TTR Tetramer Research Articles

Transthyretin Cardiac Amyloidosis: Insights into Wild-Type Variants and Familial Amyloid Cardiomyopathy

Transthyretin (TTR) cardiac amyloidosis is a progressive condition characterized by amyloid fibril deposition in the heart, leading to heart failure. This review focuses on insights into wild-type (ATTRwt) and familial (ATTRm) variants of TTR amyloidosis. The pathophysiology involves TTR tetramer destabilization, monomer misfolding, and amyloid fibril formation. Therapeutic advances, including TTR stabilizers like tafamidis and Diflunisal, and gene-silencing agents such as patisiran and inotersen, show promise in managing the disease. Despite these advancements, early diagnosis remains challenging, and side effects of current therapies necessitate the development of safer, more effective treatments. Future research should focus on novel therapies, improved diagnostic methods, and personalized treatment strategies to enhance patient outcomes. This review underscores the importance of continued innovation and collaboration in tackling TTR cardiac amyloidosis.

Clinical and biochemical characterization of asymptomatic carriers and symptomatic patients with hereditary transthyretin amyloidosis caused by TTR V30L mutation.

Hereditary transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by amyloid fibril deposition. The TTR c.148G > T mutation (V30L) in ATTR is rarely reported, and its biochemical properties are unknown. Seven patients and two asymptomatic carriers from two unrelated families diagnosed with V30L variant of ATTR were included. Data on clinical manifestations, laboratory examination, electrophysiology, ophthalmological corneal confocal microscopy (CCM), pathology and molecular biological experiments was collected and analyzed. Most patients initially experienced paresthesia, with varying degrees of peripheral neuropathy, autonomic dysfunction, and cardiac involvement. Nerve conduction studies showed extensive motor and sensory nerve involvement in upper and lower limbs. CCM revealed reduced corneal nerve density and fiber length. Sural nerve biopsies indicated loss of myelinated nerve fibers, with neurogenic patterns in gastrocnemius muscle biopsies. Asymptomatic carriers had nearly normal electrophysiology but mild reductions in corneal nerve fiber density and length. Sural nerve biopsies in carriers showed mild reductions in small myelinated nerve fibers. V30L mutation impaired thermodynamic and kinetic stability of the mutant protein. Plasma TTR tetramer concentration was lower in ATTR V30L patients compared to healthy donors. Small molecule stabilizers failed to exhibit satisfactory inhibition on fibril formation of V30L mutation in vitro. This study highlights the multisystem involvement in ATTR V30L patients, including neuropathy and cardiac issues. Both patients and carriers showed abnormalities in nerve conduction, corneal microscopy, and pathology. The V30L mutation impaired protein stability and reduced plasma TTR tetramer levels. Small molecule stabilizers were ineffective, indicating a need for alternative treatments.

Arginine: a potential prophylactic supplement for transthyretin amyloidosis

Transthyretin (TTR) is an amyloidogenic protein associated with TTR amyloidosis (ATTR). Dissociation of TTR tetramers into TTR monomers causes TTR misfolding, resulting in amyloid fibril formation and triggering the onset of ATTR. Low-molecular-weight tetrameric TTR stabilizers are potential therapeutic agents to delay ATTR progression. However, the currently available drugs are expensive and cannot be used for prophylaxis. Therefore, in this study, we aimed to identify a prophylactic supplement that suppresses TTR amyloid formation. We investigated whether arginine, an amyloidogenic protein aggregation inhibitor, stabilizes tetrameric TTR, thereby preventing amyloid fibril formation. Immunoblotting showed that arginine mixed with wild-type TTR (TTRwt), ATTR V30M (Val30Met), and human serum samples reduced the amount of monomeric TTR but increased the tetramer/monomer ratio of TTR compared to those in the samples without arginine. Additionally, oral administration of arginine (5000 mg for five days) to healthy volunteers effectively increased the tetramer/monomer ratio of TTR in the serum. Thioflavin T test, a quantitative analysis method for amyloid fibril formation, showed that amyloid fibril formation was significantly suppressed with arginine compared to that without arginine. As arginine is a common supplement and non-toxic amino acid, it can be used as a promising prophylactic supplement to suppress amyloid fibril formation in ATTR.

Transthyretin Amyloidosis: Role of oxidative stress and the beneficial implications of antioxidants and nutraceutical supplementation

Transthyretin (ATTR) amyloidosis constitutes a spectrum of debilitating neurodegenerative diseases instigated by systemic extracellular deposition of partially unfolded/aggregated aberrant transthyretin. The homotetrameric protein, TTR, is abundant in the plasma, and to a lesser extent the cerebrospinal fluid. Rate-limiting tetramer dissociation of the native protein is regarded as the critical step in the formation of morphologically heterogenous toxic aggregates and the onset of clinical manifestations such as polyneuropathy, cardiomyopathy, disturbances in motor and autonomic functions. Over the past few decades there has been increasing evidence suggesting that in addition to destabilization in TTR tetramer structure, oxidative stress may also play an important role in the pathogenesis of ATTR amyloidosis. In this review, an update on the impact of oxidative stress in TTR amyloidogenesis as well as TTR aggregate-mediated pathologies is discussed. The counteracting effects of antioxidants and nutraceutical agents explored in the treatment of ATTR amyloidosis based on recent evidence is also critically examined. The insights unveiled could further strengthen current understanding of the mechanisms underlying ATTR amyloidosis as well as extend the range of strategies for effective management of ATTR amyloidoses.

Clinical and molecular insights into A97S variants in hereditary transthyretin amyloid polyneuropathy in South China

Objective This study aims to delineate the clinical profiles of the hereditary transthyretin amyloid polyneuropathy (ATTRv-PN) patients with A97S variant from southern China and the molecular characteristics of this mutant protein. Methods Fifteen ATTRv-PN patients with heterozygous A97S and one patient with homozygous A97S were included in the study. Serum TTR tetramer concentration was quantified through ultra-performance liquid chromatography. Stabilities of A97S-TTR were assessed through in vitro urea-mediated tryptophan fluorescence experiments, and nephelometry was employed in drug response assessment. Results All patients were late-onset (≥50 years) with a mean age of onset at 59.26 ± 5.06 years old. Patients displayed a mixed phenotype featuring sensory-motor neuropathy with autonomic dysfunction and cardiac involvement, such as palpitations and chest pain. Electrophysiological studies showed generally axonal impairment of sensory and motor nerves. Tafamidis-treated patients showed significantly higher TTR tetramer concentrations, approaching healthy controls’ levels. In vitro assessment showed that A97S-TTR was more kinetically stable than the V122I-TTR, and tetramer stabilisers inhibited A97S-TTR amyloid formation by more than 70%. Conclusion This study provides valuable insights into the clinical and molecular characteristics of ATTRv-PN patients with A97S from South China, particularly regarding the differences in disease progression and stability features.

Water Plays Key Roles in Stabilities of Wild Type and Mutant Transthyretin Complexes.

Transthyretin (TTR), a 56 kDa homotetramer that is involved in the transport of thyroxine and retinol, has been linked to amyloidosis through disassembly of tetramers to form monomers, dimers, and trimers that then reassemble into higher order oligomers and/or fibrils. Hybrid TTR (hTTR) tetramers are found in heterozygous individuals that express both wild type TTR (wt-TTR) and mutant TTR (mTTR) forms of the protein, and these states display increased rates of amyloidosis. Here we monitor subunit exchange (SUE) reactions involving homomeric and mixed tetramers using high resolution native mass spectrometry (nMS). Our results show evidence that differences in TTR primary structure alter tetramer stabilities, and hTTR products can form spontaneously by SUE reactions. In addition, we find that solution temperature has strong effects on TTR tetramer stabilities and formation of SUE products. Lower temperatures promote formation of hTTR tetramers containing L55P and V30M subunits, whereas small effects on the formation of hTTR tetramers containing F87A and T119M subunits are observed. We hypothesize that the observed temperature dependent stabilities and subsequent SUE behavior are a result of perturbations to the network of "two kinds of water": hydrating and structure stabilizing water molecules (Spyrakis et al. J. Med. Chem. 2017, 60 (16), 6781-6827; Xu et al. Soft Matter 2012, 8, 324-336) that stabilize wt-TTR and mTTR tetramers. The results presented in this work illustrate the utility of high resolution nMS for studies of the structures, stabilities, and dynamics of protein complexes that directly influence SUE reactions.

A Comprehensive Review on Chemistry and Biology of Tafamidis in Transthyretin Amyloidosis.

Transthyretin amyloid cardiomyopathy and Transthyretin amyloid peripheral neuropathy are progressive disease conditions caused by Transthyretin amyloidosis (ATTR) fibril infiltration in the tissue. Transthyretin (TTR) protein misfolding and amyloid fibril deposits are pathological biomarkers of ATTR-related disorders. There are various treatment strategies targeting different stages in pathophysiology. One such strategy is TTR tetramer stabilization. Recently, a new TTR tetramer stabilizer, tafamidis, has been introduced that reduces the protein misfolding and amyloidosis and, consequently, disease progression in ATTR cardiomyopathy and peripheral neuropathy. This review will provide a comprehensive overview of the literature on tafamidis discovery, development, synthetic methods, pharmacokinetics, analytical methods and clinical trials. Overall, 7 synthetic methods, 5 analytical methods and 23 clinical trials have been summarized from the literature.

Efficacy and Safety of Acoramidis in Transthyretin Amyloid Cardiomyopathy

BackgroundTransthyretin amyloid cardiomyopathy is characterized by the deposition of misfolded monomeric transthyretin (TTR) in the heart. Acoramidis is a high-affinity TTR stabilizer that acts to inhibit dissociation of tetrameric TTR and leads to more than 90% stabilization across the dosing interval as measured ex vivo.MethodsIn this phase 3, double-blind trial, we randomly assigned patients with transthyretin amyloid cardiomyopathy in a 2:1 ratio to receive acoramidis hydrochloride at a dose of 800 mg twice daily or matching placebo for 30 months. Efficacy was assessed in the patients who had an estimated glomerular filtration rate of at least 30 ml per minute per 1.73 m2 of body-surface area. The four-step primary hierarchical analysis included death from any cause, cardiovascular-related hospitalization, the change from baseline in the N-terminal pro–B-type natriuretic peptide (NT-proBNP) level, and the change from baseline in the 6-minute walk distance. We used the Finkelstein–Schoenfeld method to compare all potential pairs of patients within strata to generate a P value. Key secondary outcomes were death from any cause, the 6-minute walk distance, the score on the Kansas City Cardiomyopathy Questionnaire–Overall Summary, and the serum TTR level.ResultsA total of 632 patients underwent randomization. The primary analysis favored acoramidis over placebo (P<0.001); the corresponding win ratio was 1.8 (95% confidence interval [CI], 1.4 to 2.2), with 63.7% of pairwise comparisons favoring acoramidis and 35.9% favoring placebo. Together, death from any cause and cardiovascular-related hospitalization contributed more than half the wins and losses to the win ratio (58% of all pairwise comparisons); NT-proBNP pairwise comparisons yielded the highest ratio of wins to losses (23.3% vs. 7.0%). The overall incidence of adverse events was similar in the acoramidis group and the placebo group (98.1% and 97.6%, respectively); serious adverse events were reported in 54.6% and 64.9% of the patients.ConclusionsIn patients with transthyretin amyloid cardiomyopathy, the receipt of acoramidis resulted in a significantly better four-step primary hierarchical outcome containing components of mortality, morbidity, and function than placebo. Adverse events were similar in the two groups. (Funded by BridgeBio Pharma; ATTRibute-CM ClinicalTrials.gov number, NCT03860935.)

3-O-Methyltolcapone and Its Lipophilic Analogues Are Potent Inhibitors of Transthyretin Amyloidogenesis with High Permeability and Low Toxicity.

Transthyretin (TTR) is an amyloidogenic homotetramer involved in the transport of thyroxine in blood and cerebrospinal fluid. To date, more than 130 TTR point mutations are known to destabilise the TTR tetramer, leading to its extracellular pathological aggregation accumulating in several organs, such as heart, peripheral and autonomic nerves, and leptomeninges. Tolcapone is an FDA-approved drug for Parkinson's disease that has been repurposed as a TTR stabiliser. We characterised 3-O-methyltolcapone and two newly synthesized lipophilic analogues, which are expected to be protected from the metabolic glucuronidation that is responsible for the lability of tolcapone in the organism. Immunoblotting assays indicated the high degree of TTR stabilisation, coupled with binding selectivity towards TTR in diluted plasma of 3-O-methyltolcapone and its lipophilic analogues. Furthermore, in vitro toxicity data showed their several-fold improved neuronal and hepatic safety compared to tolcapone. Calorimetric and structural data showed that both T4 binding sites of TTR are occupied by 3-O-methyltolcapone and its lipophilic analogs, consistent with an effective TTR tetramer stabilisation. Moreover, in vitro permeability studies showed that the three compounds can effectively cross the blood-brain barrier, which is a prerequisite for the inhibition of TTR amyloidogenesis in the cerebrospinal fluid. Our data demonstrate the relevance of 3-O-methyltolcapone and its lipophilic analogs as potent inhibitors of TTR amyloidogenesis.

Disease-Modifying Drugs Extend Survival in Hereditary Transthyretin Amyloid Polyneuropathy.

Hereditary transthyretin (ATTRv) amyloidosis is a rare, fatal systemic disease, associated with polyneuropathy and cardiomyopathy, that is caused by mutant transthyretin (TTR). In addition to liver transplantation, several groundbreaking disease-modifying drugs (DMDs) such as tetrameric TTR stabilizers and TTR gene-silencing therapies have been developed for ATTRv amyloid polyneuropathy. They were based on a working hypothesis of the mechanisms of ATTRv amyloid formation. In this retrospective cohort study, we investigated survival of all 201 consecutive patients with ATTRv amyloidosis in our center. The effects of DMDs on survival improvements were significant not only in early-onset patients but also in late-onset patients. ANN NEUROL 2024;95:230-236.

An ultra performance liquid chromatography method for transthyretin variants screening and heart failure assisting diagnosis

BackgroundTransthyretin (TTR) gene mutations are associated with hereditary amyloidosis (ATTR) caused by mutant TTR protein dissociation, misfolding, aggregation, and insoluble fibrils deposition. Herein, we reported a chromatographic approach for quantification and identification of TTR tetramer in human blood serum by ultra performance liquid chromatography (UPLC). MethodsTTR proteins and serum were incubated with a fluorescent TTR tetramer sensor (A2). The A2 sensor specifically reacted with tetrameric TTR and released stoichiometric fluorescence that was detected by fluorescence detector coupled to UPLC. The external standard was used for quantification, the chromatographic peak parameters were used to identification certain mutation types. ResultsUPLC correctly distinguished 18 types of mutant TTR proteins from wild type. The results were consistent with follow-up analysis of two ATTR patients’ blood serum samples. In addition, the tetrameric TTR of 30 heart failure (HF) patients showed strongly correlation (r = −0.63, p < 0.00) with NT-proBNP, a HF clinical biomarker. ConclusionsUPLC method has sufficient accuracy to eliminate the necessity of sequencing for certain types of TTR mutations and allows for facile initial screening of ATTR amyloidosis patients, carriers, and healthy individuals for time-saving and economical purposes. TTR tetramer may serve as a diagnostic biomarker to evaluate the risk of HF diseases.

Nanoscale structural characterization of transthyretin aggregates formed at different time points of protein aggregation using atomic force microscopy-infrared spectroscopy.

Transthyretin (TTR) amyloidosis is a progressive disease characterized by an abrupt aggregation of misfolded protein in multiple organs and tissues TTR is a tetrameric protein expressed in the liver and choroid plexus. Protein misfolding triggers monomerization of TTR tetramers. Next, monomers assemble forming oligomers and fibrils. Although the secondary structure of TTR fibrils is well understood, there is very little if anything is known about the structural organization of TTR oligomers. To end this, we used nano-infrared spectroscopy, also known as atomic force microscopy infrared (AFM-IR) spectroscopy. This emerging technique can be used to determine the secondary structure of individual amyloid oligomers and fibrils. Using AFM-IR, we examined the secondary structure of TTR oligomers formed at the early (3-6 h), middle (9-12 h), and late (28 h) of protein aggregation. We found that aggregating, TTR formed oligomers (Type 1) that were dominated by α-helix (40%) and β-sheet (~30%) together with unordered protein (30%). Our results showed that fibril formation was triggered by another type of TTR oligomers (Type 2) that appeared at 9 h. These new oligomers were primarily composed of parallel β-sheet (55%), with a small amount of antiparallel β-sheet, α-helix, and unordered protein. We also found that Type 1 oligomers were not toxic to cells, whereas TTR fibrils formed at the late stages of protein aggregation were highly cytotoxic. These results show the complexity of protein aggregation and highlight the drastic difference in the protein oligomers that can be formed during such processes.

Acoramidis produces near-complete TTR stabilization in blood samples from patients with variant transthyretin amyloidosis that is greater than that achieved with tafamidis

Abstract Introduction Transthyretin (TTR) amyloidosis (ATTR) is a progressive, fatal disease caused by destabilizing TTR variants (TTRv) and age-related factors. Dissociation of tetrameric TTR initiates protein misfolding, aggregation, and tissue deposition which constitutes the mechanism of disease. More destabilizing variants drive more severe clinical phenotypes. TTR stabilizers have demonstrated clinical benefits for neuropathic and cardiovascular outcomes correlated with the extent of TTR stabilization. Acoramidis (AG10) is a novel TTR stabilizer in development for the treatment of TTR amyloid cardiomyopathy. Hypothesis: Acoramidis achieves near-complete in vitro TTR stabilization, exceeding levels achieved with clinically relevant concentrations of tafamidis (a TTR stabilizer in clinical use), when added to blood samples from ATTRv patients across a spectrum of destabilizing TTR variants. Methods Two established assays assessed TTR stabilization: fluorescent probe exclusion (FPE; measures binding site occupancy), and Western blot (WB; quantifies tetrameric TTR persistence under conditions of accelerated dissociation). Over 60 individual patient samples representing 18 unique TTRv across a spectrum of intrinsic instability and clinical phenotypes were assayed. Acoramidis was added at its target clinical trough concentration (10 µM) and compared to tafamidis added at its clinical peak (26 µM) and trough (16 µM) concentrations reported for its maximal approved dose. Results Acoramidis bound serum TTR to a greater extent (103 ± 13%) than either peak (87 ± 14%) or trough (71 ± 14%) concentrations of tafamidis. WB assays showed that addition of acoramidis resulted in significantly greater and more durable TTR stabilization (93 ± 14%) than adding tafamidis (peak: 49 ± 14%, trough: 36 ± 13%) in all paired individual patient plasma samples tested (Figure 1, p &amp;lt; 0.0001). Rare variants (A97S, D38A, F64L, L58H, P24S, Y114C) also demonstrated near complete stabilization upon in vitro addition of acoramidis. Conclusions At its target therapeutic trough concentration, acoramidis achieved near-complete TTR stabilization across 18 unique genotypes; in the subset of paired samples, the stabilization was significantly greater for acoramidis than for tafamidis even at its peak clinical concentration. This observation held across a range of destabilizing mutations, including a∼2-fold greater stabilization than tafamidis for the prevalent cardiomyopathic V122I variant. Based on the mechanism of disease and the association between TTR destabilization and severity of clinical outcomes, these data suggest that acoramidis has the potential to be a clinically differentiated and efficacious treatment option for patients with ATTRv, independent of variant genotype.

The effect of tafamidis treatment on cardiovascular aging in patients with Transthyretin cardiomyopathy. An observational study

Abstract Background Sporadic cardiac transthyretin amyloidosis (wtATTR) is a degenerative age-related disease with dismal prognosis and continuously increasing incidence. ATTR may have adverse effects also on vascular function and accelerate vascular aging. Tafamidis, a new agent that stabilizes the TTR tetramer improves wtATTR prognosis; however, whether tafamidis exerts any effect on vascular aging processes is unknown. Purpose To evaluate the effect of tafamidis treatment on markers of vascular aging. Methods This is an observational, non-interventional longitudinal study. wtATTR patients receiving tafamidis were evaluated prospectively at baseline, 3 months, 6 months and 1 year for non-invasive markers of vascular aging including arterial stiffness by pulse wave velocity (PWV, primary outcome measure), and markers derived from pulse wave analysis and compared to non-amyloidosis elderly controls (N=30, age&amp;gt;70 years) who were re-assessed for vascular function 1 year after their baseline visit. Results Twenty-one patients with wtATTR and were receiving tafamidis completed all 4 visits. Linear mixed model analysis revealed that PWV progressively decreased compared to baseline, during follow-up visits in patients receiving tafamidis [baseline vs a) 3 months: -0.95 mean difference (95%CI -1.73, -0.159) p-value=0.0121, b) 6 months: -1.36 mean difference (95%CI -2.19, -0.54) p-value=0.0002, c) 1 year: -1.08 mean difference (95%CI -2.03, -0.12) p-value=0.002]. The non-amyloidosis elderly control group presented a significant increase in PWV after 1 year compared to baseline (13.63 m/s vs 11.99 m/s, p =0.001 respectively, Wilcoxon rank test), while PWV was significantly lower in wtATTR group after 1 year compared to the control group despite younger mean age of the latter (p for interaction=0.0089). Central systolic blood pressure was decreased after 6 months of treatment, while it returned to baseline levels after 1 year [mixed model analysis, baseline vs 6 months -5.95 mean difference (95%CI -10.31, -1.60) p-value=0.007, baseline vs 1 year -0.47 mean difference (95%CI -5.42, 4.49) p-value=0.854]. A similar trend was observed for central diastolic blood pressure, although not achieving statistical significance (mixed model analysis, baseline vs 6 months -2.66 mean difference (95%CI -5.72, 0.40) p-value=0.088, baseline vs 1 year 0.005 mean difference (95%CI -3.47, 3.48) p-value=0.998]. Conclusion The results of this prospective study suggest that tafamidis may delay vascular aging in addition to cardiac aging in patients with wtATTR.

EGCG-Mediated Protection of Transthyretin Amyloidosis by Stabilizing Transthyretin Tetramers and Disrupting Transthyretin Aggregates.

Transthyretin amyloidosis (ATTR) is a progressive and systemic disease caused by the misfolding and amyloid aggregation of transthyretin (TTR). Stabilizing the TTR tetramers and disrupting the formed TTR aggregation are treated as a promising strategy for the treatment of ATTR. Previous studies have reported that epigallocatechin gallate (EGCG) can participate in the whole process of TTR aggregation to prevent ATTR. However, the interaction mechanism of EGCG in this process is still obscure. In this work, we performed molecular dynamics simulations to investigate the interactions between EGCG and TTR tetramers, and between EGCG and TTR aggregates formed by the V30M mutation. The obtained results suggest that EGCG at the binding site of the V30M TTR tetramer can form stable hydrogen bonds with residues in the flexible AB-loop and EF-helix-loop, which reduces the structural mobility of these regions significantly. Additionally, the polyaromatic property of EGCG contributes to the increasement of hydrophobicity at the binding site and thus makes the tetramer difficult to be solvated and dissociated. For V30M-TTR-generated aggregates, EGCG can promote the dissociation of boundary β-strands by destroying key residue interactions of TTR aggregates. Moreover, EGCG is capable of inserting into the side-chain of residues of neighboring β-strands and disrupting the highly structured aggregates. Taken together, this study elucidates the role of EGCG in preventing TTR amyloidosis, which can provide important theoretical support for the future of drug design for ATTR.

Binding of a Pyrene-Based Fluorescent Amyloid Ligand to Transthyretin: A Combined Crystallographic and Molecular Dynamics Study.

Misfolding and aggregation of transthyretin (TTR) cause several amyloid diseases. Besides being an amyloidogenic protein, TTR has an affinity for bicyclic small-molecule ligands in its thyroxine (T4) binding site. One class of TTR ligands are trans-stilbenes. The trans-stilbene scaffold is also widely applied for amyloid fibril-specific ligands used as fluorescence probes and as positron emission tomography tracers for amyloid detection and diagnosis of amyloidosis. We have shown that native tetrameric TTR binds to amyloid ligands based on the trans-stilbene scaffold providing a platform for the determination of high-resolution structures of these important molecules bound to protein. In this study, we provide spectroscopic evidence of binding and X-ray crystallographic structure data on tetrameric TTR complex with the fluorescent salicylic acid-based pyrene amyloid ligand (Py1SA), an analogue of the Congo red analogue X-34. The ambiguous electron density from the X-ray diffraction, however, did not permit Py1SA placement with enough confidence likely due to partial ligand occupancy. Instead, the preferred orientation of the Py1SA ligand in the binding pocket was determined by molecular dynamics and umbrella sampling approaches. We find a distinct preference for the binding modes with the salicylic acid group pointing into the pocket and the pyrene moiety outward to the opening of the T4 binding site. Our work provides insight into TTR binding mode preference for trans-stilbene salicylic acid derivatives as well as a framework for determining structures of TTR-ligand complexes.

Hydrogen bonds connecting the N-terminal region and the DE loop stabilize the monomeric structure of transthyretin.

Transthyretin (TTR) is a homo-tetrameric serum protein associated with sporadic and hereditary systemic amyloidosis. TTR amyloid formation proceeds by the dissociation of the TTR tetramer and the subsequent partial unfolding of the TTR monomer into an aggregation-prone conformation. Although TTR kinetic stabilizers suppress tetramer dissociation, a strategy for stabilizing monomers has not yet been developed. Here, we show that an N-terminal C10S mutation increases the thermodynamic stability of the TTR monomer by forming new hydrogen bond networks through the side chain hydroxyl group of Ser10. Nuclear magnetic resonance spectrometry and molecular dynamics simulation revealed that the Ser10 hydroxyl group forms hydrogen bonds with the main chain amide group of either Gly57 or Thr59 on the DE loop. These hydrogen bonds prevent the dissociation of edge strands in the DAGH and CBEF β-sheets during the unfolding of the TTR monomer by stabilizing the interaction between β-strands A and D and the quasi-helical structure in the DE loop. We propose that introducing hydrogen bonds to connect the N-terminal region to the DE loop reduces the amyloidogenic potential of TTR by stabilizing the monomer.

P203 PLASMA TRANSTHYRETIN FORMS IN PATIENTS WITH WILD–TYPE TRANSTHYRETIN CARDIAC AMYLOIDOSIS: INFLUENCED OF TAFAMIDIS TREATMENT

Abstract Introduction Transthyretin (TTR) is a plasma protein that transports thyroxin and retinol complexed to retinol–binding protein (RBP). TTR misfolding and aggregation can lead to the extracellular deposition of amyloid in the myocardium, causing TTR cardiac amyloidosis (ATTR–CA). Aim of this study is to develop a native electrophoretic method to characterize circulating TTR forms in plasma samples of ATTR–CA patients, before and after administration of tafamidis, a TTR stabilizer. Material &amp; Methods Plasma samples from patients affected by wild–type ATTR amyloidosis (ATTRwt, n=10) were collected before (T0) and during tafamidis treatment at 14, 30 and 90 days. Plasma samples of sex– and age–matched healthy controls (n=6) were also collected. All samples were separated on a native 4–20% Tris–Gly polyacrylamide gel. Western blot analysis was performed with anti–TTR or anti–RBP antibodies. Proteins were detected by Clarity ECL substrate. ImageLab software was used for image acquisition and densitometric analysis of the blots. Total TTR (mg/dl) and RBP (mg/dl) were quantified by nephelometry. Results In both groups, the most represented forms were: TTR dimers or trimers (37–50kDa), TTR tetramers complexed with RBP protein in 1:1 (75kDa,) or 1:2 ratio (100kDa), and high molecular weight aggregates (&amp;gt;150kDa). RBP protein was detectable in association with TTR tetramers and some of the TTR aggregates (250kDa, 480kDa). Based on TTR electrophoretic pattern at T0, patients could be divided in 2 groups: ATTR–1, characterised by the presence of TTR tetramers (55kDa), and ATTR–2, characterised by its absence. The ATTR–1 group showed also higher levels of total TTR in comparison with ATTR–2 (mean±SD 25.5±3.8, 16.6±4.2 respectively, p=0.004), while plasma RBP levels were similar (5.1±0.7, 5.1±1.4 respectively). The electrophoretic patterns of ATTR–1 at T0, as well as TTR and RBP levels were comparable to those of controls (TTR 22.6±3.8, RBP 4.4±0.9). Tafamidis treatment was associated with a progressive increase of total TTR levels both in ATTR–1 and –2 (T90 31.4±5.8, 28.7±6.8) but the 2 groups continued to be distinguishable by the electrophoretic pattern. Conclusions The native electrophoresis allowed us to detect diverse circulating TTR forms and to classify patients in 2 groups according to their electrophoretic pattern. TTR quali/quantitative evaluation by electrophoresis could represent a valid tool to assess the individual pharmacological response to tafamidis.

P4 TRANSTHYRETIN–INDUCED CARDIAC AMYLOIDOSIS: A RARE MUTATION IN AN ITALIAN FAMILIAL CASE

Abstract A 65–year–old man came to our hospital for a subacute stroke of the left parietotemporal area. An aorto–coronary bypass for stable coronary artery disease and aortobisiliac bypass for infrarenal aortic aneurysm (with post–operative courses complicated by multiple atrial fibrillation paroxisms which were pharmacologically cardioverted) was reported. No undergoing anticoagulant therapy for CHADSVASc=1 was reported. Hypothesizing a cardioembolic origin of the stroke, echocardiographic analysis was performed which showed increased ventricular thickness and mass, inhomogeneous texture, reduced systolic function and strain with apical sparing, II degree diastolic dysfunction and severe left atrial dilatation. The strong suspicion of amyloidosis, raised by the echocardiographic findings, was confirmed by bone scintigraphy. The absence of free immunoglobulin chains in blood and urine and a normal protein electrophoretic pattern, would have instead excluded the diagnosis of AL amyloidosis. Thus we performed genetic testing to characterize the type of ATTR (transthyretin). An homozygous Val142Ile mutation in exon 4 of the TTR gene was found. This mutation, typically found in patients of African ancestry, is extremely rare in the Caucasian population. In fact, only 5 cases of patients with this mutation and with phenotypic manifestations very similar to the African ancestral ones have been so far reported, thus indicating that this mutation is not restricted to African ancestry but may be an underestimated Caucasian variant. The large GnomAD database estimates that this homozygous mutation is present in only 0.72% of the general population, while the remaining part of the carriers are heterozygous. Val142Ile leads to increased formation of amyloid fibrils and to the formation of a particularly unstable TTR tetramer. This variant is associated with a worse quality of life, a higher incidence of AF and a lower survival than the wild type TTR. In light of the worse clinical outcomes associated to this variant it is therefore essential to maintain a high level of clinical suspicion in order to perform an early diagnosis from a clinical and molecular point of view in order to be able to direct the patient as soon as possible to the current available therapies that can modify the course of the pathology and avoid premature fatal events.

Transthyretin Stabilizers and Seeding Inhibitors as Therapies for Amyloid Transthyretin Cardiomyopathy.

Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a progressive and increasingly recognized cause of heart failure which is associated with high mortality and morbidity. ATTR-CM is characterized by the misfolding of TTR monomers and their deposition within the myocardium as amyloid fibrils. The standard of care for ATTR-CM consists of TTR-stabilizing ligands, such as tafamidis, which aim at maintaining the native structure of TTR tetramers, thus preventing amyloid aggregation. However, their efficacy in advanced-staged disease and after long-term treatment is still a source of concern, suggesting the existence of other pathogenetic factors. Indeed, pre-formed fibrils present in the tissue can further accelerate amyloid aggregation in a self-propagating process known as "amyloid seeding". The inhibition of amyloidogenesis through TTR stabilizers combined with anti-seeding peptides may represent a novel strategy with additional benefits over current therapies. Finally, the role of stabilizing ligands needs to be reassessed in view of the promising results derived from trials which have evaluated alternative strategies, such as TTR silencers and immunological amyloid disruptors.