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.     

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.     

Biosensor discovery of thyroxine transport disrupting chemicals

Ubiquitous chemicals may interfere with the thyroid system that is essential in the development and physiology of vertebrates. We applied a surface plasmon resonance (SPR) biosensor-based screening method for the fast screening of chemicals with thyroxine (T4) transport disrupting activity. Two inhibition assays using the main thyroid hormone transport proteins, T4 binding globulin (TBG) and transthyretin (TTR), in combination with a T4-coated biosensor chip were optimized and automated for screening chemical libraries. The transport protein-based biosensor assays were rapid, high throughput and bioeffect-related. A library of 62 chemicals including the natural hormones, polychlorinated biphenyls (PCBs), polybrominated diphenylethers (PBDEs) and metabolites, halogenated bisphenol A (BPA), halogenated phenols, pharmaceuticals, pesticides and other potential environmentally relevant chemicals was tested with the two assays. We discovered ten new active compounds with moderate to high affinity for TBG with the TBG assay. Strikingly, the most potent binding was observed with hydroxylated metabolites of the brominated diphenyl ethers (BDEs) BDE 47, BDE 49 and BDE 99, that are commonly found in human plasma. The TTR assay confirmed the activity of previously identified hydroxylated metabolites of PCBs and PBDEs, halogenated BPA and genistein. These results show that the hydroxylated metabolites of the ubiquitous PBDEs not only target the T4 transport at the TTR level, but also, and to a great extent, at the TBG level where most of the T4 in humans is circulating. The optimized SPR biosensor-based transport protein assay is a suitable method for high throughput screening of large libraries for potential thyroid hormone disrupting compounds.     

Biochemical and structural evaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogenesis inhibitors

To develop potent transthyretin (TTR) amyloidogenesis inhibitors that also display high binding selectivity in blood, it proves useful to systematically optimize each of the three substructural elements that comprise a typical inhibitor: the two aryl rings and the linker joining them. In the first study, described herein, structural modifications to one aryl ring were evaluated by screening a library of 2-arylbenzoxazoles bearing thyroid hormone-like aryl substituents on the 2-aryl ring. Several potent and highly selective amyloidogenesis inhibitors were identified that exhibit minimal thyroid hormone nuclear receptor and COX-1 binding. High resolution crystal structures (1.3-1.5 A) of three inhibitors (2f, 4f, and 4d) in complex with TTR were obtained to characterize their binding orientation. Collectively, the results demonstrate that thyroid hormone-like substitution patterns on one aryl ring lead to potent and highly selective TTR amyloidogenesis inhibitors that lack undesirable thyroid hormone receptor or COX-1 binding.     

The degradation of salbutamol in ethanolic solutions

The degradation pathways of salbutamol in ethanolic solutions have been investigated and three potential ethyl ether degradation products have been identified. Two have been confirmed as salbutamol ethyl ethers and the third as a diethyl ether. All three degradation products have been structurally elucidated by LC–MS–MS (TOF and tandem quadrupole). The two ethyl ethers have a molecular weight of 267 Da (28 units higher than salbutamol) and are structural isomers (molecules with the same molecular weight but different structural arrangements). The molecular weight of the two ethyl ethers is consistent with the addition of one ethyl group to the salbutamol molecule and elimination of one water molecule. The molecular weight of the diethyl ether is 295 Da (56 units higher than salbutamol) and is consistent with the addition of two ethyl groups to the salbutamol molecule and elimination of two water molecules. A plausible degradation mechanism for the formation of the salbutamol ethyl ethers is the acid-catalysed dehydration of alcohols. Acidic pH is required to drive the degradation of salbutamol in ethanolic solution. Higher degradation levels of salbutamol ethyl ethers are achieved in acidic pH solutions. Levels of the two salbutamol ethyl ethers reach a maximum at an ethanol concentration of around 20%. Levels of the diethyl ether increase linearly with ethanol concentration, until it becomes the major degradation product in high concentration ethanolic solutions (≥30%).     

Toward optimization of the linker substructure common to transthyretin amyloidogenesis inhibitors using biochemical and structural studies

To develop potent and highly selective transthyretin (TTR) amyloidogenesis inhibitors, it is useful to systematically optimize the three substructural elements that compose a typical TTR kinetic stabilizer: the two aryl rings and the linker joining them. Herein, we evaluated 40 bisaryl molecules based on 10 unique linker substructures to determine how these linkages influence inhibitor potency and selectivity. These linkers connect one unsubstituted aromatic ring to either a 3,5-X 2 or a 3,5-X 2-4-OH phenyl substructure (X = Br or CH 3). Coconsideration of amyloid inhibition and ex vivo plasma TTR binding selectivity data reveal that direct connection of the two aryls or linkage through nonpolar E-olefin or -CH 2CH 2- substructures generates the most potent and selective TTR amyloidogenesis inhibitors exhibiting minimal undesirable binding to the thyroid hormone nuclear receptor or the COX-1 enzyme. Five high-resolution TTR.inhibitor crystal structures (1.4-1.8 A) provide insight into why such linkers afford inhibitors with greater potency and selectivity.     

Synthesis, structure, and activity of diclofenac analogues as transthyretin amyloid fibril formation inhibitors.

Twelve analogues of diclofenac (1), a nonsteroidal antiinflammatory drug and known inhibitor of transthyretin (TTR) amyloid formation, were prepared and evaluated as TTR amyloid formation inhibitors. High activity was exhibited by five of the compounds. Structure-activity relationships reveal that a carboxylic acid is required for activity, but changes in its position as well as the positions of other substituents are tolerated. High-resolution X-ray crystal structures of four of the active compounds bound to TTR were obtained. These demonstrate the significant flexibility with which TTR can accommodate ligands within its two binding sites.     

Transthyretin as a target of alkylation and a potential biomarker for sulfur mustard poisoning: Electrophoretic and mass spectrometric identification and characterization

For the verification of exposure to the banned blister agent sulfur mustard (SM) and the better understanding of its pathophysiology, protein adducts formed with endogenous proteins represent an important field of toxicological research. SM and its analogue 2-chloroethyl ethyl sulfide (CEES) are well known to alkylate nucleophilic amino acid side chains, for example, free-thiol groups of cysteine residues. The specific two-dimensional thiol difference gel electrophoresis (2D-thiol-DIGE) technique making use of maleimide dyes allows the staining of free cysteine residues in proteins. As a consequence of alkylation by, for example, SM or CEES, this staining intensity is reduced. 2D-thiol-DIGE analysis of human plasma incubated with CEES and subsequent matrix-assisted laser desorption/ionization time-of-flight (tandem) mass-spectrometry, MALDI-TOF MS(/MS), revealed transthyretin (TTR) as a target of alkylating agents. TTR was extracted from SM-treated plasma by immunomagnetic separation (IMS) and analyzed after tryptic cleavage by microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS). It was found that the Cys¹⁰-residue of TTR present in the hexapeptide C(-HETE)PLMVK was alkylated by the hydroxyethylthioethyl (HETE)-moiety, which is characteristic for SM exposure. It was shown that alkylated TTR is stable in plasma in vitro at 37°C for at least 14 days. In addition, C(-HETE)PLMVK can be selectively detected, is stable in the autosampler over 24 h, and shows linearity in a broad concentration range from 15.63 μM to 2 mM SM in plasma in vitro. Accordingly, TTR might represent a complementary protein marker molecule for the verification of SM exposure.     

Human receptor kinetics, tissue binding affinity, and stability of mometasone furoate

Mometasone furoate (MF) is a topically used glucocorticoid with high anti-inflammatory potency. In contrast to the wealth of data derived from clinical studies, information about the molecular pharmacology of the compound is lacking or contradictory. Thus, we elucidated the characteristics of receptor binding kinetics and receptor affinity in a bioassay. Metabolite formation was determined in human plasma and lung tissue as well as binding affinity to human lung tissue. Fast and extensive association of MF to the human glucocorticoid receptor was observed while the dissociation of the MF-receptor complex was faster compared to fluticasone propionate (FP). The relative receptor affinity of MF was calculated as 2200 (dexamethasone = 100, FP = 1800) and confirmed in a bioassay measuring the induction of the glucocorticoid regulated protein CD163 in human monocytes. In plasma and human lung tissue MF formed a 9,11-epoxy degradation product. The binding affinity of MF to human lung tissue was low compared to FP due to fast redistribution from tissue into plasma. These molecular pharmacological properties are in accordance with clinical data.     

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.     

Natural product-based phenols as novel probes for mycobacterial and fungal carbonic anhydrases

In order to discover novel probes that may help in the investigation and control of infectious diseases through a new mechanism of action, we have evaluated a library of phenol-based natural products (NPs) for enzyme inhibition against four recently characterized pathogen β-family carbonic anhydrases (CAs). These include CAs from Mycobacterium tuberculosis, Candida albicans, and Cryptococcus neoformans as well as α-family human CA I and CA II for comparison. Many of the NPs selectively inhibited the mycobacterial and fungal β-CAs, with the two best performing compounds displaying submicromolar inhibition with a preference for fungal over human CA inhibition of more than 2 orders of magnitude. These compounds provide the first example of non-sulfonamide inhibitors that display β over α CA enzyme selectivity. Structural characterization of the library compounds in complex with human CA II revealed a novel binding mode whereby a methyl ester interacts via a water molecule with the active site zinc.     

Studies on the toxicity of chlorinated p-nitrobiphenyl ether: I—Methemoglobin formation in vitro and in vivo induced by nitroso and amino derivatives of chlorinated biphenyl ether

Methemoglobin formation by nitroso and amino derivatives of chlorinated biphenyl ether was investigated. All the nitroso derivatives used induced methemoglubin formation in suspensions of human erythrocytes. Ability to induce methemoglobin formation among the nitroso derivatives was the highest for nitrosobenzene, followed by p-nitrosobiphenyl ether, 4-chloro p-nitrosobiphenyl ether, 2,4-dichloro p-nitrosobiphenyl ether and then 2,4,6-trichloro p-nitrosobiphenyl ether. The monochloro p-nitrosobiphenyl ethers showed all the same activity. Therefore, the ability to induced methemoglobin formation depended on the number of chlorines rather than on the position of chlorine substitution on the phenoxy group in the molecule. Since lactate decreased but glucose and xylitol increased the methemoglobin level produced by the nitroso compounds, this reaction would appear to require an NADPH-dependent enzyme system in erythrocytes. All of the amino derivatives used induced methemoglobin formation in suspensions of human erythrocytes with rat liver homogenates. Ability to induced methemoglobin formation among the amino derivatives was the highest for p-aminobiphenyl ether, followed by 4-chloro p-aminobiphenyl ether, 2,4,-dichloro p-aminobiphenyl ether, 2,4,6-trichloro p-aminobiphenyl ether, and then aniline. Methemoglobin formation bychlorinated p-aminobiphenyl ether was examined by intraperitoneal and oral administration to rats. After i.p. injection, methemoglobinemia was induced by p-aminobiphenyl ether, aniline, and 2,4-dichloro p-aminobiphenyl ether but not by 2,4,6-trichloro p-aminobiphenyl ether over the period tested. The order of methemoglobin formation was p-aminobiphenyl ether 4-chloro p-aminobiphenyl ether > aniline 2,4-dichlorop-aminobiphenyl ether. Oral administration gave almost the same result as i.p. injection. The maximum methemoglobin levels produced by the amino derivatives were highest for 4-chloro p-aminobiphenyl ether, followed by p-aminobiphenyl ether, 2,4-dichloro p-aminobiphenyl ether, aniline, and then 2,4,6-trichloro p-aminobiphenyl ether. However, the methemoglobin level produced by 2,4,6-trichloro p-aminobiphenyl ether was very low and not significantly greater than the background level.     

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.).     

Effect of glycol ethers on plasma osmolality.

1. Glycol ethers and their alkoxyacetic acid metabolites produce a linear increase in plasma osmolality with increasing plasma concentration. 2. This change in osmolality may be too small to be clinically useful at concentrations expected in cases of acute human glycol ether poisoning.     

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.     

Pharmacokinetic analysis of antiplatelet effect of sarpogrelate hydrochloride and its application to drug dosage regimen--modeling based on reversible inhibition of 5-HT2 serotonergic receptor in the platelet membrane by sarpogrelate hydrochloride and its metabolite

Sarpogrelate hydrochloride is an antiplatelet drug, and expected to be useful in the treatment of chronic arterial occlusive diseases. Sarpogrelate and its active metabolite (M-1) are potent inhibitors of human platelet aggregation, and selectively inhibit 5-HT2-serotonergic receptors on human platelets. However, the plasma concentrations of these inhibitors do not correlate to the inhibitory effect on platelet aggregation after administration. Sarpogrelate disappears from the plasma more rapidly in comparison to the duration of its pharmacological effect, and the plasma concentration of M-1 is very low (< 1/10 of sarpogrelate). In this paper, we describe a pharmacokinetic-pharmacodynamic model for ascertaining the antiplatelet effects of sarpogrelate and M-1, by considering both the competitive reversible inhibition and the association/dissociation process of these drugs at the 5-HT2 receptors on platelets (Most data used for analysis were obtained from the literatures, except for the serum protein binding rate of M-1). The developed model was well fitted to the actual data, and suggested that M-1 was more effective for the inhibition of platelet aggregation than sarpogrelate. On the basis of these findings, a new method was developed for predicting inhibitory effects on platelet aggregation after oral administration of sarpogrelate hydrochloride. This method is useful for planning a rational dosage regimen of sarpogrelate hydrochloride and predicting the duration of antiplatelet activity after the discontinuance of the drug.     

High-affinity binding of [3H]6-nitroquipazine to 5-hydroxytryptamine transporter in human platelets

The characteristics of the binding [3H]6-nitroquipazine, a very potent and selective inhibitor of 5-hydroxytryptamine (5-HT; serotonin) uptake, to human platelet membranes were studied at a physiological temperature of 37 degrees C. The presence of a single saturable high-affinity binding component for [3H]6-nitroquipazine was demonstrated Non-specific binding was estimated in the presence of 1 microM paroxetine. Scatchard analysis revealed an apparent equilibrium dissociation constant (Kd) of 0.450 +/- 0.04 nM and a maximal number of binding sites (Bmax) of 2508 +/- 360 fmol/mg protein (mean +/- S.D., n = 4). The kinetically derived dissociation constant (Kd) was 0.431 nM. [3H]6-Nitroquipazine binding was inhibited selectively by 5-HT uptake inhibitors, and the potency of various drugs to inhibit [3H]6-nitroquipazine binding closely correlated with their inhibitory effects on [3H]5-HT uptake into synaptosome. Moreover, Ki values for drug inhibition of [3H]6-nitroquipazine binding to human platelet membranes were significantly correlated with the corresponding Ki values for inhibition of [3H]paroxetine binding at 37 degrees C. The present results suggest that the binding sites for [3H]6-nitroquipazine are associated with the 5-HT transporter in human platelets.