Transthyretin Misfolding Research Articles

Misfolding of Transthyretin In Vivo is Controlled by the Redox Environment and Macromolecular Crowding

Transthyretin (TTR) amyloidosis is a progressive disorder characterized by peripheral neuropathy, autonomic dysfunction, and cardiomyopathy. The precise mechanism by which TTR misfolds and forms fibrils in vivo remains incompletely understood, posing challenges to the development of effective therapeutics. In this study, we reveal that the recently identified non-native pathological species (NNTTR), which is enriched in the plasma of ttr-val30met gene carriers, exhibits strong amyloidogenic properties, making it a promising therapeutic target. Notably, we demonstrate that NNTTR formation is dependent on an intermolecular disulfide bond and can be promoted by oxidative conditions while being effectively suppressed by reducing agents. The formation of this disulfide bond is incompatible with the native TTR fold, thereby necessitating structural flexibility. We further show that this required flexibility can be constrained using tetramer-stabilizing drugs, thereby suppressing NNTTR formation. Interestingly, the flexibility is also hindered by macromolecular crowding, and NNTTR formation is strongly suppressed by the high protein concentration in plasma. This suppression is released upon dilution, which thus promotes NNTTR formation in areas with lower protein content, highlighting a potential link to the interstitial space, brain, and vitreous body of the eye, where TTR-amyloid is frequently observed. In summary, we demonstrate that NNTTR displays strong amyloidogenic features, underscoring its potential as a therapeutic target. We identify the redox environment and macromolecular crowding as key modulatory factors. Our findings propose a mechanistic explanation for TTR misfolding and suggest a novel therapeutic approach.

A mini-review of Vutrisiran and Eplontersen in hereditary transthyretin-mediated amyloidosis with polyneuropathy.

Hereditary transthyretin-mediated amyloidosis (ATTRv amyloidosis), known as Corino de Andrade disease, is a rare neurodegenerative disorder with a significant global impact characterized by the misfolding of transthyretin (TTR) protein leading to amyloid aggregation, ATTRv amyloidosis, especially with polyneuropathy, poses a considerable challenge in managing its rapid progression and debilitating effects. This mini-review focuses on the recent advancements in the treatment landscape for ATTRv amyloidosis with polyneuropathy, specifically the RNA interference therapeutic Vutrisiran and the ligand-conjugated antisense oligonucleotide Eplontersen. We aim to provide a comprehensive overview of the mechanisms, current evidence from clinical trials, and future directions for these novel therapeutic agents. Vutrisiran and Eplontersen have demonstrated significant clinical efficacy in improving neuropathic impairment, quality of life, and serum TTR levels in various trials. The distinct mechanistic approaches of these therapies, coupled with their acceptable safety profiles, offer promising avenues for addressing the complexities of ATTRv amyloidosis with polyneuropathy. The introduction of Vutrisiran and Eplontersen marks a pivotal moment in the quest for effective therapies against ATTRv amyloidosis with polyneuropathy. While clinical evidence is promising, ongoing research is crucial to deepen mechanistic understanding and address research gaps. Future perspectives include the potential expansion of therapeutic options and a more inclusive approach to cater to the diverse needs of individuals globally. This mini-review provides valuable insights into the evolving landscape of ATTRv amyloidosis management and sets the stage for further exploration in this challenging domain.

Evolution of Disease-modifying Therapy for Transthyretin Cardiac Amyloidosis.

Transthyretin cardiac amyloidosis (ATTR-CA) represents an inexorably progressive and fatal cardiomyopathy. Increased understanding of the underlying pathogenesis responsible for the misfolding of transthyretin and the subsequent accumulation of amyloid fibrils within the myocardium has led to the development of several disease-modifying therapies that act on different stages of the disease pathway. Tafamidis is the first, and to date remains the only, therapy approved for the treatment of ATTR-CA, which, alongside acoramidis, stabilizes the transthyretin tetramer, preventing disaggregation, misfolding and formation of amyloid fibrils. Gene-silencing agents, such as patisiran, vutrisian and eplontersen, and novel gene-editing therapies, such as NTLA-2001, act to reduce the hepatic synthesis of transthyretin. Anti-amyloid therapies represent another strategy in the treatment of ATTR-CA and are designed to bind amyloid fibril epitopes and stimulate macrophage-mediated removal of amyloid fibrils from the myocardium. Many of these treatments are at an early investigational stage but represent an important area of unmet clinical need and could potentially reverse disease and restore cardiac functions even in patients with advanced disease.

Musculoskeletal manifestations associated with transthyretin-mediated (ATTR) amyloidosis: a systematic review

BackgroundHereditary and wild-type transthyretin-mediated (ATTRv and ATTRwt) amyloidoses result from the misfolding of transthyretin and aggregation of amyloid plaques in multiple organ systems. Diagnosis of ATTR amyloidosis is often delayed due to its heterogenous and non-specific presentation. This review investigates the association of musculoskeletal (MSK) manifestations with ATTR amyloidosis and the delay from the onset of these manifestations to the diagnosis of ATTR amyloidosis.MethodsThis systematic review utilized Medline and EMBASE databases. Search criteria were outlined using a pre-specified patient, intervention, comparator, outcome, time, study (PICOTS) criteria and included: amyloidosis, ATTR, and MSK manifestations. Publication quality was assessed utilizing Joanna Briggs Institute (JBI) critical appraisal checklists.The search initially identified 7,139 publications, 164 of which were included. PICOTS criteria led to the inclusion of epidemiology, clinical burden and practice, pathophysiology, and temporality of MSK manifestations associated with ATTR amyloidosis. 163 publications reported on ATTR amyloidosis and MSK manifestations, and 13 publications reported on the delay in ATTR amyloidosis diagnosis following the onset of MSK manifestations.ResultsThe MSK manifestation most frequently associated with ATTR amyloidosis was carpal tunnel syndrome (CTS); spinal stenosis (SS) and osteoarthritis (OA), among others, were also identified. The exact prevalence of different MSK manifestations in patients with ATTR amyloidosis remains unclear, as a broad range of prevalence estimates were reported. Moreover, the reported prevalence of MSK manifestations showed no clear trend or distinction in association between ATTRv and ATTRwt amyloidosis.MSK manifestations precede the diagnosis of ATTR amyloidosis by years, and there was substantial variation in the reported delay to ATTR amyloidosis diagnosis. Reports do suggest a longer diagnostic delay in patients with ATTRv amyloidosis, with 2 to 12 years delay in ATTRv versus 1.3 to 1.9 years delay in ATTRwt amyloidosis.ConclusionThese findings suggest that orthopedic surgeons may play a role in the early diagnosis of and treatment referrals for ATTR amyloidosis. Detection of MSK manifestations may enable earlier diagnosis and administration of effective treatments before disease progression occurs.

P2 A RARE CASE OF MUTANT TRANSTHYRETIN CARDIAC AMYLOIDOSIS

Abstract A 65–year–old man with a history of multiple hospitalizations in the past 12 months for heart failure with preserved left ventricular ejection fraction (HFpEF) attended the emergency department for exertional dyspnea, peripheral edema, abdominal bloating, and weight gain. An ECG showed sinus bradycardia with first degree AV block, RBBB, and inferior pseudo–q. Blood tests showed NT–proBNP 8985 pg/ml, troponin hs–cTnI 106 ng/L. An echocardiogram demonstrated severe left ventricular hypertrophy, EF 50%, GLS –11% with apical sparing, and restrictive transmitral pattern, raising suspicion of infiltrative disease (Figure 1). A cardiac MRI corroborated the suspicion, showing altered gadolinium kinetics, diffuse subendocardial LGE, and increased native T1 and extracellular volume (Figure 2). A search for monoclonal components on serum and urine was negative. A bone tracer scintigraphy showed cardiac uptake (Perugini grade 2). A genetic test showed the presence of p.Ile88Leu (Ile68Leu) mutation in heterozygosity, so a non–biopsy diagnosis of mutant transthyretin cardiac amyloidosis (ATTRv) was posed. A thorough neurological evaluation did not document signs of amyloid neuropathy. The patient was then started on tafamidis therapy. Transthyretin cardiac amyloidosis (ATTR) is still an underdiagnosed but increasingly recognized condition, causing up to 13% of HFpEF cases. ATTR is caused by extracellular accumulation of amyloid substance resulting from transthyretin misfolding. In Italy ATTRv is rare and Ile68Leu is among the most frequent mutations, especially in the Tuscan–Emilian Apennines. Ile68Leu is usually associated with an almost exclusively cardiac phenotype, clinically indistinguishable from ATTR wild type, except for an earlier age of onset. Because effective treatments are available for ATTRv with cardiac and/or neurological involvement, it is important to suspect the diagnosis of ATTR in patients with HFpEF and to proceed with genetic testing to identify individuals with ATTRv.

P289 EYESHOT OF CARDIAC AMYLOIDOSIS CASES PRESENTED TO A CARDIOLOGY UO IN CENTRAL ITALY IN 2021

Abstract Introduction ATTR–related cardiac amyloidosis, caused by the accumulation of insoluble fibrillar proteins derived from transthyretin misfolding, is an often underdiagnosed pathology, especially when neurological involvement is absent or minor. Transthoracic echocardiography represents the first fundamental step in the diagnostic process. Abstract During the year 2021, a total of 9 patients with an echocardiographic feature of hypertrophic heart disease suggestive of infiltrative pathology were referred to our Cardiology unit. Of these patients, 89% were male (8 out of 9), aged between 60 and 88 years (average age of 72 years). The clinic presentation was heart failure in 5 cases (55% of the total), arrhythmic storm in 1 case, history of amyloid polyneuropathy in 1 case and it was an occasional finding in 2 patients who required a cardiological evaluation for other reasons. The 12–lead ECG documented atrial fibrillation arrhythmia in 4 out of 9 cases (44%) and sinus rhythm in the remaining 5 cases (56%). Transthoracic echocardiography revealed left ventricular hypertrophy with granular sparkling aspect of the SIV in all patients, with indexes of global systolic function preserved in 4 out of 9 cases (44%), moderately reduced in 3 out of 9 cases (33%) and severely reduced in the remaining 2 cases (22%). Diastolic dysfunction was present in all patients, with a restrictive pattern in 4 of 9 cases (44%). Myocardial scintigraphy with DPD was performed in 7 patients, which confirmed the suspicion of aTTR amyloidosis in 6 cases (Perugini Score = 2), in the remaining 2 patients the diagnostic procedure was not performed due to severe comorbidities. At present, diagnostic confirmation of aTTR amyloidosis has been confirmed by biopsy in 2 patients; a patient‘s biopsy is currently under analysis; 1 patient is on Patisiran therapy. Conclusions Cardiac amyloidosis represents a frequent clinical condition especially in the elderly population. In the light of the most recent therapeutic strategies, a correct and early etiopathogenetic diagnosis is of fundamental importance.

Prevalence of cardiac amyloidosis in patients with unexplained left ventricle hypertrophy

Abstract Background Amyloid cardiomyopathy (CA) it is more frequently diagnosed due to rapidly developing imaging modalities. Misfolding of transthyretin (TTR) is the source of two distinct forms of amyloidosis (ATTR): acquired wild-type (ATTRwt) and hereditary (ATTRm). Types of TTR gene variants display genetic and ethnic variability. The aim of this prospective study was to assess the prevalence and types of pathogenic genetic variants associated with ATTRm amyloidosis in patients with unexplained left ventricle (LV) hypertrophy. Methods We evaluated prospectively 101 consecutive patients (37 (37%) females, age: 69.7±13.6 y.o.) in years 2016–2021. Analysis included clinical data, free light chain blood immunoglobulins and urine immunofixation, transthoracic echocardiography (TTE), single-photon emission computed tomography (SPECT) with 3,3-disphono-1,2-propanodicarboxylic acid (DPD), and in selected cases cardiac or soft tissue biopsy. Patients with DPD cardiac uptake in SPECT or positive histopathology or family history of ATTR were subjected to genetic testing by an amplicon-based next-generation TTR sequencing approach. Results Enrolled patients presented with marked LV hypertrophy with maximum wall thickness of 18.9±4 mm. Based on performed tests 34 patients (33.7%) were diagnosed with amyloidosis, including 17 cases of light-chain amyloidosis (AL), 16 of ATTR, and 1 case of other type of amyloidosis. Overall, patients with CA presented with mean 2.6±0.9 NYHA class. In TTE there was LV maximum wall thickness of 19.5±4 mm and LV mass value index value of 182±48 g/m2, decreased global longitudinal strain value (GLS, −14.1±5%) and advanced diastolic dysfunction (EA 2.2±1.1, E/E' 22±9). Overall, there were 8 cases of ATTRwt and eight patients were diagnosed ATTRm. There were detected following types of TTR variants - c.302C>T p.(Ala101Val) in a single male patient, c.325G>A p.(Glu109Lys) in a single male patient and c.157T>C p.(Phe53Leu) in six patients. Overall, 10 patients with ATTR presented with polyneuropathy, additionally six had diagnosed carpal tunnel syndrome. All patients with ATTRm had positive family history for CA. Conclusions In patients with unexpected LV hypertrophy evaluated in a cardiology referral center a high number of patients may suffer from underlying CA. Types of detected TTR gene variants associated with CA included most often Phe53Leu, and rare variants Ala101Val, Glu109Lys. Funding Acknowledgement Type of funding sources: None.

The Structural Understanding of Transthyretin Misfolding and the Inspired Drug Approaches for the Treatment of Heart Failure Associated With Transthyretin Amyloidosis.

Substantial controversies exist in the exploration of the molecular mechanism of heart failure (HF) and pose challenges to the diagnosis of HF and the discovery of specific drugs for the treatment. Recently, cardiac transthyretin (TTR) amyloidosis is becoming recognized as one of major causes of underdiagnosed HF. The investigation and modulation of TTR misfolding and amyloidal aggregation open up a new revenue to reveal the molecular mechanisms of HF and provide new possibilities for the treatment of HF. The aim of this review is to briefly introduce the recent advances in the study of TTR native and misfolding structures, discuss the correlation between the genotype and phenotype of cardiac TTR amyloidosis, and summarize the therapeutic applications of TTR structural stabilizers in the treatment of TTR amyloidosis-associated HF.

Current Emerging Therapy for Amyloidosis Neuropathy

Peripheral neuropathy is a type of neurological disorder in which patients with complex inherited neurological defects present significant phenotype in the peripheral nervous system. Hereditary amyloidogenic transthyretin (hATTR) neuropathy is typical polyneuropathy caused by single-nucleotide variants in the gene encoding transthyretin (TTR) and leads to transthyretin misfolding and systemic deposition of amyloid. One of the clinical hallmarks of hATTR neuropathy is polyneuropathy of the destruction of the somatic and autonomic peripheral nervous system, leading to loss of autonomy. Progressive amyloid accumulation also causes multi-organ dysfunction and death. There are many therapeutics that have been proposed and developed in these years. These therapies aim to reduce or eliminate hATTR from the plasma, stabilize the hATTR to prevent deposition, and dissolute the amyloid misfolding matrix. Recently, gene therapy strategy is being deployed to treat recessive genetic disorders by eliminating the expression of the mutated genes. Thus, gene-silencing approaches have been used to manage this amyloidosis neuropathy in the broad stages and produce some degree of improvement of this disease. Food and Drug Administration (FDA) approved Inotersen (an antisense oligonucleotide (ASO)) and patisiran (a small interfering ribonucleic acid (siRNA) for the treatment of hATTR polyneuropathy to suppress hATTR expression. Inotersen, a 2’-O-methoxyethylmodified ASO, which acts by reducing the production of transthyretin, and has been demonstrated to improve the quality of life in early hereditary transthyretin amyloidosis polyneuropathy. I here focus on the RNA-targeted therapy with particular emphasis on the molecular mechanisms by which antisense oligonucleotide can be designed to modulate transthyretin RNA function for being a novel therapy for hereditary amyloidosis neuropathy.

Disruption of the CD Loop by Enzymatic Cleavage Promotes the Formation of Toxic Transthyretin Oligomers through a Common Transthyretin Misfolding Pathway.

Amyloid formation of full-length TTR involves dissociation of the native tetramers into misfolded monomers that self-assemble into amyloid. In addition to the full-length TTR, C-terminal fragments including residues 49-127 were also observed in vivo, implying the presence of additional misfolding pathways. It was previously proposed that a proteolytic cleavage might lead to the formation of the C-terminal fragment TTR amyloid. Here, we report mechanistic studies of misfolding and aggregation of a TTR variant (G53A) in the absence and presence of a serine protease. A proteolytic cleavage of G53A in the CD loop (K48 and T49) with agitation promoted TTR misfolding and aggregation, suggesting that the proteolytic cleavage may lead to the aggregation of the C-terminal fragment (residues 49-127). To gain more detailed insights into TTR misfolding promoted by proteolytic cleavage, we investigated structural changes in G53A TTR in the presence and absence of trypsin. Our combined biophysical analyses revealed that the proteolytic cleavage accelerated the formation of spherical small oligomers, which exhibited cytotoxic activities. However, the truncated TTR appeared to maintain native-like structures, rather than the C-terminal fragment (residues 49-127) being released and unfolded from the native state. In addition, our solid-state nuclear magnetic resonance and Fourier transform infrared structural studies showed that the two aggregates derived from the full-length and cleaved TTR exhibited nearly identical molecular structural features, suggesting that the proteolytic cleavage in the CD loop destabilizes the native tetrameric structure and accelerates oligomer formation through a common TTR misfolding and aggregation mechanism rather than through a distinct molecular mechanism.

Structural Characterization of Cardiac Ex Vivo Transthyretin Amyloid: Insight into the Transthyretin Misfolding Pathway In Vivo.

Structural characterization of misfolded protein aggregates is essential to understanding the molecular mechanism of protein aggregation associated with various protein misfolding disorders. Here, we report structural analyses of ex vivo transthyretin aggregates extracted from human cardiac tissue. Comparative structural analyses of in vitro and ex vivo transthyretin aggregates using various biophysical techniques revealed that cardiac transthyretin amyloid has structural features similar to those of in vitro transthyretin amyloid. Our solid-state nuclear magnetic resonance studies showed that in vitro amyloid contains extensive nativelike β-sheet structures, while other loop regions including helical structures are disrupted in the amyloid state. These results suggest that transthyretin undergoes a common misfolding and aggregation transition to nativelike aggregation-prone monomers that self-assemble into amyloid precipitates in vitro and in vivo.

AN UNDERDIAGNOSED CAUSE OF CONGESTIVE HEART FAILURE

Transthyretin amyloid cardiomyopathy (ATTR-CM) is caused by mutations in the gene encoding transthyretin that induce transthyretin misfolding and deposition in cardiac tissue. Its true prevalence is not known but is estimated to be 13% in patients with heart failure with preserved ejection fraction

Two distinct aggregation pathways in transthyretin misfolding and amyloid formation

Misfolding and amyloid formation of transthyretin (TTR) is implicated in numerous degenerative diseases. TTR misfolding is greatly accelerated under acidic conditions, and thus most of the mechanistic studies of TTR amyloid formation have been conducted at various acidic pH values (2–5). In this study, we report the effect of pH on TTR misfolding pathways and amyloid structures. Our combined solution and solid-state NMR studies revealed that TTR amyloid formation can proceed via at least two distinct misfolding pathways depending on the acidic conditions. Under mildly acidic conditions (pH 4.4), tetrameric native TTR appears to dissociate to monomers that maintain most of the native-like β-sheet structures. The amyloidogenic protein undergoes a conformational transition to largely unfolded states at more acidic conditions (pH 2.4), leading to amyloid with distinct molecular structures. Aggregation kinetics is also highly dependent upon the acidic conditions. TTR quickly forms moderately ordered amyloids at pH 4.4, while the aggregation kinetics is dramatically reduced at a lower pH of 2.4. The effect of the pathogenic mutations on aggregation kinetics is also markedly different under the two different acidic conditions. Pathogenic TTR variants (V30M and L55P) aggregate more aggressively than WT TTR at pH 4.4. In contrast, the single-point mutations do not affect the aggregation kinetics at the more acidic condition of pH 2.4. Given that the pathogenic mutations lead to more aggressive forms of TTR amyloidoses, the mildly acidic condition might be more suitable for mechanistic studies of TTR misfolding and aggregation.

Inotersen Treatment for Patients with Hereditary Transthyretin Amyloidosis

BackgroundHereditary transthyretin amyloidosis is caused by pathogenic single-nucleotide variants in the gene encoding transthyretin (TTR) that induce transthyretin misfolding and systemic deposition of amyloid. Progressive amyloid accumulation leads to multiorgan dysfunction and death. Inotersen, a 2′-O-methoxyethyl–modified antisense oligonucleotide, inhibits hepatic production of transthyretin.MethodsWe conducted an international, randomized, double-blind, placebo-controlled, 15-month, phase 3 trial of inotersen in adults with stage 1 (patient is ambulatory) or stage 2 (patient is ambulatory with assistance) hereditary transthyretin amyloidosis with polyneuropathy. Patients were randomly assigned, in a 2:1 ratio, to receive weekly subcutaneous injections of inotersen (300 mg) or placebo. The primary end points were the change in the modified Neuropathy Impairment Score+7 (mNIS+7; range, −22.3 to 346.3, with higher scores indicating poorer function; minimal clinically meaningful change, 2 points) and the change in the score on the patient-reported Norfolk Quality of Life–Diabetic Neuropathy (QOL-DN) questionnaire (range, −4 to 136, with higher scores indicating poorer quality of life). A decrease in scores indicated improvement.ResultsA total of 172 patients (112 in the inotersen group and 60 in the placebo group) received at least one dose of a trial regimen, and 139 (81%) completed the intervention period. Both primary efficacy assessments favored inotersen: the difference in the least-squares mean change from baseline to week 66 between the two groups (inotersen minus placebo) was −19.7 points (95% confidence interval [CI], −26.4 to −13.0; P<0.001) for the mNIS+7 and −11.7 points (95% CI, −18.3 to −5.1; P<0.001) for the Norfolk QOL-DN score. These improvements were independent of disease stage, mutation type, or the presence of cardiomyopathy. There were five deaths in the inotersen group and none in the placebo group. The most frequent serious adverse events in the inotersen group were glomerulonephritis (in 3 patients [3%]) and thrombocytopenia (in 3 patients [3%]), with one death associated with one of the cases of grade 4 thrombocytopenia. Thereafter, all patients received enhanced monitoring.ConclusionsInotersen improved the course of neurologic disease and quality of life in patients with hereditary transthyretin amyloidosis. Thrombocytopenia and glomerulonephritis were managed with enhanced monitoring. (Funded by Ionis Pharmaceuticals; NEURO-TTR ClinicalTrials.gov number, NCT01737398.)

Corrigendum: Substoichiometric inhibition of transthyretin misfolding by immune-targeting sparsely populated misfolding intermediates: a potential diagnostic and therapeutic for TTR amyloidoses.

Scientific Reports 6: Article number: 25080; published online: 28 April 2016; updated: 10 June 2016 The Acknowledgements section in this Article is incomplete: “This study was supported by a grant from the Canadian Institutes of Health Research to A.C and support from Prothena Inc. PW was supported by FAMY, FAMY Norrbotten and Amyl, and Erik, Karin and Gösta Selander’s Foundation.

Substoichiometric inhibition of transthyretin misfolding by immune-targeting sparsely populated misfolding intermediates: a potential diagnostic and therapeutic for TTR amyloidoses.

Wild-type and mutant transthyretin (TTR) can misfold and deposit in the heart, peripheral nerves, and other sites causing amyloid disease. Pharmacological chaperones, Tafamidis® and diflunisal, inhibit TTR misfolding by stabilizing native tetrameric TTR; however, their minimal effective concentration is in the micromolar range. By immune-targeting sparsely populated TTR misfolding intermediates (i.e. monomers), we achieved fibril inhibition at substoichiometric concentrations. We developed an antibody (misTTR) that targets TTR residues 89–97, an epitope buried in the tetramer but exposed in the monomer. Nanomolar misTTR inhibits fibrillogenesis of misfolded TTR under micromolar concentrations. Pan-specific TTR antibodies do not possess such fibril inhibiting properties. We show that selective targeting of misfolding intermediates is an alternative to native state stabilization and requires substoichiometric concentrations. MisTTR or its derivative may have both diagnostic and therapeutic potential.

Structural Changes Associated with Transthyretin Misfolding and Amyloid Formation Revealed by Solution and Solid-State NMR.

Elucidation of structural changes involved in protein misfolding and amyloid formation is crucial for unraveling the molecular basis of amyloid formation. Here we report structural analyses of the amyloidogenic intermediate and amyloid aggregates of transthyretin using solution and solid-state nuclear magnetic resonance (NMR) spectroscopy. Our solution NMR results show that one of the two main β-sheet structures (CBEF β-sheet) is maintained in the aggregation-competent intermediate, while the other DAGH β-sheet is more flexible on millisecond time scales. Magic-angle-spinning solid-state NMR revealed that AB loop regions interacting with strand A in the DAGH β-sheet undergo conformational changes, leading to the destabilized DAGH β-sheet.

A Substructure Combination Strategy To Create Potent and Selective Transthyretin Kinetic Stabilizers That Prevent Amyloidogenesis and Cytotoxicity

Transthyretin aggregation-associated proteotoxicity appears to cause several human amyloid diseases. Rate-limiting tetramer dissociation and monomer misfolding of transthyretin (TTR) occur before its aggregation into cross-beta-sheet amyloid fibrils. Small molecule binding to and preferential stabilization of the tetrameric state of TTR over the dissociative transition state raises the kinetic barrier for dissociation, imposing kinetic stabilization on TTR and preventing aggregation. This is an effective strategy to halt neurodegeneration associated with polyneuropathy, according to recent placebo-controlled clinical trial results. In three recent papers, we systematically ranked possibilities for the three substructures composing a typical TTR kinetic stabilizer, using fibril inhibition potency and plasma TTR binding selectivity data. Herein, we have successfully employed a substructure combination strategy to use these data to develop potent and selective TTR kinetic stabilizers that rescue cells from the cytotoxic effects of TTR amyloidogenesis. Of the 92 stilbene and dihydrostilbene analogues synthesized, nearly all potently inhibit TTR fibril formation. Seventeen of these exhibit a binding stoichiometry of >1.5 of a maximum of 2 to plasma TTR, while displaying minimal binding to the thyroid hormone receptor (<20%). Six analogues were definitively categorized as kinetic stabilizers by evaluating dissociation time-courses. High-resolution TTR.(kinetic stabilizer)(2) crystal structures (1.31-1.70 A) confirmed the anticipated binding orientation of the 3,5-dibromo-4-hydroxyphenyl substructure and revealed a strong preference of the isosteric 3,5-dibromo-4-aminophenyl substructure to bind to the inner thyroxine binding pocket of TTR.

Genistein, a natural product from soy, is a potent inhibitor of transthyretin amyloidosis

The misfolding of transthyretin (TTR), including rate-limiting tetramer dissociation and partial monomer denaturation, is sufficient for TTR misassembly into amyloid and other abnormal quaternary structures associated with three amyloid diseases: senile systemic amyloidosis, familial amyloid polyneuropathy, and familial amyloid cardiomyopathy. Small molecules can bind to one or both of the unoccupied TTR thyroid hormone-binding sites, stabilizing the native tetramer more than the dissociative transition state, thereby raising the kinetic barrier for tetramer dissociation. Herein we demonstrate that genistein, the major isoflavone natural product in soy, works in this fashion and is an excellent inhibitor of transthyretin tetramer dissociation and amyloidogenesis, reducing acid-mediated fibril formation to <10% of that exhibited by TTR alone. Genistein also inhibits the amyloidogenesis of the most common familial amyloid polyneuropathy and familial amyloid cardiomyopathy mutations in TTR: V30M and V122I, respectively. Genistein additionally inhibits tetramer dissociation under physiological conditions thought to lead to slow amyloidogenesis in humans. Furthermore, this natural product exhibits highly selective binding to TTR in plasma over all of the other plasma proteins. Isothermal titration calorimetry shows that genistein binds to TTR with negative cooperativity (K(d1) = 40 nM, K(d2) = 1.4 microM). The benefits of using a nutraceutical such as genistein to treat orphan diseases such as the TTR amyloidoses include known oral bioavailability and safety data. It is conceivable that some patients could benefit from simply increasing their intake of soy products or supplements.

Synthesis and characterization of potent bivalent amyloidosis inhibitors that bind prior to transthyretin tetramerization.

The misfolding of transthyretin (TTR), including rate-limiting tetramer dissociation and partial monomer denaturation, is sufficient for TTR misassembly into amyloid and other abnormal quaternary structures associated with senile systemic amyloidosis, familial amyloid polyneuropathy, and familial amyloid cardiomyopathy. Monovalent small molecules that bind to one or both of the unoccupied thyroid hormone binding sites at the TTR quaternary structure interface stabilize the native state, raising the kinetic barrier for tetramer dissociation sufficiently that the rate of dissociation, and therefore amyloidosis, becomes slow. Bivalent amyloid inhibitors that bind to both binding sites simultaneously are reported herein. The candidate bivalent inhibitors are generally unable to bind to the native TTR tetramer and typically do not engage in monovalent binding owing to a strong inhibitor orientation preference. However, the TTR quaternary structure can assemble around several of the bivalent inhibitors if the inhibitor intercepts the protein before assembly occurs. Some of the wild-type TTR.bivalent inhibitor complexes prepared in this fashion retain a tetrameric structure when subjected to substantial denaturation stresses (8 M urea, 120 h). The best bivalent inhibitor reduced acid-mediated TTR (3.6 microM) amyloid fibril formation to 6% of that exhibited by TTR in the absence of inhibitor, a significant improvement over the approximately 30% observed for the best monovalent inhibitors (3.6 microM, 72 h). The apparent dissociation rate of the best bivalent inhibitor is effectively zero, consistent with the idea that TTR tetramer dissociation and inhibitor dissociation are linked-as a result of the inhibitor-templating tetramer assembly. X-ray cocrystal structures of two of the complexes demonstrate that the bivalent inhibitors simultaneously occupy both sites in TTR, consistent with the 1:1 binding stoichiometry derived from HPLC analysis. The purpose of this study was to demonstrate that bivalent inhibitors could be useful; what resulted are the best inhibitors produced to date. In this context, molecules capable of intercepting TTR during folding and assembly in the lumen of the endoplasmic reticulum would be of obvious interest.