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| StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=''
| | '''HUMAN TRANSTHYRETIN IN COMPLEX WITH DIBENZOFURAN-4,6-DICARBOXYLIC ACID''' |
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| | == Human TTR == |
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| == TTR transport functions == | | === Functions === |
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| Human transthyretin (TTR) ([[4tlt]]) is a highly conserved homotetrameric transport protein. Identified in 1942, it was originally called prealbumin as it runs faster than albumin ([[1bm0]]) during SDS-PAGE <ref> Seibert FB, Nelson JW. Electrophoretic study of the blood protein response in tuberculosis. J Biol Chem 1942; 143: 29–38. </ref>. After discovering its binding and transport ability to thyroid hormones, it was given the name of “thyroxine-binding prealbumin” (TBPA). Finally, its actual name refers to an additional carrier function: '''trans'''ports '''thyr'''oxine (T4) and '''ret'''inol (vitamin A).
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| It is mainly present in the plasma and synthetized by the liver, but also in the cerebrospinal fluid produced by the choroid plexus of the brain, and in retinal pigment epithelium.
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| The TTR gene is located on chromosome 18 <ref> Wallace MR, Naylor SL, Kluve-Beckerman B, Long GL, McDonald L, Shows TB, Benson MD, Localization of the human prealbumin gene to chromosome 18 [archive], Biochem Biophys Res Commun, 1985;129:753–758</ref>.
| | Identified on 1942, Human transthyretin (TTR) ([[1dvq]]) is a transport protein encoded by the TTR gene, located on chromosome 18 <ref> Wallace MR, Naylor SL, Kluve-Beckerman B, Long GL, McDonald L, Shows TB, Benson MD, Localization of the human prealbumin gene to chromosome 18, Biochem Biophys Res Commun, 1985;129:753–758 doi:https://doi.org/10.1016/0006-291X(85)91956-4 </ref>. It is mainly present in the plasma and synthetized by the liver, but also in the cerebrospinal fluid produced by the choroid plexus of the brain, and in retinal pigment epithelium. This protein was originally called prealbumin as it runs faster than albumin ([[1bm0]]) during SDS-PAGE <ref> Seibert FB, Nelson JW. Electrophoretic study of the blood protein response in tuberculosis. J Biol Chem 1942; 143: 29–38. </ref>. After discovering its binding and transport ability to thyroxine (T4), it was given the name of “thyroxine-binding prealbumin” (TBPA). Indeed, its role is crucial as it allows essential thyroid hormones, implied in cell differenciation, growth and metabolic regulation<ref> Wikipedia contributors. "Thyroid hormones." Wikipedia, The Free Encyclopedia. [https://en.wikipedia.org/wiki/Thyroid_hormones] (accessed on Jan. 16 2020)</ref>, to pass through the bloodstream. Finally, its actual name refers to an additional carrier function: '''trans'''ports '''thyr'''oxine and '''retin'''ol (one form of vitamin A). Recently, it has been found that TTR also has a role in proteolysis, and in the nervous system, implicated axonal growth, neurogenesis, and nerve regeneration<ref name= "Vieira">Vieira M, Saraiva MJ, Transthyretin: a multifaceted protein, Biomol Concepts, 2014;5:45–54 doi: https://doi.org/10.1515/bmc-2013-0038</ref>. |
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| | <Structure load='1dvq' size='350' frame='true' align='right' caption='Crystal Structure of human transthyretin (TTR) from homo sapiens gene in Escherichia coli, resolution 2Å (PDB entry : [[1dvq]])' scene='3D structure of human transthyretin' /> |
| | === Structure === |
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| == Human TTR structure with natural ligand : T4 and retinol ==
| | Human TTR is a 54 kDa homo-tetramer, described as a dimer of dimer, rich in β-sheet. It is composed of 127 amino acids assembled around the central channel of the protein, resulting in a 222 symmetry protein. This tetramer contains a channel divided into two symmetry-related L-T4-binding sites. |
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| === Human TTR ===
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| Human TTR is a 54 kDa homo-tetramer, described as a dimer of dimer, rich in β-sheet. It is composed of 127 amino acids assembled around the central channel of the protein, resulting in a 222 symmetry protein. This tetramer contains a channel divided into two symmetry-related L-T4-binding sites. The channel has three sets of small depressions, termed halogen binding pockets (HBPs). They have a two-fold symmetry and confer a hydrophobic surface to the protein. But then, when the side chain of the TTR changes of conformation, these pockets can realise more hydrogen bonds with other molecules, they can be donor or acceptor. Thus, they are involved in the binding of the natural ligand, the thyroxine (T4).
| | The channel has three sets of small hydrophobic depressions, termed '''halogen binding pockets (HBPs)'''. But then, when the side chain of the TTR changes of conformation, these pockets can realise more hydrogen bonds with other molecules, they can be donor or acceptor. Thus, they had this name due to their ability to bind the iodines of thyroxine (T4) its natural ligand <ref name="Labaudinière">Labaudinière R. Chapter 9 Discovery and Development of Tafamidis for the Treatment of TTR Familial Amyloid Polyneuropathy. Orphan Drugs and Rare Diseases. Aug 2014 202-229. doi:https://doi.org/10.1039/9781782624202-00202</ref>. |
| | At the entry of the binding site, the TTR has a hydrophilic tail, into which the four iodine atoms of the ligand are placed. The dimer interface of the TTR is divided in two part, the inner and the outer binding cavity. The outermost pockets <scene name='82/829358/Hbp1/3'>HBP1</scene> and <scene name='82/829358/Hbp1prim/3'>HBP1'</scene> are located between the side chains of <scene name='82/829358/Hbp1aa/1'>Ala 108, Thr 106, Met 13 and Lys 15</scene> <ref name= "Klabunde">PMID: 10742177</ref>. The central <scene name='82/829358/Hbp2/2'>HBP2</scene> and <scene name='82/829358/Hbp2prime/1'>HBP2'</scene> are formed by the side chains of <scene name='82/829358/Hbp2aa/1'>Leu 110, Ala 109, Lys 15, and Leu 17</scene>, it is primarily hydrophobic with polar or electrostatic contributions from the carbonyl groups of Lys 15, Ala 108 and Ala 109. The innermost binding pockets, <scene name='82/829358/Hbp3/3'>HBP3</scene> and <scene name='82/829358/Hbp3prim/2'>HBP3'</scene>, are located between the side chains of <scene name='82/829358/Hbp3aa/2'>Ser 117, Thr 119, Ala 108 and Leu 110</scene>. Their surfaces are composed of aliphatic methyl and methylene groups, as well as the Ser 117 hydroxyl group, the carbonyl groups of Ser 117, Thr 118 and Ala 108, and the main chain NH groups of Thr 119, Ala 109 and Leu 110. |
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| | Each monomer is composed of an <scene name='82/829358/Helix/1'>α-helix</scene> and <scene name='82/829358/Sheet/1'>two four stranded β-sheets</scene>, which results in two eight-stranded β-sheets per dimer <ref name="seb">Sebastião, M. P., Lamzin, V., Saraiva, M. J., & Damas, A. M. (2001). Transthyretin stability as a key factor in amyloidogenesis: X-ray analysis at atomic resolution. Journal of Molecular Biology, 306(4), 733–744. doi:http://dx.doi.org/10.1006/jmbi.2000.4415 </ref>. There is a large solvent channel which passes between the two sheets in which two molecules of T4 can bind. Monomers associate via the formation of an eight-stranded anti-parallel β-sheet to which each monomer contributes four β-strands. These β-sheets are situated at the center of the tetramer and positioned back to back. <scene name='82/829358/Interacab/1'>Ile107</scene> and <scene name='82/829358/Interacab/1'>Val122</scene> of monomer A are in direct van der Waals contact with the phenol ring of <scene name='82/829358/Interacab/1'>Phe87</scene> from monomer B. |
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| Each monomer is composed of an α-helix and two four stranded β-sheets, which results in two eight-stranded β-sheets per dimer <ref name="seb">Sebastião, M. P., Lamzin, V., Saraiva, M. J., & Damas, A. M. (2001). Transthyretin stability as a key factor in amyloidogenesis: X-ray analysis at atomic resolution. Journal of Molecular Biology, 306(4), 733–744. doi:10.1006/jmbi.2000.4415 </ref>. There is a large solvent channel which passes between the two sheets in which two molecules of T4 can bind. Monomers associate via the formation of an eight-stranded anti-parallel β-sheet to which each monomer contributes four β-strands. These β-sheets are situated at the center of the tetramer and positioned back to back. Ile107 and Val122 of monomer A are in direct van der Waals contact with the phenol ring of Phe87 from monomer B. * And Phe64 is in van der Waals contact with Cys10 via Pro11 in monomer A.** Each monomer contain a single cysteine (Cys10), which is usually bound to various sulfhydryls or sulfite from plasma. S-oxidation of Cys10 to cysteic acid has a stabilizing effect on the monomer. It may be derived from hydrogen bonds between the sulfonic oxygens of Cys10–SO3 – and Gly57 N, His56 NE and Arg104 NH1 <ref name= "Altland"> Altland, K., Benson, M. D., Costello, C. E., Ferlini, A., Hazenberg, B. P. C., Hund, E., … Winter, P. (2007). Genetic microheterogeneity of human transthyretin detected by IEF. ELECTROPHORESIS, 28(12), 2053–2064. doi:10.1002/elps.200600840</ref>.
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| Monomers of TTR will be called A, B, C, D. And as we consider the TTR as a dimer of dimer, we have A and B the upper part of the protein and C and D the lower part. The contact between upper and lower dimers is made via β-sheet contacts, creating hydrogen bonds between main-chain atoms. In contrast to the monomer associations, contacts between the upper and lower parts are much less important so that the dimer assembly unit of TTR is best defined as the monomers which are joined by β-strand hydrogen bonding. Two funnel-shaped hormone binding sites are located at the dimer–dimer region.
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| At the entry of the binding site, the TTR has a hydrophilic tail, into which the four iodine atoms of the ligand are placed. The innermost binding pocket, HBP-3, is located between the side chains of Ser 117, Thr 119, Ala 108 and Leu 110. Its surface is composed of aliphatic methyl and methylene groups, as well as the Ser 117 hydroxyl group, the carbonyl groups of Ser 117, Thr 118 and Ala 108, and the main chain NH groups of Thr 119, Ala 109 and Leu 110. The central HBP-2 is formed by the side chains of Leu 110, Ala 109, Lys 15, and Leu 17, it is primarily hydrophobic with polar or electrostatic contributions from the carbonyl groups of Lys 15, Ala 108 and Ala 109. The outermost pocket HBP-1 is located between the side chains of Ala 108, Thr 106, Met 13 and Lys 15. This pocket is lined with the methyl and methylene groups of Lys 15, Ala 108 and Thr 106 <ref name= "Klabunde">PMID: 10742177</ref>.
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| === TTR-ligand complex ===
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| The TTR – ligand interaction provides kinetic stabilization the protein. The more the affinity is high, the more the ligand stabilizes the complex. The dissociation constants with T4 and retinol-binding protein (RBP) are respectively from 1,1.10-7 to 1,5.10-7 M <ref name = "Monaco"> Monaco, H., Rizzi, M., & Coda, A. (1995). Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein. Science, 268(5213), 1039–1041. doi:10.1126/science.7754382</ref>.
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| ==== TTR-T4 complex ====
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| The crystal structure of this complex is orthorhombic <ref name="wo">Wojtczak, A., Cody, V., Luft, J. R., & Pangborn, W. (1996). Structures of Human Transthyretin Complexed with Thyroxine at 2.0 Å Resolution and 3’,5’-Dinitro-N-acetyl-L-thyronine at 2.2 Å Resolution. Acta Crystallographica Section D Biological Crystallography, 52(4), 758–765. doi:10.1107/s0907444996003046 </ref>.
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| Two hormone binding sites are located at the dimer–dimer region bind T4 with negative cooperativity. Under physiological conditions, the bound between the natural ligand and the tetramer can’t be broken down. Moreover, there is only one hormone bound per tetramer. The negative cooperativity mechanism explain the fact that the affinity constants (Ka) for the binding of the first and the second T4 changes, they are respectively 108 and 106 M-1 <ref name= "Klabunde">PMID: 10742177</ref>.
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| For the TTR-T4 complex, the HBP play a key role. The HBP interact with the four iodine groups of the thyroxine. HBPs bind the iodine of the ligands in two different ways: 3 2’ 1 1’ or 3’ 2 1 1’ with prime indicating the HBP symmetry <ref name= "Klabunde">PMID: 10742177</ref>. PICTURE
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| A significant contribution of T4 binding to TTR comes from charged groups near the periphery of the binding site. Glu 54 and Lys15 are located near the HBP-1 pocket allowing potential electrostatic interactions with the ligands <ref name= "Klabunde">PMID: 10742177</ref>.
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| P1, 2 and 3 represent the three HBPs.
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| Thyroxine is bound deep in the cleft of the channel surface between the side chains of residues Leu17, Alal8 and Leull0, with interactions of its phenolic ring with Ser117 and Thr119 in the P2 pocket which has a more nucleophilic character than the P1 pocket, and with its alanyl moiety interacting with Glu54 and Lysl5 near the channel entrance.
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| T 4 interactions with TTR side chains shows that it can make good hydrogen-bonding contacts with Lysl5 and Glu54.
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| The P3 pocket forms close contacts between iodine and Leu110 backbone N atom, while the shortest contacts for 3'-I are formed with the carbonyl of Alal09. These contacts for the low-occupancy model of the hormone are shifted toward the tetramer center and toward the carbonyl and hydroxyl of Ser117, as well as Alal09.
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| ==== TTR-RBP complex ====
| | [[Image:2.png|thumb|left]] |
| TTR is a specific carrier of retinol-binding protein (RBP). RBPs have a molecular mass of 21 kDa. They are composed of an eight-stranded β-barrel and a C-terminal α-helix.
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| One tetramer of TTR can bind two molecules of RBP in vitro (1:2 stoichiometry). However, when we isolate the TTR-RBP complex from the plasma (in vivo) we find a 1:1 stoichiometry <ref name= "Naylor"> "Naylor, H. M., & Newcomer, M. E. (1999). The Structure of Human Retinol-Binding Protein (RBP) with Its Carrier Protein Transthyretin Reveals an Interaction with the Carboxy Terminus of RBP†,‡. Biochemistry, 38(9), 2647–2653. doi:10.1021/bi982291i"</ref> . The β-barrel entrance loop involved in A-B strands binding (amino acids from 31 to 38, hairpin1) is also implicated in the TTR-RBP interaction.
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| RBP-TTR complex stale at high ionic strength and dissociate at low ionic strength <ref name ="Zanetti">Zanotti, G., Ottonello, S., Berni, R., & Monaco, H. L. (1993). Crystal Structure of the Trigonal Form of Human Plasma Retinol-binding Protein at 2·5 Å Resolution. Journal of Molecular Biology, 230(2), 613–624. doi:10.1006/jmbi.1993.1173 </ref>. It is explained by the presence of a hydrophobic surface in the contact region, represented by hairpin 1,2,3 (include Leu35, 63, 64 and 67). Trp67 (close to hairpin1) seems to be involved in the binding<ref name="Zanetti"/>. The dissociation constant of this complex is around 0.4 µM <ref name= "Naylor"/>.
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| [[Image:images.png|thumb|left|alt=Puzzle globe|Caption for the image|Human RBP : Human TTR structure in "opposite dimer" model|200px]]
| | And <scene name='82/829358/Aabis/1'>Phe64</scene> is in van der Waals contact with <scene name='82/829358/Aabis/1'>Cys10</scene> via <scene name='82/829358/Aabis/1'>Pro11</scene> in monomer A. Each monomer contain a single cysteine (<scene name='82/829358/Cys/1'>Cys10</scene>), which is usually bound to various sulfhydryls or sulfite from plasma. S-oxidation of Cys10 to cysteic acid has a stabilizing effect on the monomer. It may be derived from hydrogen bonds between the sulfonic oxygens of Cys10–SO3 – and Gly57 N, His56 NE and Arg104 NH1 <ref name= "Altland"> Altland, K., Benson, M. D., Costello, C. E., Ferlini, A., Hazenberg, B. P. C., Hund, E., … Winter, P. (2007). Genetic microheterogeneity of human transthyretin detected by IEF. ELECTROPHORESIS, 28(12), 2053–2064. doi: http://dx.doi.org/10.1002/elps.200600840</ref>. |
| RBP has two polypeptide chains, E and F, which are bound to “opposite dimers” of TTR, the main TTR-RBP contact region is made between TTR-D / RBP-E and TTR-B / RBP-F. Thus, we observe an asymmetry in RBP-TTR relationship: RBP-E has more extensive interactions with TTR than molecule RBP-F<ref name= "Naylor"/>.
| | Monomers of TTR will be called A, B, AA, BA. And as we consider the TTR as a dimer of dimer, we have A and AA the upper part of the protein and B and BA the lower part. The contact between upper and lower dimers is made via β-sheet contacts, creating hydrogen bonds between main-chain atoms. In contrast to the monomer associations, contacts between the upper and lower parts are much less important so that the dimer assembly unit of TTR is best defined as the monomers which are joined by β-strand hydrogen bonding. Two funnel-shaped hormone binding sites are located at the dimer–dimer region. |
| The specific protein – protein recognition of this complex is classified in the three-dimensional docking model. The recognition site between RBP and TTR implies the positioning of the open end on the RBP β-barrel. For each interface 21 amino acids are involved from both proteins. At the periphery of the site there is charged amino acids. Half of the amino acid side chains are hydrophobic or aromatic in this area. Then, at the center of the site, we find hydrophobic amino acids, Leu and Ile are the predominant amino acids. RBPs present two different complementary surfaces to a dimeric surface. Indeed, at the core of the RBP-E interface, we find Ile84 from TTR-A and TTR-D, as well as Val20 and Ala81. Moreover, Trp67, Phe96, and Leu63 and 97 from RBP are surrounded by Val20, Leu82, and Ile84 from TTR-A and D.
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| All mammalian RBP’s have a carboxy-terminal extension of eight amino acids<ref name= "Naylor"/>. This region interacts with TTR and the carboxy-extension of RBP-E is deeply located in the RBP-TTR interface. Indeed, the two terminal Leu182 and Leu183 are embedded in a hydrophobic region which includes Leu82 from TTR-A monomer and Val69 of RBP-E. Moreover, the terminal carboxylate group of RBP-E extension adopts a position to be neutralized by Arg21 of TTR-A. This interaction allows to bury more than 40% of the surface area, compared to the area buried without the carboxy terminal group of RBP-E<ref name= "Naylor"/>.
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| == Disease ==
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| ===Type of disease===
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| The most known defect related to TTR is the formation of amyloid fibrils, which can engender several diseases such as familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), and senile systemic amyloidosis (SSA) also called wild-type transthyretin amyloid (WTTA or ATTR)<ref> Faria TQ, Almeida ZL, Cruz PF, Jesus CS, Castanheira P, Brito RM. A look into amyloid formation by transthyretin: aggregation pathway and a novel kinetic model. Phys Chem Chem Phys. 2015 Mar 4;17(11):7255-63. doi: 10.1039/c4cp04549a. PMID:25694367 doi:http://dx.doi.org/10.1039/c4cp04549a </ref>. Another type of disease possibly engendered due to TTR amyloid fibrils is the central nervous system selective amyloidosis (CNSA) including familial oculoleptomeningeal amyloidosis characterized by an eye injury, or meningocerebrovascular amyloidosis if the eye is not affected. <ref> ARTICLE Human brain amyloidoses</ref>
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| ===TTR amyloid fibril===
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| Inappropriate TTR foldings cause amyloidosis. Indeed, aggregates formation can be explained by a destabilization of the TTR’s native conformation, namely the tetramer dissociation into an alternative folded monomeric intermediate. The final result is a protein self-assembly. A particular beta-pleated-sheet structure characterizes the proteins with amyloidogenic potential. <ref name="Klabunde" />
| | === TTR with natural ligands === |
| TTR aggregation into amyloid fibrils leads to insolubility. Consequently, it creates abnormal deposits in the peripheral nerves in the case of FAP, in the central nerves for CNSA, and in heart tissues for FAC and SSA. Therefore, the insoluble proteins alter the corresponding organ and tissue functions, and are unable to be subjected to a proper degradation by cell metabolism.
| | The TTR – ligand interaction provides kinetic stabilization the protein. The more the affinity is high, the more the ligand stabilizes the complex. The dissociation constants with T4 and retinol-binding protein (RBP) are respectively from 1,1.10-7 to 1,5.10-7 M <ref name = "Monaco"> Monaco, H., Rizzi, M., & Coda, A. (1995). Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein. Science, 268(5213), 1039–1041. doi: http://dx.doi.org/10.1126/science.7754382</ref>. |
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| In most of the cases, autosomal dominant mutations of the TTR gene are at the origin of the Human familial amyloidosis (FAP, FAC, CNSA) through TTR conformational disorder. Val30Met is the most recensed amyloidogenic point mutation observed ([[4tl4]]). However, SSA differentiates from these TTR-related hereditary amyloidosis by usually affecting patients in advanced age, as it involves an aggregate formation due to a progressive accumulation of wild-type TTR proteins mainly associated to misshaping and beta-strand lacking <ref> Pinney JH, Whelan CJ, Petrie A, Dungu J, Banypersad SM, Sattianayagam P, Wechalekar A, Gibbs SD, Venner CP, Wassef N, McCarthy CA, Gilbertson JA, Rowczenio D, Hawkins PN, Gillmore JD, Lachmann HJ (April 2013). "Senile systemic amyloidosis: clinical features at presentation and outcome". Journal of the American Heart Association. 2 (2): e000098. PMC 3647259. PMID 23608605 doi: http://dx.doi.org/10.1161/JAHA.113.000098 </ref><ref> Amyloid fibril composition and transthyretin gene structure in senile systemic amyloidosis.
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| Gustavsson A1, Jahr H, Tobiassen R, Jacobson DR, Sletten K, Westermark P., of Pathology I, Linköping University, Sweden</ref>
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| | [[Image:t4.png|thumb|left|alt=Puzzle globe|Caption for the image|Structure of thyroxine (T4)|200px]] |
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| ==Drug development==
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| === First drugs developed: Non-steroidal anti-inflammatory drugs=== | | Thyroxine (T4) is the main precursor of T3 hormone (triiodothyronine). They both control physiological processes like urinary water absorption and also cell metabolism. T4 is produced by the thyroid gland. Its concentration determination is used to diagnose thyroid diseases <ref name= "t4">Eshar, D., Nau, M. R., & Pohlman, L. M. (2017). PLASMA THYROXINE (T4) CONCENTRATION IN ZOO-KEPT BLACK-TAILED PRAIRIE DOGS (CYNOMYS LUDOVICIANUS). Journal of Zoo and Wildlife Medicine, 48(1), 116–120. doi: http://dx.doi.org/10.1638/2016-0073.1 </ref>. |
| | Two hormone binding sites are located at the dimer–dimer region bind T4 with negative cooperativity. Under physiological conditions, the bound between the natural ligand and the tetramer can’t be broken down. Moreover, there is only one hormone bound per tetramer. The negative cooperativity mechanism |
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| [[Image: Drugstructure.png | thumb | left | alt=Puzzle globe| Structures of thyroxine (T4), the natural ligand of TTR and other designed TTR fibril formation inhibitors| 200 px]]
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| Drug research is based on the inhibition of amyloidogenic TTR by stabilization of native tetrameric conformation, using binding ligands to prevent TTR dissociation.
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| The fibril formation inhibitors studied are ligands that resemble to the natural ligand T4 but more efficient in binding TTR, leading to a decrease of the amyloidogenic potential. The first potent amyloid inhibitors developed were non-steroidal anti-inflammatory drugs (NSAID), such as flufenamic acid ([[1bm7]]), diclofenac ([[1dvx]]), flurbiprofen ([[1dvt]]), indomethacin, diflunisal, meclofenamic acid, mefenamic acid, or fenoprofen.
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| However, regardless of a noticeable decrease of the TTR’s amyloidogenic potential <ref name="Klabunde" />, prolonged NSAIDs administration could provoke renal failure, cardiac side effects, and gastrointestinal ulcers. <ref>Bally, M; Dendukuri, N; Rich, B; Nadeau, L; Helin-Salmivaara, A; Garbe, E; Brophy, JM (9 May 2017). "Risk of acute myocardial infarction with NSAIDs in real world use: bayesian meta-analysis of individual patient data". BMJ (Clinical Research Ed.). 357: j1909. PMC 5423546. PMID 28487435 doi: http://dx.doi.org/10.1136/bmj.j1909</ref> Gastric toxicity is linked to NSAID’s binding to a cyclooxygenase isoform, resulting in an inhibition of the activity of COX-1 and/or COX-2 associated to prostaglandin’s negative regulation. <ref name="Klabunde" />
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| | [[Image:1ggl.jpg|thumb|left|alt=Puzzle globe|Caption for the image|Structure of retinol binding protein (RBP)|200px]] |
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| | TTR is a specific carrier of RBP ([[1ggl]]). RBP are specific carriers circulating in complex with TTR in the plasma. It transports vitamin A (retinol), mainly synthesized in the liver, to its final tissue, the carrier is needed fo retinol secretion<ref name= "Naylor"/>. Also, they are markers for acute undernutrition. For instance, it is associated to insulin-resistance for type 2 diabetes. RBP concentration in the serum allows to diagnose several diseases. |
| | RBPs have a molecular mass of 21 kDa. They are composed of an eight-stranded β-barrel and a C-terminal α-helix. |
| | One tetramer of TTR can bind two molecules of RBP in vitro (1:2 stoichiometry). However, when we isolate the TTR-RBP complex from the plasma (in vivo) we find a 1:1 stoichiometry <ref name= "Naylor"> "Naylor, H. M., & Newcomer, M. E. (1999). The Structure of Human Retinol-Binding Protein (RBP) with Its Carrier Protein Transthyretin Reveals an Interaction with the Carboxy Terminus of RBP†,‡. Biochemistry, 38(9), 2647–2653. doi:http://dx.doi.org/10.1021/bi982291i"</ref> . |
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| === Dibenzofuran-4,6-dicarboxylic acid ===
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| <Structure load='1dvu' size='340' frame='true' align='right' caption='Crystal Structure of human transthyretin in complex with dibenzofuran-4,6-dicarboxylic acid from homo sapiens gene in Escherichia coli [[resolution 2.05Å]] (PDB entry : [[1dvu]]) ' scene='Insert optional scene name here' />
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| | == Disease == |
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| | Abnormal TTR levels are found in neuropathologies such as Guillain-Barré syndrome (GBS), frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), amd Parkinson’s disease (PD)<ref name= "Vieira" />. |
| | Additionally, there is another defect related to TTR, which is more probable and known: the formation of amyloid fibrils. It can engender several diseases such as familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), and senile systemic amyloidosis (SSA) also called wild-type transthyretin amyloid (WTTA or ATTR)<ref> Faria TQ, Almeida ZL, Cruz PF, Jesus CS, Castanheira P, Brito RM. A look into amyloid formation by transthyretin: aggregation pathway and a novel kinetic model. Phys Chem Chem Phys. 2015 Mar 4;17(11):7255-63. PMID 25694367 doi:http://dx.doi.org/10.1039/c4cp04549a </ref>. Another type of disease possibly engendered due to TTR amyloid fibrils is the central nervous system selective amyloidosis (CNSA) including familial oculoleptomeningeal amyloidosis characterized by an eye injury, or meningocerebrovascular amyloidosis if the eye is not affected. <ref> P.Gambetti, C. Russo. Human brain amyloidoses. Neuphrol Dial Transplant. 1998; 13 [Suppl 7] : 33-40</ref> |
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| | Inappropriate TTR foldings cause amyloidosis. Indeed, aggregates formation can be explained by a destabilization of the TTR’s native conformation, namely the tetramer dissociation into an alternative folded monomeric intermediate. The final result is a protein self-assembly. A particular beta-pleated-sheet structure characterizes the proteins with amyloidogenic potential. <ref name="Klabunde" /> |
| | TTR aggregation into amyloid fibrils leads to insolubility. Consequently, it creates abnormal deposits in the peripheral nerves in the case of FAP, in the central nerves for CNSA, and in heart tissues for FAC and SSA. Therefore, the insoluble proteins alter the corresponding organ and tissue functions, and are unable to be subjected to a proper degradation by cell metabolism. |
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| | In most of the cases, autosomal dominant mutations of the TTR gene are at the origin of the Human familial amyloidosis (FAP, FAC, CNSA) through TTR conformational disorder. Val30Met is the most recensed amyloidogenic point mutation observed ([[4tl4]]). However, SSA differentiates from these TTR-related hereditary amyloidosis by usually affecting patients in advanced age, as it involves an aggregate formation due to a progressive accumulation of wild-type TTR proteins mainly associated to misshaping and beta-strand lacking <ref> Pinney JH, Whelan CJ, Petrie A, Dungu J, Banypersad SM, Sattianayagam P, Wechalekar A, Gibbs SD, Venner CP, Wassef N, McCarthy CA, Gilbertson JA, Rowczenio D, Hawkins PN, Gillmore JD, Lachmann HJ (April 2013). "Senile systemic amyloidosis: clinical features at presentation and outcome". Journal of the American Heart Association. 2 (2): e000098. PMC 3647259. PMID 23608605 doi:http://dx.doi.org/10.1161/JAHA.113.000098 </ref><ref> Gustavsson A. Jahr H, Tobiassen R, Jacobson DR, Sletten K, Westermark P. Amyloid fibril composition and transthyretin gene structure in senile systemic amyloidosis. 1995 Nov; 73(5):703-8 </ref> |
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| The dimer interface of the TTR is divided in two part, the inner and the outer binding cavity. The channel forming by dimerization has three symmetric binding pockets on each dimer parts. These pockets are called Halogen-binding pocket (HBPs) due to their ability to bind the iodines of thyroxine <ref name="Labaudinière">Labaudinière R. Chapter 9 Discovery and Development of Tafamidis for the Treatment of TTR Familial Amyloid Polyneuropathy. Orphan Drugs and Rare Diseases. Aug 2014 202-229. DOI:https://doi.org/10.1039/9781782624202-00202</ref>. In the outer cavity are positioned the <scene name='82/829358/Hbp1/2'>HBP1</scene> and <scene name='82/829358/Hbp1prim/2'>HBP1'</scene> pockets, in the inner cavity are placed the <scene name='82/829358/Hbp3/2'>HBP3</scene> and <scene name='82/829358/Hbp3prim/1'>HBP3’</scene> pockets, and the <scene name='82/829358/Hbp2/1'>HBP2</scene> and <scene name='82/829358/Hbp2prim/1'>HBP2’</scene> pockets are at the interface between the inner and outer cavity
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| DDBF is bounded according two symmetric equivalent modes <ref name="Petrassi">PMID:15869287</ref>. Indeed, DDBF wears a tricyclic ring system, with 2 hydrogen bond donors and 5 hydrogen bond acceptors, allowing to bound the dimer-dimer interface of the TTR cavity <ref name="National Center for Biotechnology Information">[https://pubchem.ncbi.nlm.nih.gov/compound/Dibenzofuran-4_6-dicarboxylic-acid Link text], PubChem Database. CID:3022(accessed on Dec. 26, 2019).</ref><ref name="Klabunde">PMID:10742177</ref>. Thanks to the complementarity of shape and hydrophobicity, DDBF enters nicely the outer portion of HBPs pockets <ref name="Petrassi "/>. Besides, the tricyclic ring system interacts with <scene name='83/832920/Lys15_leu17_and_ala108/1'>Lys15, Leu17 and Ala108</scene> from two adjacent TTR subunits <ref name="Petrassi"/>. Additionally, carboxylates at the position 4 and 6 of DDBF make electrostatic interactions at the entrance of <scene name='82/829358/Hbp1/2'>HBP1</scene> and <scene name='82/829358/Hbp1prim/2'>HBP1'</scene> with <scene name='83/832920/Lys15/3'>Lys15</scene> on the ε-NH3+ groups <ref name="Petrassi"/>.
| | ==Drug development== |
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| | === Non-steroidal anti-inflammatory drugs=== |
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| TTR in complex with dibenzofuran-4,6-dicarboxylic acid keeps the general apo-structure, with water molecules bind to <scene name='82/829358/Hbp3/2'>HBP3</scene> and <scene name='82/829358/Hbp3prim/1'>HBP3’</scene> cavities of TTR <ref name="Klabunde">PMID:10742177</ref>. There is not conformational change of <scene name='83/832920/Ser117/1'>Ser117</scene> and <scene name='83/832920/Thr119/1'>Thr119</scene> of TTR, contrary to other inhibitor, such as FLU.
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| Consequently, DDBF creates a bridge between two adjacent subunits stabilized by ionic and hydrophobic interactions.
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| | [[Image:nat_lig.jpg|thumb|left|alt=Puzzle globe|Caption for the image|Structures of thyroxine (T4), the natural ligand of TTR and other designed TTR fibril formation inhibitors|200px]] |
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| | Drug research is based on the inhibition of amyloidogenic TTR by stabilization of native tetrameric conformation, using binding ligands to prevent TTR dissociation. |
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| | The fibril formation inhibitors studied are ligands that resemble to the natural ligand T4 but more efficient in binding TTR, leading to a decrease of the amyloidogenic potential. The first potent amyloid inhibitors developed were non-steroidal anti-inflammatory drugs (NSAID), such as flufenamic acid ([[1bm7]]), resveratrol ([[1dvs]]), diclofenac ([[1dvx]]), flurbiprofen ([[1dvt]]), indomethacin, diflunisal, meclofenamic acid, mefenamic acid, or fenoprofen. |
| | However, regardless of a noticeable decrease of the TTR’s amyloidogenic potential <ref name="Klabunde" />, prolonged NSAIDs administration could provoke renal failure, cardiac side effects, and gastrointestinal ulcers. <ref>Bally, M; Dendukuri, N; Rich, B; Nadeau, L; Helin-Salmivaara, A; Garbe, E; Brophy, JM (9 May 2017). "Risk of acute myocardial infarction with NSAIDs in real world use: bayesian meta-analysis of individual patient data". BMJ (Clinical Research Ed.). 357: j1909. PMC 5423546. PMID 28487435 doi: http://dx.doi.org/10.1136/bmj.j1909</ref> Gastric toxicity is linked to NSAID’s binding to a [[cyclooxygenase]] isoform, resulting in an inhibition of the activity of COX-1 and/or COX-2 associated to prostaglandin’s negative regulation. <ref name="Klabunde" /> |
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| ====Improvements====
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| <Structure load='1dvy' size='340' frame='true' align='left' caption='Crystal Structure of human transthyretin in complex with N-(M-trifluoromethylphenyl) phenoxazine-4,6-dicarboxylic acid from homo sapiens gene in Escherichia coli [[resolution 1.09Å]] (PDB entry : [[1dvy]]) ' scene='Insert optional scene name here' />
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| To exploit the TTR inner cavity, DDBR can be ameliorate <ref name="Klabunde">PMID:10742177</ref>. An additional substituent like an aryl ring could be link at DDBR thought a heteroatom or directly via a covalent bond <ref name="Petrassi"/>.
| | === Dibenzofuran-4,6-dicarboxylic acid === |
| For example, a N-phenyl phenoxazine-4,6-dicarboxylate, called Phenox (PDB entry : [[1dvy]]), allows to create additional bonds, which increase the kinetic stabilization of TTR. Besides, Van der Waals interactions are established with Thr106, Lys15, Leu17 from to adjacent TTR subunits. Moreover, the carboxylate groups link not only Lys15 but also Glu54 (carboxylate groups must to be protonated at physiological pH). In the side chain Leu17, Leu110 and Thr119 hydrophobic interactions take place with the trifluoromethyl group <ref name="Klabunde">PMID:10742177</ref>.
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| Unlike to a single DDBR, these substituted molecules make conformational change on side chains Thr119 and Ser117 with the formation of additional hydrogen bond. Furthermore, the water molecule in HBP3 pocket is displaced.
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| <ref name="Klabunde">PMID:10742177</ref>
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| | <Structure load='1dvu' size='340' frame='true' align='right' caption='Crystal Structure of human transthyretin in complex with dibenzofuran-4,6-dicarboxylic acid from homo sapiens gene in Escherichia coli, resolution 2.05Å (PDB entry : [[1dvu]]) ' scene='Insert optional scene name here' /> |
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| | 4,6-dicarboxylate derivative of dibenzofuran (DDBF) is bounded according two symmetric equivalent modes <ref name="Petrassi">PMID:15869287</ref>. Indeed, DDBF wears a tricyclic ring system, with 2 hydrogen bond donors and 5 hydrogen bond acceptors, allowing to bound the dimer-dimer interface of the TTR cavity <ref name="National Center for Biotechnology Information">[https://pubchem.ncbi.nlm.nih.gov/compound/Dibenzofuran-4_6-dicarboxylic-acid Link text], PubChem Database. CID:3022(accessed on Dec. 26, 2019).</ref><ref name="Klabunde">PMID:10742177</ref>. Thanks to the complementarity of shape and hydrophobicity, DDBF enters nicely the outer portion of HBPs pockets <ref name="Petrassi "/>. Besides, the tricyclic ring system interacts with <scene name='83/832920/Lys15_leu17_and_ala108/1'>Lys15, Leu17 and Ala108</scene> from two adjacent TTR subunits <ref name="Petrassi"/>. Additionally, carboxylates at the position 4 and 6 of DDBF make electrostatic interactions at the entrance of HBP1 and HBP1' with <scene name='83/832920/Lys15/3'>Lys15</scene> on the ε-NH3+ groups <ref name="Petrassi"/>. |
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| | TTR in complex with dibenzofuran-4,6-dicarboxylic acid keeps the general apo-structure, with water molecules bind to HBP3 and HBP3’ cavities of TTR <ref name="Klabunde">PMID:10742177</ref>. There is not conformational change of <scene name='83/832920/Ser117/1'>Ser117</scene> and <scene name='83/832920/Thr119/1'>Thr119</scene> of TTR, contrary to other inhibitor, unlike most NSAID. |
| | Consequently, DDBF creates a bridge between two adjacent subunits stabilized by ionic and hydrophobic interactions. |
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| | ====Improvements==== |
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| | To exploit the TTR inner cavity, DDBF can be ameliorated <ref name="Klabunde">PMID:10742177</ref>. An additional substituent like an aryl ring could be link at DDBF thought a heteroatom or directly via a covalent bond <ref name="Petrassi"/>. |
| | For example, a N-phenyl phenoxazine-4,6-dicarboxylate, called Phenox (PDB entry : [[1dvy]]), allows to create additional bonds, which increase the kinetic stabilization of TTR. Besides, Van der Waals interactions are established with Thr106, Lys15, Leu17 from to adjacent TTR subunits. Moreover, the carboxylate groups link not only Lys15 but also Glu54 (carboxylate groups must to be protonated at physiological pH). In the side chain Leu17, Leu110 and Thr119 hydrophobic interactions take place with the trifluoromethyl group <ref name="Klabunde">PMID:10742177</ref>. |
| | Unlike to a single DDBF, these substituted molecules make conformational change on side chains Thr119 and Ser117 with the formation of additional hydrogen bond. Furthermore, the water molecule in HBP3 pocket is displaced. |
| | <ref name="Klabunde">PMID:10742177</ref> |
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| ====Advantages==== | | ====Advantages==== |
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| The substituted DDBR possess numerous advantages. In vivo, at a molar ratio of 1, there is more of 50% of fibril inhibition activity <ref name="Labaudinière"/> | | The substituted DDBF possess numerous advantages. In vivo, at a molar ratio of 1, there is more of 50% of fibril inhibition activity <ref name="Labaudinière"/> |
| <ref name="National Center for Biotechnology Information">CID:3022,https://pubchem.ncbi.nlm.nih.gov/compound/Dibenzofuran-4_6-dicarboxylic-acid</ref>. The occupancy of the TTR and the energetically favourable interactions reduce tetrameric dissociation by 70%. In vitro, the tetrameric structure of TTR is retained during 7 days with the N-phenyl phenoxazine-4,6-dicarboxylate, whereas without inhibitor TTR fibril formation takes place after 72h.<ref name="Klabunde">PMID:10742177</ref> Additionally, these drugs don’t affect the cyclooxygenase activities <ref name="Labaudinière"/>. Consequently, DDBR and its derivatives are potent drug for human transthyretin amyloid disease. | | <ref name="National Center for Biotechnology Information">CID:3022,https://pubchem.ncbi.nlm.nih.gov/compound/Dibenzofuran-4_6-dicarboxylic-acid</ref>. The occupancy of the TTR and the energetically favourable interactions reduce tetrameric dissociation by 70%. In vitro, the tetrameric structure of TTR is retained during 7 days with the N-phenyl phenoxazine-4,6-dicarboxylate, whereas without inhibitor TTR fibril formation takes place after 72h.<ref name="Klabunde">PMID:10742177</ref> Additionally, these drugs don’t affect the cyclooxygenase activities <ref name="Labaudinière"/>. Consequently, DDBF and its derivatives are potent drug for human transthyretin amyloid disease. |
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| == References == | | == References == |
| <references/> | | <references/> |