Small transthyretin (TTR) ligands as possible therapeutic agents in TTR amyloidoses

In transthyretin (TTR) amyloidosis TTR variants deposit as amyloid fibrils giving origin, in most cases, to peripheral polyneuropathy, cardiomyopathy, carpal tunnel syndrome and/or amyloid deposition in the eye. More than eighty TTR variants are known, most of them being pathogenic. The mechanism of TTR fibril formation is still not completely elucidated. However it is widely accepted that the amino acid substitutions in the TTR variants contribute to a destabilizing effect on the TTR tetramer molecule, which in particular conditions dissociate into non native monomeric intermediates that aggregate and polymerize in amyloid fibrils that further elongate. Since this is a multi-step process there is the possibility to impair TTR amyloid fibril formation at different stages of the process namely by tetramer stabilization, inhibition of fibril formation or fibril disruption. Till now the only efficient therapy available is liver transplant when performed in an early phase of the onset of the disease symptoms. Since this is a very invasive therapy alternatives are desirable. In that sense, several compounds have been proposed to impair amyloid formation or disruption. Based on the proposed mechanism for TTR amyloid fibril formation we discuss the action of some of the proposed TTR stabilizers such as derivatives of some NSAIDs (diflunisal, diclofenac, flufenamic acid, and derivatives) and the action of amyloid disrupters such as 4'-iodo-4'-deoxydoxorubicin (I-DOX) and tetracyclines. Among all these compounds, TTR stabilizers seem to be the most interesting since they would impair very early the process of amyloid formation and could also have a prophylactic effect.     

Diflunisal analogues stabilize the native state of transthyretin. Potent inhibition of amyloidogenesis

Analogues of diflunisal, an FDA-approved nonsteroidal antiinflammatory drug (NSAID), were synthesized and evaluated as inhibitors of transthyretin (TTR) aggregation, including amyloid fibril formation. High inhibitory activity was observed for 26 of the compounds. Of those, eight exhibited excellent binding selectivity for TTR in human plasma (binding stoichiometry >0.50, with a theoretical maximum of 2.0 inhibitors bound per TTR tetramer). Biophysical studies reveal that these eight inhibitors dramatically slow tetramer dissociation (the rate-determining step of amyloidogenesis) over a duration of 168 h. This appears to be achieved through ground-state stabilization, which raises the kinetic barrier for tetramer dissociation. Kinetic stabilization of WT TTR by these eight inhibitors is further substantiated by the decreasing rate of amyloid fibril formation as a function of increasing inhibitor concentration (pH 4.4). X-ray cocrystal structures of the TTR.18(2) and TTR.20(2) complexes reveal that 18 and 20 bind in opposite orientations in the TTR binding site. Moving the fluorines from the meta positions in 18 to the ortho positions in 20 reverses the binding orientation, allowing the hydrophilic aromatic ring of 20 to orient in the outer binding pocket where the carboxylate engages in favorable electrostatic interactions with the epsilon-ammonium groups of Lys 15 and 15'. The hydrophilic aryl ring of 18 occupies the inner binding pocket, with the carboxylate positioned to hydrogen bond to the serine 117 and 117' residues. Diflunisal itself appears to occupy both orientations based on the electron density in the TTR.1(2) structure. Structure-activity relationships reveal that para-carboxylate substitution on the hydrophilic ring and dihalogen substitution on the hydrophobic ring afford the most active TTR amyloid inhibitors.     

Methods to evaluate the inhibition of TTR fibrillogenesis induced by small ligands

Transthyretin is an amyloidogenic protein associated with several amyloidosis, namely familial amyloidotic polyneuropathy, familial amyloidotic cardiomyopathy, and central nervous system selective amyloidosis, familial rare diseases caused by single point mutants, and senile systemic amyloidosis associated with wild-type TTR. The current model for amyloid fibril formation involves initial dissociation of the native TTR tetramer into non-native monomers which associate into soluble oligomers and protofibrils that evolve to mature amyloid deposits. A number of efforts are addressed to identify small molecules targeting the formation, clearance, or assembly of toxic aggregates as a promising therapeutic strategy to treat amyloidosis. This review classifies and summarizes the different strategies and assays that have been developed in vitro, ex vivo, and in vivo as tools to screen libraries of compounds or to test compounds from rational design in the search of drug candidates for the treatment of TTR-associated amyloidosis. Depending on the property they measure, the assays are classified as: a) in vitro assays that monitor protein aggregation and/or fibril formation, b) in vitro assays that monitor binding to native protein, c) ex vivo TTR plasma selectivity assays, d) in vitro assays for tetrameric TTR stabilization, e) cellular assays, and f) animal models to evaluate amyloidosis inhibitors.     

Transthyretin deposition in familial amyloidotic polyneuropathy

The subject of the review is on hereditary transthyretin (TTR) amyloidosis which is a genetically transmitted disease that results from a mutation in the gene encoding the plasma TTR protein. TTR is a transport protein for thyroid hormones and vitamin A and is predominantly synthesised in the liver. Although originally regarded as a rare disease, it is now becoming clear that many kindreds exist worldwide. Current knowledge and hypotheses on the biology of TTR, mechanisms of TTR amyloid fibril formation, phenotypic consequences TTR amyloid deposition and pre-clinical models of the disease will be discussed.     

Bisaryloxime ethers as potent inhibitors of transthyretin amyloid fibril formation

Amyloid fibril formation by the plasma protein transthyretin (TTR), requiring rate-limiting tetramer dissociation and monomer misfolding, is implicated in several human diseases. Amyloidogenesis can be inhibited through native state stabilization, mediated by small molecule binding to TTR's primarily unoccupied thyroid hormone binding sites. New native state stabilizers have been discovered herein by the facile condensation of arylaldehydes with aryloxyamines affording a bisarylaldoxime ether library. Of the library's 95 compounds, 31 were active inhibitors of TTR amyloid formation in vitro. The bisaryloxime ethers selectively stabilize the native tetrameric state of TTR over the dissociative transition state under amyloidogenic conditions, leading to an increase in the dissociation activation barrier. Several bisaryloxime ethers bind selectively to TTR in human blood plasma over the plethora of other plasma proteins, a necessary attribute for efficacy in vivo. While bisarylaldoxime ethers are susceptible to degradation by N-O bond cleavage, this process is slowed by their binding to TTR. Furthermore, the degradation rate of many of the bisarylaldoxime ethers is slow relative to the half-life of plasma TTR. The bisaryloxime ether library provides valuable structure-activity relationship insight for the development of structurally analogous inhibitors with superior stability profiles, should that prove necessary.     

Amyloid disease prevention by transthyretin native state complexation with carborane derivatives lacking cyclooxygenase inhibition

Misfolding and subsequent aggregation of any of a number of proteins leads to the accumulation of amyloid fibrils, which have been associated with a variety of diseases. One such amyloidogenic protein is transthyretin (TTR), a 55-kDa homotetrameric protein found in the blood plasma and cerebrospinal fluid where it binds and transports thyroxine. In humans, the T119M-TTR variant has been shown to be protective against familial amyloid polyneuropathy, a TTR amyloid disease, through kinetic stabilization of the unliganded tetrameric structure. Studies have indicated that a diverse range of small molecules may also bind TTR in the thyroxine-binding pocket and subsequently kinetically stabilize the protein's native conformation in vitro, preventing the misfolding that has been implicated in the progression of several diseases. However, cyclooxygenase inhibition is a common unwanted side effect among such small-molecule kinetic stabilizers. The recent development of transthyretin stabilizers not subject to cyclooxygenase inhibition may prove attractive for the long-term treatment of TTR misfolding diseases in humans. Such compounds are attained by incorporating aromatic carborane icosahedra at strategic points in their structures.     

Tafamidis for transthyretin amyloidosis

Tafamidis meglumine (Vyndaqel?, Pfizer) is a novel, first-in-class drug for the treatment of transthyretin familial amyloid polyneuropathy (TTR-FAP), a rare neurodegenerative disorder characterized by progressive sensory, motor and autonomic impairment that is ultimately fatal. Pathogenic mutations in the transthyretin (TTR) protein lead to destabilization of its tetrameric structure and subsequent formation of amyloid aggregates. Tafamidis is a small-molecule inhibitor that binds selectively to TTR in human plasma and kinetically stabilizes the tetrameric structure of both wild-type TTR and a number of different mutants. Clinical trials indicate that tafamidis slows disease progression in patients with TTR-FAP and reduces the burden of disease, demonstrating improvement in small and large nerve fiber function, modified body mass index and lower extremity neurological examination. Tafamidis has been granted marketing authorization by the European Commission for the treatment of TTR-FAP and the U.S. Food and Drug Administration is currently reviewing this drug for the same indication.     

治疗转甲状腺素蛋白介导的淀粉样变性心肌病新药：氯苯唑酸

转甲状腺素蛋白(TTR)介导的淀粉样变性心肌病主要是由野生或突变型TTR淀粉样纤维错误折叠在心肌沉积所致。氯苯唑酸对TTR的甲状腺素结合部位具有较高的亲和力,可选择性与其结合并抑制TTR降解。临床试验表明,氯苯唑酸可使90%以上患者的TTR稳定,而且与安慰剂比较,氯苯唑酸可明显降低患者的全因死亡率及心血管疾病相关住院率,患者心脏功能和生活质量也显著改善。氯苯唑酸无严重不良事件,患者耐受性好。     

Advances in the treatment of transthyretin cardiac amyloidosis: Current and emerging therapies

Transthyretin cardiac amyloidosis (ATTR-CA) has been recognized as an underdiagnosed and undertreated cause of heart failure with often unrecognized multiorgan involvement. Guideline development and the establishment of nonbiopsy criteria for diagnosis of ATTR-CA have led to an increased rate of diagnosis and hence patients referred for therapies. ATTR is a protein misfolding disorder where the TTR tetramer disassociates into monomers which form insoluble amyloid depositions in organs, including the heart. ATTR-CA can be due to autosomal dominant transmitted gene mutation or due to misfolding of wild-type TTR. Prior to 2019, there were no FDA-approved pharmacological treatments for ATTR-CA. Understanding of ATTR-CA pathogenesis has enabled development of targeted strategies with novel disease-modifying therapies. Current and emerging therapies for ATTR-CA include (1) TTR gene silencing (siRNA, ASO, CRISPR/Cas9), (2) TTR tetramer stabilization, and (3) TTR amyloid fibril degradation. This review focuses on the pathophysiology of ATTR-CA, diagnostic criteria, and addresses current and emerging treatments for this diverse disorder.     

Perspectives for drug intervention in amyloid diseases

Amyloid fibres are stable, persistent and highly ordered aggregates of mis-folded protein that accumulate in tissues and are a prominent feature of the pathology of a wide range of human diseases. The presumed role of amyloid as a causative factor of tissue damage is based largely on 'guilt by association'. However, growing understanding of the nature of amyloid, its formation by a nucleated growth mechanism from destabilised and partially unfolded precursors and its persistence at sites of deposition has provided the foundation for the development of approaches to inhibit amyloid formation and enable its clearance. In spite of intensive study, our understanding of the detailed structure of amyloid itself remains incomplete although 'crossed-beta' structure is clearly a common constituent. On the other hand detailed structural understanding of transthyretin, beta-secretase and serum amyloid P component is contributing to the design of small molecule compounds to target amyloid. Thyroxin mimetics stabilise the native tetrameric protein structure, beta-secretase inhibitors will limit the production of the amyloidogenic Abeta1-42 polypeptide. Compounds that crosslink serum amyloid P component rapidly deplete the plasma and amyloid-bound pool of this protein. The efficacy of these compounds as drugs to prevent formation or enable removal of amyloid will provide a stringent test of the 'amyloid hypothesis' of disease.     

Neurodegeneration in familial amyloidotic polyneuropathy

Familial amyloid polyneuropathies (FAP) constitute a group of inherited amyloidoses that affect peripheral nerves. One common form of FAP is caused by transthyretin (TTR) misfolding and deposition in the peripheral nervous system, leading to neuronal toxicity and death. The molecular mechanisms responsible for this toxicity are unclear; however, there is good biochemical and histopathological evidence that the toxicity of TTR mutations is correlated to their aggregation state. In addition, neuronal calcium dysregulation is a mechanism that has been suggested to drive the pathogenesis of FAP. Amyloidogenic TTR mutations cause significant calcium influx via L-type calcium channels in neuronal cell lines, while in primary sensory neurons, TTR mediates a calcium influx via a novel mechanism of transient receptor potential melanostatin (TRPM8) and voltage-gated sodium and calcium channel activation. Significantly, calcium dysregulation is a pathological hallmark of other neurodegenerative diseases involving amyloidosis, for example Alzheimer's disease, and this mechanism could explain the molecular events that drive amyloid toxicity in other neurodegenerative diseases.     

Effects of transthyretin on thyroxine and β‐amyloid removal from cerebrospinal fluid in mice

Transthyretin (TTR) is a binding protein for the thyroid hormone thyroxine (T₄), retinol and β‐amyloid peptide. TTR aids the transfer of T₄ from the blood to the cerebrospinal fluid (CSF), but also prevents T₄ loss from the blood‐CSF barrier. It is, however, unclear whether TTR affects the clearance of β‐amyloid from the CSF. This study aimed to investigate roles of TTR in β‐amyloid and T₄ efflux from the CSF. Eight‐week‐old 129sv male mice were anaesthetized and their lateral ventricles were cannulated. Mice were infused with artificial CSF containing ¹²⁵I‐T₄/³H‐mannitol, or ¹²⁵I‐Aβ40/³H‐inulin, in the presence or absence of TTR. Mice were decapitated at 2, 4, 8, 16, 24 minutes after injection. The whole brain was then removed and divided into different regions. The radioactivities in the brain were determined by liquid scintillation counting. At baseline, the net uptake of ¹²⁵I‐T₄ into the brain was significantly higher than that of ¹²⁵I‐Aβ40, and the half time for efflux was shorter (¹²⁵I‐T₄, 5.16; ³H‐mannitol, 7.44; ¹²⁵I‐Aβ40, 8.34; ³H‐inulin, 10.78 minutes). The presence of TTR increased the half time for efflux of ¹²⁵I‐T₄ efflux, and caused a noticeable increase in the uptake of ¹²⁵I‐T₄ and ¹²⁵I‐Aβ40 in the choroid plexus, whilst uptakes of ³H‐mannitol and ³H‐inulin remained similar to control experiments. This study indicates that thyroxine and amyloid peptide effuse from the CSF using different transporters. TTR binds to thyroxine and amyloid peptide to prevent the loss of thyroxine from the brain and redistribute amyloid peptide to the choroid plexus.     

TTR fibril formation inhibitors: is there a SAR?

Transthyretin is a homotetrameric protein that carries thyroxine and retinol binding protein in plasma and is associated with a variety of amyloid diseases. One approach to the potential treatment of TTR amyloidosis is the stabilization of the native tetramer, over the dissociative transition state, through the binding of small molecules; this increases the kinetic barrier for tetramer dissociation and prevents protein misfolding. Several molecules discovered through focused screening, or created utilizing the structure-based design, were studied to identify the structural features that could make up for a good candidate drug. In this review, we examine several different chemical classes of TTR fibril formation inhibitors, highlighting the structural modifications that have led to an improvement or to a decrease of their potency and/or selectivity.     

Competitive interactions of chlorinated phenol compounds with 3,3',5-triiodothyronine binding to transthyretin: detection of possible thyroid-disrupting chemicals in environmental waste water

Chlorinated phenol compounds, such as the chlorinated derivatives of bisphenol A, have been detected in effluents from paper manufacturing plants. We investigated the effects of bisphenol A, nonylphenol, and their seven chlorinated derivatives on 3,3',5-[(125)I]triiodothyronine ([(125)I]T(3)) binding to purified chicken and bullfrog transthyretin (cTTR and bTTR) and to the ligand-binding domains of chicken and bullfrog thyroid hormone receptor beta (cTR LBD and bTR LBD). The concentrations at which the chlorinated derivatives displaced [(125)I]T(3) from TTR were 10-10(3) times less than those of their parent molecules. 2,6-Dichloro-4-nonylphenol and 3,3',5-trichlorobisphenol A were the most potent competitors of T(3) binding to cTTR and to bTTR, respectively. The interactions of the chlorinated derivatives with the cTR and the bTR LBDs were weaker than those of the chlorinated derivatives with cTTR and bTTR. Chlorinated derivatives with a greater degree of chlorination were more efficient competitors of T(3) binding to TTR and TR. A structure-activity relationship between the phenol compounds and TTR (TTR assay) and TR (TR assay) was established. Structures with chlorine in either ortho position or both ortho positions, with respect to the hydroxy group, were more efficient competitors. Chemicals that interacted with bTTR, cTTR, and Japanese quail TTR were detected in water downstream of effluents from paper manufacturing plants using the TTR assay. As some of the chlorinated bisphenols and nonylphenols were potent competitors of T(3) binding to TTRs, the TTR assay could be applied as primary screening for possible thyroid-disrupting chemicals in environmental waste water.     

Diagnosis and therapeutic approaches to transthyretin amyloidosis

Hereditary amyloidogenic transthyretin (TTR) (ATTR) amyloidosis is an autosomal dominant form of fatal hereditary amyloidosis. Owing to progress in biochemical and molecular genetic analyses, this disease is now believed to occur worldwide. As of today, reports of about 120 different points of single or double mutations, or a deletion in the TTR gene have been reported, and several different phenotypes of ATTR amyloidosis have been documented. In addition, since liver transplantation has been established to halt the progression of hereditary ATTR amyloidosis in the early stage, rapid and reliable diagnostic system for ATTR amyloidosis is needed. On the other hand, senile systemic amyloidosis (SSA) derived from wild-type (WT) TTR affects primarily in the heart and lungs and occasionally in carpal ligaments in the elderly. To perform accurate diagnosis and effective treatments, we should distinguish between hereditary ATTR amyloidosis and SSA by means of genetic and proteomic analyses. The liver transplantation for hereditary ATTR amyloidosis has become a well-established treatment, because the main source of serum variant TTR is shut out. However, this treatment has several problems, such as expensive medical costs, lifelong administration of immunosuppressants, non-indication for the mutated-TTR gene carriers without clinical symptoms, shortage of liver donors, and further development of cardiac and ocular disorders. Therefore, we and other ATTR amyloidosis research groups have been investigating the possibility of stabilization of variant TTR, gene therapy, and immunotherapy for ATTR amyloidosis on the basis of TTR amyloid formation mechanism. We present here the current diagnostic procedure and therapeutic approaches for the disease.     

Binding of a metabolite of 3,4,3',4'-tetrachlorobiphenyl to transthyretin reduces serum vitamin A transport by inhibiting the formation of the protein complex carrying both retinol and thyroxin

The mechanism of serum vitamin A reduction by polychlorinated biphenyls was studied at the level of the plasma transport protein system for vitamin A. Analysis of [3H]retinol-labeled serum proteins by polyacrylamide gel electrophoresis (PAGE) showed association of retinol with two proteins that were identified as retinol binding protein (RBP) and the RBP complex with transthyretin (TTR). The amount of [3H]retinol radioactivity in the serum as well as the label associated with the binding proteins was strongly reduced by 3,4,3',4'-tetrachlorobiphenyl (TCB). A possible interaction of TCB with the retinol binding proteins was investigated, using radiolabeled TCB. Analysis of the plasma proteins by PAGE revealed the presence of four peaks of 3H-TCB label, the major ones being associated with lipoproteins and TTR. No 3H-TCB radioactivity was found in the region of the gel where RBP or the RBP-TTR complex was located. HPLC analysis of the radioactive compound associated with TTR showed the presence of a metabolite of TCB, rather than the parent compound. These data indicate a direct interaction of a metabolite of TCB with TTR leading to an inhibition of formation of the serum transport protein complex carrying both retinol and thyroxin. A model is proposed, which may explain certain characteristic toxicopathological lesions observed in species exposed to polychlorinated biphenyls and related compounds (TCDD, PBBs, etc.).     

Synthesis and in vitro characterization of some benzothiazole- and benzofuranone-derivatives for quantification of fibrillar aggregates and inhibition of amyloid-mediated peroxidase activity

Neurodegenerative diseases are characterized by amyloid deposition. Thioflavin T (ThT) is one of the molecules considered for detection of amyloid deposits; however, its lipophilicity is too low to cross the blood–brain barrier. Therefore, there is a strong motivation to develop suitable compounds for in vitro fibril quantification as well as for in vivo amyloid imaging. Moreover, oxidative stress (particularly, uncontrolled peroxidase activity) has frequently been reported to play a critical role in the onset/progression of some neurodegenerative disorders. In this study, we describe the synthesis of some benzothiazole and benzofuranone compounds and examine their peroxidase inhibitory properties. Furthermore, to establish the potential binding of synthesized compounds to amyloid aggregates, their in vitro binding to some non-disease related amyloidogenic proteins were characterized. Analyses of the in vitro binding studies indicated that compounds 2 and 4 bind to the amyloid structures successfully while compounds 1 and 3 showed a low affinity in binding to fibrils. Furthermore, compounds 3 and 4 were observed to inhibit amyloid-mediated peroxidase activity in a reversible un-competitive manner.     

Surface plasmon resonance analysis for the screening of anti-prion compounds

The interaction of anti-prion compounds and amyloid binding dyes with a carboxy-terminal domain of prion protein (PrP121-231) was examined using surface plasmon resonance (SPR) and compared with inhibition activities of abnormal PrP formation in scrapie-infected cells. Most examined compounds had affinities for PrP121-231: antimalarials had low affinities, whereas Congo red, phthalocyanine and thioflavin S had high affinities. The SPR binding response correlated with the inhibition activity of abnormal PrP formation. Several drugs were screened using SPR to verify the findings: propranolol was identified as a new anti-prion compound. This fact indicates that drug screenings by this assay are useful.