyloidoses, which are all autosomal dominant disorders with adult age of onset. TTR is the main constituent of amyloid that deposits preferentially in peripheral nerve giving rise to familial amyloid polyneuropathy (FAP), or in the heart leading to familial amyloid cardiomyopathy. Since the beginning of this decade the central question of these types of amyloidoses has been why TTR is an amyloidogenic protein with clinically heterogeneous pathogenic consequences. As a result of amino acid substitutions, conformational changes occur in the molecule, leading to weaker subunit interactions of the tetrameric structure as revealed by X-ray studies of some amyloidogenic mutants. Modified soluble tetramers exposing cryptic epitopes seem to circulate in FAP patients as evidenced by antibody probes recognizing specifically TTR amyloid fibrils, but what triggers dissociation into monomeric and oligomeric intermediates of amyloid fibrils is largely unknown. Avoiding tetramer dissociation and disrupting amyloid fibrils are possible avenues of therapeutic intervention based on current molecular knowledge of TTR amyloidogenesis and fibril structure.     

The sequence-dependent unfolding pathway plays a critical role in the amyloidogenicity of transthyretin

Human transthyretin (TTR) is an amyloidogenic protein whose aggregation is associated with several types of amyloid diseases. The following mechanism of TTR amyloid formation has been proposed. TTR tetramer at first dissociates into native monomers, which is the rate-limiting step in fibril formation. The monomeric species then partially unfold to form amyloidogenic intermediates that subsequently undergo a downhill self-assembly process. The amyloid deposit can be facilitated by disease-associated point mutations. However, only subtle structural differences were observed between the crystal structures of the wild type and the disease-associated variants. To investigate how single-point mutations influence the effective energy landscapes of TTR monomers, molecular dynamics (MD) simulations were performed on wild-type TTR and two pathogenic variants. Principal coordinate analysis on MD-generated ensembles has revealed multiple unfolding pathways for each protein. Amyloidogenic intermediates with the dislocated C strand-loop-D strand motif were observed only on the unfolding pathways of V30M and L55P variants and not for wild-type TTR. Our study suggests that the sequence-dependent unfolding pathway plays a crucial role in the amyloidogenicity of TTR. Analyses of side chain concerted motions indicate that pathogenic mutations on "edge strands" disrupt the delicate side chain correlated motions, which in turn may alter the sequence of unfolding events.     

Identification of a subunit interface in transthyretin amyloid fibrils: evidence for self-assembly from oligomeric building blocks

Amyloid and prion diseases appear to stem from the conversion of normally folded proteins into insoluble, fiber-like assemblies. Despite numerous structural studies, a detailed molecular characterization of amyloid fibrils remains elusive. In particular, models of amyloid fibrils proposed thus far have not adequately defined the constituent protein subunit interactions. To further our understanding of amyloid structure, we employed thiol-specific cross-linking and site-directed spin labeling to identify specific protein-protein associations in transthyretin (TTR) amyloid fibrils. We find that certain cysteine mutants of TTR, when dimerized by chemical cross-linkers, still form fibers under typical in vitro fibrillogenic conditions. In addition, site-directed spin labeling of many residues at the natural dimer interface reveals that their spatial proximity is preserved in the fibrillar state even in the absence of cross-linking constraints. Here, we present the first view of a subunit interface in TTR fibers and show that it is very similar to one of the natural dimeric interchain associations evident in the structure of soluble TTR. The results clarify varied models of amyloidogenesis by demonstrating that transthyretin amyloid fibrils may assemble from oligomeric protein building blocks rather than structurally rearranged monomers.     

Intrinsic versus mutation dependent instability/flexibility: a comparative analysis of the structure and dynamics of wild-type transthyretin and its pathogenic variants

Transthyretin (TTR) is one of the about 20 known human proteins associated with amyloidosis which is characterized by the accumulation of amyloid fibrils in tissues or extracellular matrix surrounding vital organs. Unlike Alzheimer's fibrils that comprise a fragment of a large precursor protein, TTR amyloid fibrils are composed of both full-length protein and fragments of the molecule. The native state of TTR is a homotetramer with eight beta-strands organized into a beta-sandwich in each monomer. To elucidate the structural reorganization mechanisms preceding amyloid formation, it is important to characterize the dynamic features of the wild-type native state as well as to reveal the influence of disease-associated mutations on the structure and dynamics. Molecular dynamics (MD) simulations complement X-ray crystallography and D-H exchange to capture the intrinsically unstable/flexible sites of the wild-type as well as the mutation dependent unstable sites of the pathogenic variants. Our results of MD simulations have shown that the Leu55-->Pro (L55P) mutation occurs in an intrinsically unstable site, leading to substantial local and global structural changes. This observation supports the early speculation that the C-strand-loop-D-strand rearrangement leads to the formation of amyloidogenic intermediates. In addition to the D strand, the alpha-helical region and the strands at the monomer-monomer interface are also intrinsically unstable. The central channel of L55P-TTR undergoes opening and closing fluctuations, which may provide an explanation for the fact that while the mutation is far from the channel, the mutant shows a substantial low binding affinity of thyroxine.     

Review: TTR Amyloidosis—Structural Features Leading to Protein Aggregation and Their Implications on Therapeutic Strategies

Transthyretin amyloidosis represents a spectrum of clinical syndromes that, in all cases except senile systemic amyloidosis, are dependent on the mutation present in the transthyretin (TTR) protein. Although the role of amyloid deposits in the pathogenesis of the disease is not clear, preventing their formation or promoting their disaggregation is necessary to control the development of clinical symptoms. The design of therapies aiming at preventing amyloid formation or promoting its dissociation requires detailed knowledge of the fibrils' molecular structure and a complete view about the factors responsible for protein aggregation. This review is focused on the structural studies, performed on amyloid fibrils and amyloidogenic TTR variants, aiming at understanding the aggregation mechanism as well as the atomic structure of the fibril assembly. Based on the available information possible therapies are also surveyed.     

Review: TTR amyloidosis-structural features leading to protein aggregation and their implications on therapeutic strategies

Transthyretin amyloidosis represents a spectrum of clinical syndromes that, in all cases except senile systemic amyloidosis, are dependent on the mutation present in the transthyretin (TTR) protein. Although the role of amyloid deposits in the pathogenesis of the disease is not clear, preventing their formation or promoting their disaggregation is necessary to control the development of clinical symptoms. The design of therapies aiming at preventing amyloid formation or promoting its dissociation requires detailed knowledge of the fibrils' molecular structure and a complete view about the factors responsible for protein aggregation. This review is focused on the structural studies, performed on amyloid fibrils and amyloidogenic TTR variants, aiming at understanding the aggregation mechanism as well as the atomic structure of the fibril assembly. Based on the available information possible therapies are also surveyed.     

The most pathogenic transthyretin variant, L55P, forms amyloid fibrils under acidic conditions and protofilaments under physiological conditions.

The L55P transthyretin (TTR) familial amyloid polyneuropathy-associated variant is distinct from the other TTR variants studied to date and the wild-type protein in that the L55P tetramer can dissociate to the monomeric amyloidogenic intermediate and form fibril precursors under physiological conditions (pH 7.0, 37 degrees C). The activation barrier associated with L55P-TTR tetramer dissociation is lower than the barrier for wild-type transthyretin dissociation, which does not form fibrils under physiological conditions. The L55P-TTR tetramer is also very sensitive to acidic conditions, readily dissociating to form the monomeric amyloidogenic intermediate between pH 5.5-5.0 where the wild-type TTR adopts a nonamyloidogenic tetrameric structure. The formation of the L55P monomeric amyloidogenic intermediate involves subtle tertiary structural changes within the beta-sheet rich subunit as discerned from Trp fluorescence, circular dichroism analysis, and ANS binding studies. The assembly of the L55P-TTR amyloidogenic intermediate at physiological pH (pH 7.5) affords protofilaments that elongate with time. TEM studies suggest that the entropic barrier associated with filament assembly (amyloid fibril formation) is high in vitro, amyloid being defined by the laterally assembled four filament structure observed by Blake upon isolation of "fibrils" from the eye of a FAP patient. The L55P-TTR protofilaments formed in vitro bind Congo red and thioflavin T (albeit more weakly than the fibrils produced at acidic pH), suggesting that the structure observed probably represents an amyloid precursor. The structural continuum from misfolded monomer through protofilaments, filaments, and ultimately fibrils must be considered as a possible source of pathology associated with these diseases.     

X-ray absorption spectroscopy reveals a substantial increase of sulfur oxidation in transthyretin (TTR) upon fibrillization

Transthyretin (TTR) amyloid fibrils are the main component of the amyloid deposits occurring in Familial Amyloidotic Polyneuropathy patients. This is 1 of 20 human proteins leading to protein aggregation disorders such as Alzheimer's and Creutzfeldt-Jakob diseases. The structural details concerning the association of the protein molecules are essential for a better understanding of the disease and consequently the design of new strategies for diagnosis and therapeutics. Disulfide bonds are frequently considered essential for the stability of protein aggregates and since in the TTR monomers there is one cysteine residue, it is important to determine unambiguously the redox state of sulfur present in the fibrils. In this work we used x-ray spectroscopy to further characterize TTR amyloid fibrils. The sulfur K-edge absorption spectra for the wild type and some amyloidogenic TTR variants in the soluble and fibrillar forms were analyzed. Whereas in the soluble proteins the thiol group from cysteine (R-SH) and the thioether group from methionine (R-S-CH(3)) are the most abundant forms, in the TTR fibrils there is a significant oxidation of sulfur to the sulfonate form in the cysteine residue and a partial oxidation of sulfur to sulfoxide in the methionine residues. Further interpretation of the data reveals that there are no disulfide bridges in the fibrillar samples and suggest conformational changes in the TTR molecule, namely in strand A and/or in its vicinity, upon fibril formation.     

Disease-associated mutations impacting BC-loop flexibility trigger long-range transthyretin tetramer destabilization and aggregation

Hereditary transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by the extracellular deposition of the transport protein transthyretin (TTR) as amyloid fibrils. Despite the progress achieved in recent years, understanding why different TTR residue substitutions lead to different clinical manifestations remains elusive. Here, we studied the molecular basis of disease-causing missense mutations affecting residues R34 and K35. R34G and K35T variants cause vitreous amyloidosis, whereas R34T and K35N mutations result in amyloid polyneuropathy and restrictive cardiomyopathy. All variants are more sensitive to pH-induced dissociation and amyloid formation than the wild-type (WT)-TTR counterpart, specifically in the variants deposited in the eyes amyloid formation occurs close to physiological pHs. Chemical denaturation experiments indicate that all the mutants are less stable than WT-TTR, with the vitreous amyloidosis variants, R34G and K35T, being highly destabilized. Sequence-induced stabilization of the dimer–dimer interface with T119M rendered tetramers containing R34G or K35T mutations resistant to pH-induced aggregation. Because R34 and K35 are among the residues more distant to the TTR interface, their impact in this region is therefore theorized to occur at long range. The crystal structures of double mutants, R34G/T119M and K35T/T119M, together with molecular dynamics simulations indicate that their strong destabilizing effect is initiated locally at the BC loop, increasing its flexibility in a mutation-dependent manner. Overall, the present findings help us to understand the sequence-dynamic-structural mechanistic details of TTR amyloid aggregation triggered by R34 and K35 variants and to link the degree of mutation-induced conformational flexibility to protein aggregation propensity.     

Surface exposed epitopes and structural heterogeneity of in vivo formed transthyretin amyloid fibrils

We have investigated the structure of in vivo formed transthyretin (TTR) amyloid deposits by using antisera raised against short linear sequences of the TTR molecule. In immunohistochemistry, antisera anti-TTR41-50 and anti-TTR115-124-a reacted specifically with both wildtype ATTR and ATTR V30M material, whereas only anti-TTR41-50 recognized ATTR Y114C material. Similar results were obtained by ELISA analysis of ATTR V30M and ATTR Y114C vitreous amyloid, where the anti-TTR115-124-a antiserum failed to react with ATTR Y114C material. Moreover, neither of the antisera recognized natively structured TTR present in pancreatic alpha cells. Our results strongly indicate that the TTR molecule undergoes structural changes during fibrillogenesis in vivo. The finding of a structural difference between wildtype ATTR and ATTR V30M material on one hand and ATTR Y114C material on the other suggests that the fibril formation pathway of these ATTR variants may differ in vivo.     

Clusterin regulates transthyretin amyloidosis

Transthyretin (TTR) is a human disease-associated amyloidogenic protein that has been implicated in senile systemic amyloidosis (SSA) and familial amyloidotic polyneuropathy (FAP). FAP typically results in severe and early-onset disease, and the only therapy established so far is liver transplantation; thus, developing new strategies for treating FAP is of paramount interest. Clusterin has recently been proposed to play a role as an extracellular molecular chaperone, affecting the fibril formation of amyloidogenic proteins. The ability of clusterin to influence amyloid fibril formation prompted us to investigate whether clusterin is capable of inhibiting TTR amyloidosis. Here, we report that clusterin strongly interacts with wild-type TTR and TTR variants V30M and L55P under acidic conditions, and blocks the amyloid fibril formation of TTR variants. In particular, the amyloid fibril formation of V30M TTR in the presence of clusterin is reduced to level similar to wild-type TTR. We also demonstrated that clusterin is an effective inhibitor of L55P TTR amyloidosis, the most aggressive form of TTR diseases. The mechanism by which clusterin inhibits TTR amyloidosis appears to be through stabilization of TTR tetrameric structure. These findings suggest the possibility of using clusterin as a therapeutic agent for TTR amyloidosis.     

Initial conformational changes of human transthyretin under partially denaturing conditions

Human transthyretin (TTR) is an amyloidogenic protein. The pathway of TTR amyloid formation has been proposed based on lines of evidence: TTR tetramer first dissociates into native monomers, which is shown to be a rate-limiting step in the formation of fibrils. Subsequently, the monomeric species partially unfold to form the aggregation intermediates. Once such intermediates are formed, the following self-assembly process is a downhill polymerization. Hence, tertiary structural changes within the monomers after the dissociation are essential for the amyloid formation. These tertiary structural changes can be facilitated by partial denaturation. To probe the conformational changes under the partially denaturing conditions, five independent trajectories were collected for the wild-type (WT) and its pathogenic variants at 300 and 350 K, resulting in simulations that totaled 59 ns. Under these conditions, L55P variant is more labile than the wild-type and V30M variant. We have observed that the D strand of WT-TTR is trapped in two local minima: the native conformation and the amyloidogenic fold that resembles the surface loop of residues 54-55 of L55P variant. In the tetrameric state, the F strand is bent with large separations at the F-F' interface. This strand becomes flatter in the monomeric state, which may facilitate the formation of new F-F' interface with possible prolonged hydrogen bonds and/or shift in beta-strand register in the fibril state. During the unfolding process, the anticorrelated motion between the strands H and G as well as the strands H and A pulls the H strand out of the inner sheet plane, leading to a more twisted inner sheet. Our simulation has provided important detailed structural information about the partially unfolded state of TTR that may be related to the amyloidogenic intermediates.     

The crystal structure of transthyretin in complex with diethylstilbestrol: a promising template for the design of amyloid inhibitors

Transthyretin (TTR) is a homotetrameric plasma protein that, in conditions not yet completely understood, may aggregate, forming the fibrillar material associated with TTR amyloidosis. A number of reported experiments indicate that dissociation of the TTR tetramer occurs prior to fibril formation, and therefore, studies aiming at the discovery of compounds that stabilize the protein quaternary structure, thereby acting as amyloid inhibitors, are being performed. The ability of diethylstilbestrol (DES) to act as a competitive inhibitor for the thyroid hormone binding to TTR indicated a possible stabilizing effect of DES upon binding. Here we report the crystallographic study of DES binding to TTR. The structural data reveal two different binding modes, both located in the thyroxine binding channel. In both cases, DES binds deeply in the channel and establishes interactions with the equivalent molecule present in the adjacent binding site. The most remarkable features of DES interaction with TTR are its hydrophobic interactions within the protein halogen binding pockets, where its ethyl groups are snugly fitted, and the hydrogen bonds established at the center of the tetramer with Ser-117. Experiments concerning amyloid formation in vitro suggest that DES is effectively an amyloid inhibitor in acid-mediated fibrillogenesis and may be used for the design of more powerful drugs. The present study gave us further insight in the molecular mechanism by which DES competes with thyroid hormone binding to TTR and highlights key interactions between DES and TTR that oppose amyloid formation.     

Mass spectrometric immunoassay for quantitative determination of transthyretin and its variants

Transthyretin (TTR, or prealbumin) is a tetrameric protein found in plasma and cerebrospinal fluid. Its major role is to transport thyroid hormones (thyroxin-T4) and retinol (through association with retinol-binding protein). TTR has been studied extensively due to the great number of point mutations that result in sequence heterogeneity. Many of these variants are associated with pathological conditions that result in extracellular deposition of amyloid fibers in tissues. In this work, we have developed a rapid mass spectrometric immunoassay for determination and quantification of TTR and its variants from human serum and plasma samples. The assay was fully characterized in terms of its precision, linearity and recovery characteristics. The new assay was also compared with a conventional TTR ELISA. Furthermore, we have applied the optimized method to analyze TTR and its modifications in 44 human plasma samples, and in the process optimized a method for TTR proteolytic digestion and identification of point mutations.     

Iodine Atoms: A New Molecular Feature for the Design of Potent Transthyretin Fibrillogenesis Inhibitors

The thyroid hormone and retinol transporter protein known as transthyretin (TTR) is in the origin of one of the 20 or so known amyloid diseases. TTR self assembles as a homotetramer leaving a central hydrophobic channel with two symmetrical binding sites. The aggregation pathway of TTR into amiloid fibrils is not yet well characterized but in vitro binding of thyroid hormones and other small organic molecules to TTR binding channel results in tetramer stabilization which prevents amyloid formation in an extent which is proportional to the binding constant. Up to now, TTR aggregation inhibitors have been designed looking at various structural features of this binding channel others than its ability to host iodine atoms. In the present work, greatly improved inhibitors have been designed and tested by taking into account that thyroid hormones are unique in human biochemistry owing to the presence of multiple iodine atoms in their molecules which are probed to interact with specific halogen binding domains sitting at the TTR binding channel. The new TTR fibrillogenesis inhibitors are based on the diflunisal core structure because diflunisal is a registered salicylate drug with NSAID activity now undergoing clinical trials for TTR amyloid diseases. Biochemical and biophysical evidence confirms that iodine atoms can be an important design feature in the search for candidate drugs for TTR related amyloidosis.