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Transthyretin amyloidosis is a fatal disorder caused by transthyretin amyloid aggregation. Stabilizing the native structure of transthyretin is an effective approach to inhibit amyloid aggregation. To develop kinetic stabilizers of transthyretin, it is crucial to explore compounds that selectively bind to transthyretin in plasma. Our recent findings demonstrated that the uricosuric agent benziodarone selectively binds to transthyretin in plasma. Here, we report the development of benziodarone analogues with enhanced potency for selective binding to transthyretin in plasma compared to benziodarone. These analogues featured substituents of chlorine, bromine, iodine, a methyl group, or a trifluoromethyl group, at the 4-position of the benzofuran ring. X-ray crystal structure analysis revealed that CH···O hydrogen bonds and a halogen bond are important for the binding of the compounds to the thyroxine-binding sites. The bioavailability of benziodarone analogues with 4-Br, 4-Cl, or 4-CH3was comparable to that of tafamidis, a current therapeutic agent for transthyretin amyloidosis.

Misfolding and aggregation of transthyretin are implicated in the fatal systemic disease known as transthyretin amyloidosis. Here, we report the development of a naringenin derivative bearing two chlorine atoms that will be efficacious for preventing aggregation of transthyretin in the eye. The amyloid inhibitory activity of the naringenin derivative was as strong as that of tafamidis, which is the first therapeutic agent targeting transthyretin in the plasma. X-ray crystal structures of the compounds in complex with transthyretin demonstrated that the naringenin derivative with one chlorine bound to the thyroxine-binding site of transthyretin in the forward mode and that the derivative with two chlorines bound to it in the reverse mode. An ex vivo competitive binding assay showed that naringenin derivatives exhibited more potent binding than tafamidis in the plasma. Furthermore, an in vivo pharmacokinetic study demonstrated that the dichlorinated derivative was significantly delivered to the eye.

Intrinsically disordered proteins (IDPs) are ubiquitous proteins that are disordered entirely or partly and play important roles in diverse biological phenomena. Their structure dynamically samples a multitude of conformational states, thus rendering their structural analysis very difficult. Here we explore the potential of high-speed atomic force microscopy (HS-AFM) for characterizing the structure and dynamics of IDPs. Successive HS-AFM images of an IDP molecule can not only identify constantly folded and constantly disordered regions in the molecule, but can also document disorder-to-order transitions. Moreover, the number of amino acids contained in these disordered regions can be roughly estimated, enabling a semiquantitative, realistic description of the dynamic structure of IDPs.

Transthyretin (TTR) is a homotetrameric protein in human plasma. The dissociation of the TTR tetramer and misfolding of the TTR monomer result in the formation of amyloid fibrils. Hereditary TTR amyloidosis is characterized by the extracellular deposition of amyloid fibrils containing TTR variants. The development of small molecules that kinetically stabilize the TTR tetramer is one of the effective strategies for the treatment of hereditary TTR amyloidosis. So far, several stabilizers have been discovered. Tafamidis is the only approved stabilizer for treatment of hereditary TTR amyloidosis, although two nucleic acid medicines that inhibit TTR synthesis, inotersen and patisiran, were recently approved for treatment of this disorder. In this Perspective, we seek to describe the representative kinetic stabilizers from discovery to development, interweaving the crystallographic study of the complex structures.

We have achieved the divergent total synthesis of nine isoflavone natural products 1–9starting from commercially available 2,4,6-trihydroxyacetophenone as a starting material. The isoflavone skeleton of 1–9was constructed by the Suzuki–Miyaura coupling reaction as the key reaction. Investigation of the potential of 1–9as therapeutic agents for transthyretin (TTR) amyloidosis revealed that millexatin F (3) showed the best efficacy in ex vivocompetitive binding experiments and thioflavin-T fluorescence studies. Therefore, millexatin F (3) is promising as a seed compound for a novel TTR amyloidosis therapeutic agent.

The highly sulfated domains of heparan sulfate (HS), alias HS S-domains, are made up of repeated trisulfated disaccharide units [iduronic acid (2S)-glucosamine (NS, 6S)] and are selectively remodeled by extracellular endoglucosamine 6-sulfatases (Sulfs). Although HS S-domains are critical for signal transduction of several growth factors, their roles in amyloidoses are not yet fully understood. Herein, we found HS S-domains in the kidney of a patient with transthyretin amyloidosis. In in vitroassays with cells stably expressing human Sulfs, heparin, a structural analog of HS S-domains, promoted aggregation of transthyretin in an HS S-domain–dependent manner. Interactions of cells with transthyretin fibrils and cytotoxicity of these fibrils also depended on HS S-domains at the cell surface. Furthermore, glypican-5, encoded by the susceptibility gene for nephrotic syndrome GPC5, was found to be accumulated in the transthyretin amyloidosis kidney. Our study, thus, provides a novel insight into the pathologic roles of HS S-domains in amyloidoses, and we propose that enzymatic remodeling of HS chains by Sulfs may offer an effective approach to inhibiting formation and cytotoxicity of amyloid fibrils.

Transthyretin (TTR) is a tetrameric protein found in human serum and associated with amyloid diseases. Because the tetramer dissociation and misfolding of the monomer precede amyloid fibril formation, development of a small molecule that binds to TTR and stabilizes the TTR tetramer is an efficient strategy for the treatment of amyloidosis. Here, we report our discovery of the anti-TTR amyloidogenesis activities of crown ethers. X-ray crystallographic analysis, binding assay, and chemical cross-linking assay showed that 4′-carboxybenzo-18C6 (4) stabilized the TTR tetramer by binding to the allosteric sites on the molecular surface of the TTR tetramer. In addition, 4synergistically increased the stabilization activity of diflunisal, one of the most potent TTR amyloidogenesis inhibitors. These experimental evidences establish that 4is a valuable template compound as an allosteric inhibitor of TTR amyloidogenesis.

T-705 (favipiravir) is a potent inhibitor of RNA-dependent RNA polymerases of influenza viruses and no favipiravir-resistant virus has been isolated. Poliovirus RNA polymerase has been well characterized and isolation of resistant virus was examined in poliovirus.

Hereditary ATTR amyloidosis is a disease caused by the deposition of amyloid fibrils formed by mutated transthyretin (TTR), a protein that binds to thyroid hormone in the serum, in the organs. The development of a small molecule that binds to and stabilizes TTR is a promising strategy for the treatment of ATTR amyloidosis. In the present study, we demonstrated that the resveratrol derivatives including pterostilbene available as a dietary supplement inhibit the fibrillization of V30M-TTR to the same extent as the approved drug tafamidis. Furthermore, based on a thermodynamic and X-ray crystallographic analysis, the binding of the resveratrol derivative to TTR was shown to be enthalpy-driven, with the binding enthalpy being acquired by hydrogen bonding to S117. Moreover, direct observation of hydrogen atoms by neutron crystallography provided details of the hydrogen bond network by S117 and emphasized the importance of the CH···π interaction by L110 in the ligand binding.

Familial amyloid polyneuropathy (FAP) is a genetic, adult-onset, neurodegenerative disorder caused by amyloid formation of transthyretin (TTR), a thyroxine-binding protein. Mutation in TTR causes a propensity of TTR tetramer to dissociate to monomer, which is the first step to amyloidosis. Thus, a drug that can stabilize the tetramer structure will have therapeutic benefit. Here, by virtual screening and biochemical assays, we identified small molecule 6-benzoyl-2-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione (L6) that can prevent the dissociation of TTR to monomer. X-ray crystallography reveals that L6 binds to the T4 binding pocket of TTR. These findings show that L6 is a candidate TTR stabilizer.

ABSTRACTTo confirm the magnitude of an echovirus type 13 (E13) outbreak in 2002 and to evaluate whether genetic and antigenic changes in E13 influenced the occurrence of the outbreak, we measured titers of neutralizing (NT) antibody against the Toyama, 2002-240-SF, and prototype Del Carmen E13 strains among inhabitants of Toyama before and after 2002. The rate of positivity for NT antibodies against both 2002-240-SF and Del Carmen in 2003 made a remarkable upturn in children 0 to 14 years old, compared to that in 2000. Titers of NT antibody against strain 2002-240-SF of inhabitants were slightly higher than those against Del Carmen, whereas anti-E13 rabbit serum raised against either strain Del Carmen or 2002-240-SF showed almost the same titer of NT antibody against both strains. These data indicate that the antigenic properties of the strains may be slightly different. Differences in amino acids between strains 2002-240-SF and Del Carmen in the VP4, VP2, VP3, and VP1 regions may affect both antigenic and receptor binding properties, even though they do not seem to be significant enough to escape widespread immunity. One of the factors of the outbreak was thought to be the increase in susceptibility in the young generation.

Amyloidosis pathologically proceeds via production of amyloidogenic proteins by organs, formation of protein aggregates through structural changes, and their deposition on tissues. A growing body of evidence demonstrates that amyloidosis generally develops through three critical pathological steps: (1) production of amyloid precursor proteins, (2) amyloid formation, and (3) amyloid deposition. However, no clinically effective therapy that is capable of targeting each pathological step of amyloidosis independently is currently available. Here, we combined therapeutic effects and developed a short hairpin RNA expression vector (shRNA) complex with a cyclodextrin-appended cationic dendrimer (CDE) as a novel multitarget therapeutic drug that is capable of simultaneously suppressing these three steps. We evaluated its therapeutic effects on systemic transthyretin (ATTR) amyloidosis and Alzheimer’s disease (AD) as localized amyloidosis, by targeting TTR and amyloid β, respectively. CDE/shRNA exhibited RNAi effects to suppress amyloid protein production and also achieved both inhibition of amyloid formation and disruption of existing amyloid fibrils. The multitarget therapeutic effects of CDE/shRNA were confirmed by evaluating TTR deposition reduction in early- and late-onset human ATTR amyloidosis model rats and amyloid β deposition reduction in AppNL-G-F/NL-G-FAD model mice. Thus, the CDE/shRNA complex exhibits multifunctional therapeutic efficacy and may reveal novel strategies for establishing curative treatments for both systemic and localized amyloidosis.

Hereditary ATTR amyloidosis is caused by the point mutation in serum protein transthyretin (TTR) that destabilizes its tetrameric structure to dissociate into monomer. The monomers form amyloid fibrils, which are deposited in peripheral nerves and organs, resulting in dysfunction. Therefore, a drug that dissolves amyloid after it has formed, termed amyloid disruptor, is needed as a new therapeutic drug. Here, we first established a high throughput screening system to find TTR interactors from the LOPAC1280 compound library. Among the hit compounds, thioflavin T-based post-treatment assay determined lead compounds for TTR amyloid disruptors, NSC95397 and Gossypol, designated as B and R, respectively. Because these compounds have a naphthoquinone structure, we tested 100 naphthoquinone derivatives, and found 10 candidate compounds that disrupted TTR amyloid. Furthermore, to determine whether these 10 compounds are selective for TTR amyloid, we evaluated them against beta-amyloid (Aβ1-42). We found two compounds that were selective for TTR and did not disrupt Aβ-derived amyloid. Therefore, we succeeded in identifying TTR-selective amyloid disruptors, and demonstrated that naphthoquinone compounds are useful structures as amyloid disruptors. These findings contribute to the on-going efforts to discover new therapeutic tools for TTR amyloidosis. (189 words).

GBP, a small insect cytokine isolated from lepidopterans, has a variety of functions. We constructed a series of mutants focusing on the unstructured N-terminal residues of GBP by acetylation, deletion, and elongation in order to investigate the interaction between GBP and its receptor in plasmatocytes. The 1H NMR spectra showed no significant changes in the tertiary structures of these peptides, which indicated that all the mutants maintained their core -sheet structures. The deletion and acetylated mutants, 2-25GBP, Ac2-25GBP, and AcGBP, lost their activity. 2-25GBP was the strongest antagonist, while Ac2-25GBP and AcGBP were moderate. In contrast, the elongated mutants, (-1R)GBP, (- 1A)GBP, and (-2G,-1R)GBP maintained their plasmatocyte-spreading activity. These results demonstrate the importance of the GBP N-terminal charged amine and length of N-terminal GBP-peptide backbone for plasmatocyte-spreading activity. Next, we analyzed other mutant peptides, 1-25(N2A)GBP and 2-25(N2A)GBP, focusing on Asn2. Surprisingly, 2- 25(N2A)GBP had slight plasmatocyte-spreading activity, whereas 2-25GBP lost its activity. Finally, substituted mutant, F3AGBP, had neither plasmatocyte-spreading activity nor antagonistic activity. These results demonstrate the function of each N-terminal residue in the interaction between GBP and its receptor in plasmatocytes.

The structure, stability, and unfolding‐refolding kinetics of a chimeric protein, in which the amino acid sequence of the flexible loop region (residues 105–110) comes from equine lysozyme and the remainder of the sequence comes from bovine α‐lactalbumin were studied by circular dichroism spectroscopy and stopped‐flow measurements, and the results were compared with those of bovine α‐lactalbumin. The substitution of the flexible loop in bovine α‐lactalbumin with the helix D of equine lysozyme destabilizes the molten globule state, although the native state is significantly stabilized by substitution of the flexible loop region. The kinetic refolding and unfolding experiments showed that the chimeric protein refolds significantly faster and unfolds substantially slower than bovine α‐lactalbumin. To characterize the transition state between the molten globule and the native states, we investigated the guanidine hydrochloride concentration dependence of the rate constants of refolding and unfolding. Despite the significant differences in the stabilities of both the molten globule and native states between the chimeric protein and bovine α‐lactalbumin, the free energy level of the transition state is not affected by the amino acid substitution in the flexible loop region. Our results suggest that the destabilization in the molten globule state of the chimeric protein is caused by the disruption of the non‐native interaction in the flexible loop region and that the disruption of the non‐native interaction reduces the free energy barrier of refolding. We conclude that the non‐native interaction in the molten globule state may act as a kinetic trap for the folding of α‐lactalbumin. Proteins 2002;49:95–103. © 2002 Wiley‐Liss, Inc.

The molten globule state of α-lactalbumin has ordered secondary structure in the α-domain, which comprises residues 1 to 34 and 86 to 123. In order to investigate which part of a polypeptide is important for stabilizing the molten globule state of α-lactalbumin, we have produced and studied three chimeric proteins of bovine and human α-lactalbumin. The stability of the molten globule state formed by domain-exchanged α-lactalbumin, in which the amino acid sequence in the α-domain comes from human α-lactalbumin and that in the β-domain comes from bovine α-lactalbumin, is the same as that of human α-lactalbumin and is substantially greater than that of bovine α-lactalbumin. Therefore, our results show that the stability of the molten globule state of α-lactalbumin is determined by the α-domain and the β-domain is not important for stabilizing the molten globule state. The substitution of residues 1 to 34 of bovine α-lactalbumin with those of human α-lactalbumin substantially increases the stability of the molten globule state, while the substitution of residues 86 to 123 of bovine α-lactalbumin with those of human α-lactalbumin decreases the stability of the molten globule state. Therefore, residues 1 to 34 in human α-lactalbumin is more important for the stability of the human α-lactalbumin molten globule state than residues 86 to 123. The stabilization of the molten globule state due to substitution of both residues 1 to 34 and 86 to 123 is not identical with the sum of the two individual substitutions, demonstrating the non-additivity of the stabilization of the molten globule state. This result indicates that there is a long-range interaction between residues 1 to 34 and 86 to 123 in the molten globule state of human α-lactalbumin. The differences in the stabilities of the molten globule states are well correlated with the averaged helical propensity values in the α-domain when the long-range interactions are negligible, suggesting that the local interaction is the dominant term for determining the stability of the molten globule state. Our results also indicate that the apparent cooperativity is closely linked to the stability of the molten globule state, even if the molten globule state is weakly cooperative.

We have investigated the thermal unfolding of bovine α-lactalbumin by means of circular dichroism spectroscopy in the far- and near-ultraviolet regions, and shown that the native α-lactalbumin undergoes heat and cold denaturation. The guanidine hydrochloride–induced unfolding of α-lactalbumin was also investigated by circular dichroism spectroscopy at various temperatures from 261 to 318 K. It is shown that the population of the molten globule state is strongly dependent on temperature and that the molten globule state does not accumulate during the guanidine hydrochloride–induced unfolding transition at 261 K. Our results indicate that the molten globule state of α-lactalbumin undergoes cold denaturation as the native α-lactalbumin does, and that the heat capacity change of unfolding from the molten globule to the unfolded state is positive and significant. The present results further support the idea that the molten globule and the unfolded states do not belong to the same thermodynamic state, and that the native, molten globule and unfolded states are sufficient for interpreting the guanidine hydrochloride–induced unfolding behavior of α-lactalbumin. Proteins 2000;38:407–413. © 2000 Wiley-Liss, Inc.

We have investigated the thermal unfolding of bovine α‐lactalbumin by means of circular dichroism spectroscopy in the far‐ and near‐ultraviolet regions, and shown that the native α‐lactalbumin undergoes heat and cold denaturation. The guanidine hydrochloride–induced unfolding of α‐lactalbumin was also investigated by circular dichroism spectroscopy at various temperatures from 261 to 318 K. It is shown that the population of the molten globule state is strongly dependent on temperature and that the molten globule state does not accumulate during the guanidine hydrochloride–induced unfolding transition at 261 K. Our results indicate that the molten globule state of α‐lactalbumin undergoes cold denaturation as the native α‐lactalbumin does, and that the heat capacity change of unfolding from the molten globule to the unfolded state is positive and significant. The present results further support the idea that the molten globule and the unfolded states do not belong to the same thermodynamic state, and that the native, molten globule and unfolded states are sufficient for interpreting the guanidine hydrochloride–induced unfolding behavior of α‐lactalbumin. Proteins 2000;38:407–413. © 2000 Wiley‐Liss, Inc.

We have investigated the thermal unfolding of bovine α-lactalbumin by means of circular dichroism spectroscopy in the far- and near-ultraviolet regions, and shown that the native α-lactalbumin undergoes heat and cold denaturation. The guanidine hydrochloride–induced unfolding of α-lactalbumin was also investigated by circular dichroism spectroscopy at various temperatures from 261 to 318 K. It is shown that the population of the molten globule state is strongly dependent on temperature and that the molten globule state does not accumulate during the guanidine hydrochloride–induced unfolding transition at 261 K. Our results indicate that the molten globule state of α-lactalbumin undergoes cold denaturation as the native α-lactalbumin does, and that the heat capacity change of unfolding from the molten globule to the unfolded state is positive and significant. The present results further support the idea that the molten globule and the unfolded states do not belong to the same thermodynamic state, and that the native, molten globule and unfolded states are sufficient for interpreting the guanidine hydrochloride–induced unfolding behavior of α-lactalbumin. Proteins 2000;38:407–413. © 2000 Wiley-Liss, Inc.

We have investigated the thermal unfolding of bovine α-lactalbumin by means of circular dichroism spectroscopy in the far- and near-ultraviolet regions, and shown that the native α-lactalbumin undergoes heat and cold denaturation. The guanidine hydrochloride–induced unfolding of α-lactalbumin was also investigated by circular dichroism spectroscopy at various temperatures from 261 to 318 K. It is shown that the population of the molten globule state is strongly dependent on temperature and that the molten globule state does not accumulate during the guanidine hydrochloride–induced unfolding transition at 261 K. Our results indicate that the molten globule state of α-lactalbumin undergoes cold denaturation as the native α-lactalbumin does, and that the heat capacity change of unfolding from the molten globule to the unfolded state is positive and significant. The present results further support the idea that the molten globule and the unfolded states do not belong to the same thermodynamic state, and that the native, molten globule and unfolded states are sufficient for interpreting the guanidine hydrochloride–induced unfolding behavior of α-lactalbumin. Proteins 2000;38:407–413. © 2000 Wiley-Liss, Inc.

The molten globule state of equine lysozyme is more stable than that of α-lactalbumin and is stabilized by non-specific hydrophobic interactions and native-like hydrophobic interactions. We constructed a chimeric protein which is produced by replacing the flexible loop (residues 105–110) in human α-lactalbumin with the helix D (residues 109–114) in equine lysozyme to investigate the possible role of the helix D for the high stability and native-like packing interaction in the molten globule state of equine lysozyme. The stability of the molten globule state formed by the chimeric protein to guanidine hydrochloride-induced unfolding is the same as that of equine lysozyme and is substantially greater than that of human α-lactalbumin, although only six residues come from equine lysozyme. Our results also suggest that the non-native interaction in the molten globule state of α-lactalbumin changes to the native-like packing interaction due to helix substitution. The solvent-accessibility of the Trp residues in the molten globule state of the chimeric protein is similar to that in the molten globule state of equine lysozyme in which packing interaction around the Trp residues in the native state is partially preserved. Therefore, the helix D in equine lysozyme is one of the contributing factors to the high stability and native-like packing interaction in the molten globule state of equine lysozyme. Our results indicate that the native-like packing interaction can stabilize the rudimentary intermediate which is stabilized by the non-specific hydrophobic interactions.

Fourier-transform infrared spectroscopy (FT-IR) was applied to examine relationships between the type of coordination and the COO −antisymmetric and symmetric stretches of the COO −groups in the Ca 2+-binding site of bovine α-lactalbumin. The peaks at 1593, 1578, 1425, and 1403 cm −1were assigned to the COO −groups of Asp-82, 87, and 88 coordinating to Ca 2+in the pseudo bridging mode, according to the results of X-ray crystallography. The bands due to the COO −groups were quite similar to each other between α-lactalbumin and EDTA which is the model compound for the pseudo bridging state.

Densities and sound velocities in dilute aqueous solutions ofN-acetyl-DL-serinamide andN-acetyl-L-threoninamide were measured at 5, 15, 25, 35, and 45°C. Partial molar volumes and partial molar adiabatic compressibilities of these amino acid derivatives at infinite dilution were determined. The partial molar quantities for the parent amino acids, serine and threonine, were also determined and compared with the acetyl amide derivatives. The contribution of the side chain of theN-acetyl amino acid amide or amino acid to the partial molar quantities were estimated from the difference between the partial molar quantities for the solute studied and those for the corresponding species,N-acetyl-glycinamide or glycine, without the side chain.

The equilibrium unfolding and the kinetics of unfolding and refolding of equine lysozyme, a Ca2+-binding protein, were studied by means of circular dichroism spectra in the far and near-ultraviolet regions. The transition curves of the guanidine hydrochloride-induced unfolding measured at 230 nm and 292.5 nm, and for the apo and holo forms of the protein have shown that the unfolding is well represented by a three-state mechanism in which the molten globule state is populated as a stable intermediate. The molten globule state of this protein is more stable and more native-like than that of α-lactalbumin, a homologous protein of equine lysozyme. The kinetic unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by stopped-flow circular dichroism. The observed unfolding and refolding curves both agreed well with a single-exponential function. However, in the kinetic refolding reactions below 3 M guanidine hydrochloride, a burst-phase change in the circular dichroism was present, and the burst-phase intermediate in the kinetic refolding is shown to be identical with the molten globule state observed in the equilibrium unfolding. Under a strongly native condition, virtually all the molecules of equine lysozyme transform the structure from the unfolded state into the molten globule, and the subsequent refolding takes place from the molten globule state. The transition state of folding, which may exist between the molten globule and the native states, was characterized by investigating the guanidine hydrochloride concentration-dependence of the rate constants of refolding and unfolding. More than 80% of the hydrophobic surface of the protein is buried in the transition state, so that it is much closer to the native state than to the molten globule in which only 36% of the surface is buried in the interior of the molecule. It is concluded that all the present results are best explained by a sequential model of protein folding, in which the molten globule state is an obligatory folding intermediate on the pathway of folding.

Fourier‐transform infrared spectroscopy (FT‐IR) was applied to examine relationships between the type of coordination and the COO−antisymmetric and symmetric stretches of the COO−groups in the Ca2+‐binding site of bovine α‐lactalbumin. The peaks at 1593, 1578, 1425, and 1403 cm−1were assigned to the COO−groups of Asp‐82, 87, and 88 coordinating to Ca2+in the pseudo bridging mode, according to the results of X‐ray crystallography. The bands due to the COO−groups were quite similar to each other between α‐lactalbumin and EDTA which is the model compound for the pseudo bridging state.

Transthyretin (TTR) is a causative protein of TTR amyloidosis (ATTR amyloidosis), a general term for diseases characterized by deposition of TTR amyloid fibrils in specific organs. ATTR amyloidosis can be ameliorated by stabilization of the TTR tetramer through the binding of small molecules. Here, we show that the clinical anthelmintic drugs bithionol (42) and triclabendazole (43) potently inhibit aggregation of the amyloidogenic variant V30M-TTR. A competitive binding assay using a fluorescence probe showed that the binding affinity of 42with V30M-TTR was significantly higher than that of the first-in-class drug tafamidis (1), and the binding affinity of 43was similar to that of 1. The crystallographic and thermodynamic analysis revealed that 42efficiently occupied the halogen-binding grooves of TTR, resulting in the favorable binding entropy. Multifaceted in vitrostudies of anthelmintic drugs have the potential to reposition these drugs as ATTR amyloidosis inhibitors.