Familial Amyloid Cardiomyopathy Research Articles

Structural and thermodynamic characterization of a highly amyloidogenic dimer of transthyretin involved in a severe cardiomyopathy

Transthyretin (TTR) is a homotetrameric protein involved in the transport of thyroxine. More than 150 different mutations have been described in the TTR gene, several of them associated with familial amyloid cardiomyopathy (FAC). Recently, our group described a new variant of TTR in Brazil, namely A39D-TTR, which causes a severe cardiac condition. Position 39 is in the AB loop, a region of the protein that is located within the thyroxine-binding channels and is involved in tetramer formation. In the present study we solved the structure and characterize the thermodynamic stability of this new variant of TTR using urea and high hydrostatic pressure (HHP). Interestingly, during the process of purification, A39D-TTR turned out to be a dimer and not a tetramer, a variation that might be explained by the close contact of the four aspartic acids at position 39, where they face each other inside the thyroxine channel. In the presence of sub-denaturing concentrations of urea, bis-ANS binding and dynamic light scattering revealed A39D-TTR in the form of a molten-globule dimer. Co-expression of A39D and WT isoforms in the same bacterial cell did not produce heterodimers or heterotetramers, suggesting that somehow a negative charge at the AB loop precludes tetramer formation. A39D-TTR proved to be highly amyloidogenic, even at mildly acidic pH values where WT-TTR does not aggregate. Interestingly, despite being a dimer, aggregation of A39D-TTR was inhibited by diclofenac, which binds to the thyroxine channel in the tetramer, suggesting the existence of other pockets in A39D-TTR able to accommodate this molecule.

Structures and Dynamics of β-Rich Oligomers of ATTR (105-115) Assembly.

Transthyretin (TTR) is a tetrameric homologous protein that can dissociate into monomers. Misfolding and aggregation of TTR can lead to amyloid transthyretin amyloidosis (ATTR), which can cause many diseases (e.g., senile systemic amyloidosis, familial amyloid cardiomyopathy, and familial amyloid polyneuropathy). Despite growing evidence indicating that small oligomers play a critical role in regulating cytotoxicity, the structures of these oligomeric intermediates and their conformational transformations are still unclear, impeding our understanding of neurodegenerative mechanisms and the development of therapeutics targeting early aggregation species. The TTR monomer protein consists of various fragments prone to self-aggregation, including the residue 105-115 sequence. Therefore, our study investigated the assembly progress of ATTR (105-115) peptides using all-atom molecular dynamics simulations. The findings indicate that the probability of β-sheet content increases with increasing numbers of peptides. Additionally, interactions between hydrophobic residues L110 and L111 are crucial for the formation of a β-rich oligomer formation. These β-rich oligomers may adopt β-barrel conformations, potentially toxic oligomer species. Free-energy analysis reveals that β-barrel conformations serve as intermediates for these β-rich oligomers. Our insights into the structural ensemble dynamics of ATTR (105-115) contribute to understanding the physical mechanisms underlying the β-barrel oligomers of ATTR. These findings may shed light on the pathological role of ATTR in neurodegenerative diseases and offer potential therapeutic targets.

Generation of an induced pluripotent stem cell line (ESi107-A) from a transthyretin amyloid cardiomyopathy (ATTR-CM) patient carrying a p.Ser43Asn mutation in the TTR gene

Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a life-threatening disease caused by the abnormal production of misfolded TTR protein by liver cells, which is then released systemically. Its amyloid deposition in the heart is linked to cardiac toxicity and progression toward heart failure. A human induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells (PBMCs) from a patient suffering familial transthyretin amyloid cardiomyopathy carrying a c.128G>A (p.Ser43Asn) mutation in the TTR gene. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for therapeutic discovery.

Synthesis and biological evaluation of quinolone derivatives as transthyretin amyloidogenesis inhibitors and fluorescence sensors

Under certain conditions, numerous soluble proteins possess an inherent tendency to convert into insoluble amyloid aggregates, which are associated with several sporadic and genetic human diseases. Transthyretin (TTR) is one of the more than 30 human amyloidogenic proteins involved in conditions such as senile systemic amyloidosis, familial amyloid polyneuropathy, and familial amyloid cardiomyopathy. Considerable effort has been focused on identifying the native tetrameric TTR stabilizers to inhibit rate-limiting tetramer dissociation and, consequently, ameliorate TTR amyloidogenesis. Here, we describe the design and synthesis of quinolin-2(1H)-one derivatives that could be structurally complementary to the thyroxine-binding site within tetrameric TTR. Among these quinolin-2(1H)-one derivatives, compound 7a allowed 16.7% of V30M-TTR (3.6 μM) fibril formation at the same concentration and 49.6% at a concentration of 1.8 μM. Compound 7a exhibited much greater potency in complex biological samples like human plasma than that observed with tafamidis, the drug approved for the treatment of TTR amyloid cardiomyopathy for wild-type or hereditary TTR-mediated amyloidosis. Furthermore, the unique spectral properties of compound 7a demonstrated its high potential for TTR quantification, imaging sensors, and fluorescent tools to study the mechanism of TTR amyloidogenesis.

P-PN022. Transthyretin familial amyloid polyneuropathy in multi-ethnic Malaysians

Introduction . Familial amyloid polyneuropathy (FAP) is a rare autosomal dominant peripheral neuropathy. We report a case series of Malaysian FAP patients. Methods . Patients with definite (genetically confirmed) FAP seen at the University of Malaya Medical Centre between 2008 till 2019 were included in the series. The patients’ demographic and clinical features and investigations including nerve conduction studies and electromyography (NCS/EMG), echocardiography, sural nerve biopsy and hATTR mutation are reported. Results . A total 21 index cases were seen. Nineteen (90%) were ethnic Chinese, one ethnic (5%) Malay and one (5%) ethnic Indian; Fourteen (67%) were men. Mean age of onset of symptoms was 56.1 ± 10.5 years (range: 31-73). Nineteen (90%) patients presented with numbness and weakness of the limbs while two had postural hypotension. Fifteen (71%) patients had positive family history. NCS/EMG showed sensorimotor polyneuropathy in 19 (90%) patients; fifteen (71%) showing axonal neuropathy. Amyloid deposits were seen on nerve biopsy in ten patients and rectal, leptomeningeal and endomyocardial biopsies in 1 patient respectively. The latter was diagnosed as familial amyloid cardiomyopathy (FAC). Sixteen patients (76%) had abnormal cardiac echocardiogram with amyloid deposits. Fifteen Chinese patients (71%) carried the Ala97Ser hATTR mutation while the FAC patient has Asp39Val mutation. Our Malay patient had Glu54Lys mutation and Indian patient had Gly67Val mutation. Two patients carried Val30Met mutation. Autonomic function test was abnormal in all 7 patients in whom they were carried out. Conclusion . FAP in Malaysians is seen mainly among ethnic Chinese, in whom Ala97Ser may be a common mutation. This is also the reported “hotspot” mutation for FAP reported in Taiwan but not in China. Similar to Taiwan, Chinese-Malaysians are descended from immigrants from Southern China. Similar to Taiwanese patients, our FAP patients with Ala97Ser mutation, present with late-onset progressive sensorimotor polyneuropathy and cardiac involvement was common.

Transthyretin Misfolding, A Fatal Structural Pathogenesis Mechanism.

Transthyretin (TTR) is an essential transporter of a thyroid hormone and a holo-retinol binding protein, found abundantly in human plasma and cerebrospinal fluid. In addition, this protein is infamous for its amyloidogenic propensity, causing various amyloidoses in humans, such as senile systemic amyloidosis, familial amyloid polyneuropathy, and familial amyloid cardiomyopathy. It has been known for over two decades that decreased stability of the native tetrameric conformation of TTR is the main cause of these diseases. Yet, mechanistic details on the amyloidogenic transformation of TTR were not clear until recent multidisciplinary investigations on various structural states of TTR. In this review, we discuss recent advancements in the structural understanding of TTR misfolding and amyloidosis processes. Special emphasis has been laid on the observations of novel structural features in various amyloidogenic species of TTR. In addition, proteolysis-induced fragmentation of TTR, a recently proposed mechanism facilitating TTR amyloidosis, has been discussed in light of its structural consequences and relevance to acknowledge the amyloidogenicity of TTR.

Non-biopsy diagnosis of familial amyloid cardiomyopathy

Non-biopsy diagnosis of familial amyloid cardiomyopathy

Design of Small Molecules to Modulate Transthyretin Aggregation

Transthyretin (TTR) amyloid deposits have been causally implicated in familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC). An effective means to block formation and disaggregate transthyretin amyloid deposits of patients with FAP and FAC does not exist. We have designed and synthesized small molecules to stop formation of, as well as disaggregate these deposits. Our hypothesis is that TTR fibrils can be abrogated by TTR‐interactive compounds. The specific aim of this study is to define the activity of our novel small molecules on TTR aggregation and disaggregation with biophysical methods. Thioflavin T (ThT) fluorescence assay was used to monitor fibril formation with synthetic TTR truncated peptide (TTR81–127) treated with small molecules. At the end of a time‐course study, fibrils were directly detected by transmission electron microscopy. Preliminary results showed a few molecules that were able to abrogate fibril formation via the ThT fluorescence assay. The percentages of fibril formation measured at the plateau phase of the kinetics of fibril formation obtained with control and the most potent molecules were compared. One of our best compounds, JF‐19‐029, was ten time more potent than the positive control, resveratrol in inhibiting fibril formation. When TTR81–127 was incubated for 24h with JF‐19‐029, rarely fibrils were observed on the copper grid visualized with the electron microscope. This compound is also being studied to determine the potential for disaggregation of mature TTR81–127 fibrils. This project discovered novel small molecules that potentially reduce TTR fibril formation, with the eventual goal to improve the therapeutic arsenal for FAP and FAC.

S-Homocysteinylation effects on transthyretin: worsening of cardiomyopathy onset

BackgroundL-Homocysteine (Hcy) is a non-proteinogenic α-amino acid synthesized from dietary methionine. In healthy humans, high Hcy levels are a risk factor for cardiovascular diseases, stroke and type 2 diabetes. A recent study reports that Hcy reacts with Cys10 of transthyretin (TTR), generating a stable covalent adduct. However, to date the effect of S-homocysteinylation on TTR conformational stability remains unknown. MethodsThe effect of Hcy on the conformational properties of wt- and L55P-TTR were analysed using a set of biophysical techniques. The cytotoxicity of S-homocysteinylated L55P-TTR was also evaluated in the HL-1 cardiomyocyte cell line, while the effects of the assemblies on kinematic and dynamics properties of cardiac muscle cells were analysed in cardiomyocyte syncytia. ResultsWe found that Hcy stabilizes tetrameric wt-TTR, while it destabilizes the tetrameric structure of the L55P mutant, promoting the accumulation of self-assembly-prone monomeric species. ConclusionsOur study demonstrated that S-homocysteinylation of the L55P-TTR mutant impairs protein stability, favouring the appearance of toxic monomers. Interestingly, S-homocysteinylation affected only mutant, not wt-TTR. Moreover, we also show that assemblies of S-homocysteinylated L55P-TTR impair cardiomyocytes functional parameters. General significanceOur study offers new insights on the negative impact of S-homocysteinylation on L55P-TTR stability, whose aggregation is considered the causative agent of a form of early-onset familial amyloid polyneuropathy and cardiomyopathy. Our results suggest that high homocysteine levels are a further risk factor for TTR cardiomyopathy in patients harbouring the L55P-TTR mutation.

Inotersen for the treatment of adults with polyneuropathy caused by hereditary transthyretin-mediated amyloidosis

ABSTRACTIntroduction: Hereditary transthyretin-mediated amyloidosis (ATTRv; v for variant) is an underdiagnosed, progressive, and fatal multisystemic disease with a heterogenous clinical phenotype that is caused by TTR gene mutations that destabilize the TTR protein, resulting in its misfolding, aggregation, and deposition in tissues throughout the body.Areas covered: Inotersen, an antisense oligonucleotide inhibitor, was recently approved in the United States and Europe for the treatment of the polyneuropathy of ATTRv based on the positive results obtained in the pivotal phase 3 trial, NEURO-TTR. This review will discuss the mechanism of action of inotersen and its pharmacology, clinical efficacy, and safety and tolerability. A PubMed search using the terms ‘inotersen,’ ‘AG10,’ ‘antisense oligonucleotide,’ ‘hereditary transthyretin amyloidosis,’ ‘familial amyloid polyneuropathy,’ and ‘familial amyloid cardiomyopathy’ was performed, and the results were screened for the most relevant English language publications. The bibliographies of all retrieved articles were manually searched to identify additional studies of relevance.Expert opinion: Inotersen targets the disease-forming protein, TTR, and has been shown to improve quality of life and neuropathy progression in patients with stage 1 or 2 ATTRv with polyneuropathy. Inotersen is well tolerated, with a manageable safety profile through regular monitoring for the development of glomerulonephritis or thrombocytopenia.

Monoallelic expression of the TTR gene as a contributor to the age at onset and penetrance of TTR-related amyloidosis

TTR-related amyloidosis (ATTR) is manifested in two allelic forms: familial amyloid polyneuropathy (TTR-FAP) and cardiomyopathy (TTR-FAC), both caused by mutations in the TTR gene. The most prevalent mutation in Bulgaria is p.Glu89Gln. Markedly different age at onset and disease penetrance is noticed in Bulgarian p.Glu89Gln cases even in a single family or between genetically identical twins. The present study aimed to evaluate the transcription profile of the TTR gene in order to better understand the difference in disease onset and penetrance. Six p.Glu89Gln positive families were selected from our registry, based on intrafamilial differences in disease onset and penetrance. Plasma and urine specimens were collected from 13 patients and subjected to transcription analysis. Both mutant and wild type transcripts were visualized in a mixed transcription profile, which is the traditional model of autosomal gene expression. The results from a relative quantification of the mutant versus wild type transcript showed presence of the mutant transcript between 0.14 and 1.14 times against the wild type.In addition, monoallelic expression signature was also detected. Based on our results we propose a model of natural selection, which includes age-related allele exclusion or suppression: predominant expression of a wild type (at an early age) and mutant (at the process of ageing) alleles. The intrafamilial differences in disease onset and penetrance need to be considered in genetic counselling and in follow-up of mutation carriers.

Contributions of Animal Models to the Mechanisms and Therapies of Transthyretin Amyloidosis

Transthyretin amyloidosis (ATTR amyloidosis) is a fatal systemic disease caused by amyloid deposits of misfolded transthyretin, leading to familial amyloid polyneuropathy and/or cardiomyopathy, or a rare oculoleptomeningeal amyloidosis. A good model system that mimic the disease phenotype is crucial for the development of drugs and treatments for this devastating degenerative disorder. The present models using fruit flies, worms, rodents, non-human primates and induced pluripotent stem cells have helped researchers understand important disease-related mechanisms and test potential therapeutic options. However, the challenge of creating an ideal model still looms, for these models did not recapitulates all symptoms, particularly neurological presentation, of ATTR amyloidosis. Recently, knock-in techniques was used to generate two humanized ATTR mouse models, leading to amyloid deposition in the nerves and neuropathic manifestation in these models. This review gives a recent update on the milestone, progress, and challenges in developing different models for ATTR amyloidosis research.

Characterizing the High Disease Burden of Transthyretin Amyloidosis for Patients and Caregivers.

IntroductionTransthyretin amyloidosis (ATTR amyloidosis), whether manifesting as familial amyloid polyneuropathy (ATTR-PN) or cardiomyopathy (ATTR-CM), is a progressive, debilitating, and often fatal, rare disease requiring significant caregiver support. This study aims to better characterize the burden of disease for ATTR amyloidosis patients and caregivers.MethodsPatients and caregivers in the USA and Spain were recruited through patient advocacy groups to complete a cross-sectional survey. Assessments included the 12-Item Short Form Health Survey, the Work Productivity and Activity Impairment Questionnaire, the Zarit Burden Interview, pain and symptom measures, health care resource use measures, and caregiving burden measures.ResultsRespondents included 60 ATTR amyloidosis patients and 32 caregivers. Patients registered scores up to two standard deviations below normal for physical health, impairment in quality of life, and reduced work productivity. Patients with liver transplant versus without liver transplant reported better overall outcomes, and those without liver transplant reported a greater impact on physical versus mental health. A high rate of health care utilization in the 3 months prior to the survey was reported by patients. Caregivers reported substantial burden, including poor mental health, work impairment, and much time spent providing care (mean 45.9 h/week). Work productivity was impacted for employed patients and caregivers.ConclusionATTR amyloidosis was associated with high levels of impairment in many domains, including physical health, quality of life, and reduced productivity. Providing care for ATTR amyloidosis patients is associated with a negative impact on the mental health of caregivers. These results highlight the substantial burden of ATTR amyloidosis for patients and caregivers.Trial RegistrationClinicalTrials.gov: NCT01604122.FundingPfizer.

Serum Proteomic Variability Associated with Clinical Phenotype in Familial Transthyretin Amyloidosis (ATTRm).

Transthyretin (TTR), normally a plasma circulating protein, can become misfolded and aggregated, ultimately leading to extracellular deposition of amyloid fibrils usually targeted to heart or nerve tissues. Referred to as TTR-associated amyloidoses (ATTR), this group of diseases is frequently life threatening and fatal if untreated. ATTR, caused by amyloid-forming variant TTR proteins (ATTRm) that arise from point mutations in the TTR gene, were classically referred to as familial amyloid cardiomyopathy (FAC) or familial amyloid polyneuropathy (FAP), reflecting the clinical phenotype. FAC and FAP are pathologies that can be challenging to diagnose as there are no definitive biomarkers of disease; moreover, disease-specific measures of progression are lacking, and treatment options are limited. Thus, the discovery of sensitive and specific indicators of disease has the potential to improve recognition, enable accurate measurement of amyloid progression and response to treatment, and reveal key information regarding FAC and FAP pathobiological mechanisms. In this study, the goal was to investigate serum proteomic features unique to FAC and FAP types of ATTRm. Multiple-reaction monitoring mass spectrometry (MRM-MS), a powerful technique in profiling proteomes, was used to measure the serum concentrations of 160 proteins in samples from FAC and FAP patients. Results were compared to data from healthy control sera obtained from individuals matched to age (≥60 years), gender (male), and race (Caucasian). Proteomic analyses of ATTRm (FAC and FAP) and control samples showed significant concentration differences in 107 of 192 (56%) of the serum proteins that were studied. In comparing FAC to FAP, differences in concentrations as well as interactions and functions of several proteins were identified as unique to each disease; significantly lower levels of TTR were specific to FAC, but not to FAP. Annotated functional clustering identified extracellular region, signal, and signal peptide as terms common to FAC and FAP. Conversely, disulfide bond was unique to FAC; secreted, glycosylation site: N-linked, glycosylation, glycoprotein, polymorphism, and sequence variant were associated solely with FAP. Predicted protein-protein associations in FAC were seen for reaction, binding, and activation processes; no associations were found in FAP. This study demonstrates significant proteomic differences between ATTRm patient and control sera, as well as ATTRm phenotype-associated variations in the circulating levels of several proteins including TTR. The identification of serum proteins unique to FAC and FAP may have diagnostic and prognostic utility and could possibly provide important clues about disease mechanisms.

Amyloid Cardiomyopathy: Disease on the Rise.

Amyloidoses represent a group of human degenerative diseases characterized by the deposition of aggregates of abnormally folded proteins in single or multi-organs. Whereas neurological amyloidoses, such as Alzheimer and Parkinson disease, have received the greatest recognition, there also exists many systemic amyloidoses that affect many target organs, including the heart. Cardiac amyloidosis is primarily associated with the systemic production and release of many amyloidogenic proteins, notably immunoglobulin light chain or transthyretin (TTR). AL (light chain amyloidosis) is the result of a clonal plasma cell dyscrasia and production of amyloidgenic light chain proteins. TTR amyloidosis may result from normal wild-type amyloidogenic TTR protein as seen in the elderly (known as senile systemic amyloidosis or ATTRwt) or from mutations in the TTR protein (ATTRm) as in familial amyloid cardiomyopathy.1,2 Cardiac deposition of amyloidogenic proteins often results in an aggressive form of heart disease with resulting cardiac failure that is largely resistant to many common heart failure therapies. Although once thought to be a rare disease, cardiac amyloidosis is more recently acknowledged to be much more common. For instance, AL amyloidosis, the most frequent cause of systemic amyloidosis in the developed world, has an incidence similar to that of Hodgkin lymphoma or chronic myelogenous leukemia and was widely underdiagnosed because of its ambiguous presentation and rapid mortality. Similarly, autopsy studies have identified significant cardiac deposition of wild-type TTR deposition in over 25% of individuals >80 years of age.3 Physician education, along with now widely accessible assays for diagnosis and monitoring (such as the Freelite serum-free LC assay), and improved cardiac imaging have contributed to the increase in diagnosis. In addition to ultrasound echocardiography and late gadolinium enhancement by cardiac magnetic resonance imaging,4 newer imaging modalities with 99mTc-pyrophosphate4,5 and florbetapir6 have improved detection, monitoring …

FRET studies of various conformational states adopted by transthyretin.

Transthyretin (TTR) is an extracellular protein able to deposit into well-defined protein aggregates called amyloid, in pathological conditions known as senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and leptomeningeal amyloidosis. At least three distinct partially folded states have been described for TTR, including the widely studied amyloidogenic state at mildly acidic pH. Here, we have used fluorescence resonance energy transfer (FRET) experiments in a monomeric variant of TTR (M-TTR) and in its W41F and W79F mutants, taking advantage of the presence of a unique, solvent-exposed, cysteine residue at position 10, that we have labelled with a coumarin derivative (DACM, acceptor), and of the two natural tryptophan residues at positions 41 and 79 (donors). Trp41 is located in an ideal position as it is one of the residues of β-strand C, whose degree of unfolding is debated. We found that the amyloidogenic state at low pH has the same FRET efficiency as the folded state at neutral pH in both M-TTR and W79F-M-TTR, indicating an unmodified Cys10-Trp41 distance. The partially folded state populated at low denaturant concentrations also has a similar FRET efficiency, but other spectroscopic probes indicate that it is distinct from the amyloidogenic state at acidic pH. By contrast, the off-pathway state accumulating transiently during refolding has a higher FRET efficiency, indicating non-native interactions that reduce the Cys10-Trp41 spatial distance, revealing a third distinct conformational state. Overall, our results clarify a negligible degree of unfolding of β-strand C in the formation of the amyloidogenic state and establish the concept that TTR is a highly plastic protein able to populate at least three distinct conformational states.

Biochemical and Electrophysiological Modification of Amyloid Transthyretin on Cardiomyocytes

Transthyretin (TTR) amyloidoses are familial or sporadic degenerative conditions that often feature heavy cardiac involvement. Presently, no effective pharmacological therapy for TTR amyloidoses is available, mostly due to a substantial lack of knowledge about both the molecular mechanisms of TTR aggregation in tissue and the ensuing functional and viability modifications that occur in aggregate-exposed cells. TTR amyloidoses are of particular interest regarding the relation between functional and viability impairment in aggregate-exposed excitable cells such as peripheral neurons and cardiomyocytes. In particular, the latter cells provide an opportunity to investigate in parallel the electrophysiological and biochemical modifications that take place when the cells are exposed for various lengths of time to variously aggregated wild-type TTR, a condition that characterizes senile systemic amyloidosis. In this study, we investigated biochemical and electrophysiological modifications in cardiomyocytes exposed to amyloid oligomers or fibrils of wild-type TTR or to its T4-stabilized form, which resists tetramer disassembly, misfolding, and aggregation. Amyloid TTR cytotoxicity results in mitochondrial potential modification, oxidative stress, deregulation of cytoplasmic Ca2+ levels, and Ca2+ cycling. The altered intracellular Ca2+ cycling causes a prolongation of the action potential, as determined by whole-cell recordings of action potentials on isolated mouse ventricular myocytes, which may contribute to the development of cellular arrhythmias and conduction alterations often seen in patients with TTR amyloidosis. Our data add information about the biochemical, functional, and viability alterations that occur in cardiomyocytes exposed to aggregated TTR, and provide clues as to the molecular and physiological basis of heart dysfunction in sporadic senile systemic amyloidosis and familial amyloid cardiomyopathy forms of TTR amyloidoses.

Synthesis and structural analysis of halogen substituted fibril formation inhibitors of Human Transthyretin (TTR)

Transthyretin (TTR), a β-sheet-rich tetrameric protein, in equilibrium with an unstable amyloidogenic monomeric form is responsible for extracellular deposition of amyloid fibrils, is associated with the onset of neurodegenerative diseases, such as senile systemic amyloidosis, familial amyloid polyneuropathy and familial amyloid cardiomyopathy. One of the therapeutic strategies is to use small molecules to stabilize the TTR tetramer and thus curb amyloid fibril formation. Here, we report the synthesis, the in vitro evaluation of several halogen substituted 9-fluorenyl- and di-benzophenon-based ligands and their three-dimensional crystallographic analysis in complex with TTR. The synthesized compounds bind TTR and stabilize the tetramer with different potency. Of these compounds, 2c is the best inhibitor. The dual binding mode prevalent in the absence of substitutions on the fluorenyl ring, is disfavored by (2,7-dichloro-fluoren-9-ylideneaminooxy)-acetic acid (1b), (2,7-dibromo-fluoren-9-ylideneaminooxy)-acetic acid (1c) and (E/Z)-((3,4-dichloro-phenyl)-methyleneaminooxy)-acetic acid (2c), all with halogen substitutions.

The polyphenol Oleuropein aglycone hinders the growth of toxic transthyretin amyloid assemblies

Transthyretin (TTR) is involved in a subset of familial or sporadic amyloid diseases including senile systemic amyloidosis (SSA), familial amyloid polyneuropathy and cardiomyopathy (FAP/FAC) for which no effective therapy has been found yet. These conditions are characterized by extracellular deposits primarily found in the heart parenchyma and in peripheral nerves whose main component are amyloid fibrils, presently considered the main culprits of cell sufferance. The latter are polymeric assemblies grown from misfolded TTR, either wt or carrying one out of many identified mutations. The recent introduction in the clinical practice of synthetic TTR-stabilizing molecules that reduce protein aggregation provides the rationale to search natural effective molecules able to interfere with TTR amyloid aggregation by hindering the appearance of toxic species or by favoring the growth of harmless aggregates. Here we carried out an in depth biophysical and morphological study on the molecular features of the aggregation of wt- and L55P-TTR involved in SSA or FAP/FAC, respectively, and on the interference with fibril aggregation, stability and toxicity to cardiac HL-1 cells to demonstrate the ability of Oleuropein aglycone (OleA), the main phenolic component of the extra virgin olive oil. We describe the molecular basis of such interference and the resulting reduction of TTR amyloid aggregate cytotoxicity. Our data offer the possibility to validate and optimize the use of OleA or its molecular scaffold to rationally design promising drugs against TTR-related pathologies that could enter a clinical experimental phase.

Heart transplantation in hereditary ATTR amyloidosis

Systemic amyloidosis related to mutation of TTR gene can be complicated with Familial Amyloid Cardiomyopathy (TTR-FAC), a severe and life threatening form of heart failure with preserved ejection fraction, with a poor prognosis. To date there is no proven effective specific treatment against TTR-FAC, and the usual treatments of chronic heart failure are either ineffective or contra indicated, except for diuretics. Progression of the disease results in reduction of physical capacity, repeated hospitalizations, increase in diuretics dose, and ultimately in refractory heart failure. Cardiac transplantation has been performed in TTR-FAC since 2003, and reported in 40 patients in the literature with acceptable results in highly selected patients. The results of combined heart and liver transplantation appear to be similar or even better than for isolated heart transplantation, due to a “tolerance” phenomenon, with less cardiac rejection and less graft coronary artery disease. We performed heart transplantation for TTR-FAC in 10 pts (9 men; mean age 59 years range 49 to 70), NYHA class II to IV, isolated in 6 and combined with a liver transplantation in 4. Patients had received a pacemaker (n=3) or a defibrillator (n=3) during their preoperative course. Mutations of patients were TYR77 in 5, SER24 in 1, GLU62 in 1, MET30 in 1. In hospital mortality was 40%. Patients can be considered as good candidates to heart transplantation for TTR-FAC if they have symptomatic heart failure persisting under optimal medical therapy, disregarding their ejection fraction, normal pulmonary resistance, no major comorbidities, an acceptable physical and psychological condition, and limited neurologic involvement (ability to engage in a post-operative rehabilitation program is mandatory). In the future, indications of combined heart and liver transplantations will have to be weighed against isolated heart transplantation in association with specific medical treatments (Tafamidis, or SiRNA).