Sandbox Reserved 1105

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This Sandbox is Reserved from 25/11/2019, through 30/9/2020 for use in the course "Structural Biology" taught by Bruno Kieffer at the University of Strasbourg, ESBS. This reservation includes Sandbox Reserved 1091 through Sandbox Reserved 1115.
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StructureSection load='1stp' size='340' side='right' caption='Caption for this structure' scene=


TTR transport functions

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 [1]. 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: transports thyroxine (T4) and retinol (vitamin A). 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.

The TTR gene is located on chromosome 18 [2].


Human TTR structure with natural ligand : T4 and retinol

Human TTR:

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).

Each monomer is composed of an α-helix and two four stranded β-sheets, which results in two eight-stranded β-sheets per dimer (5). 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 (6). 

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.


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 Cite error: Invalid parameter in <ref> tag. 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. [3]

TTR amyloid fibril

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. [4] 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.

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 [5][6]


Drug development

First drugs developed: Non-steroidal anti-inflammatory drugs

Puzzle globe
Structures of thyroxine (T4), the natural ligand of TTR and other designed TTR fibril formation inhibitors

Drug research is basée on the inhibition of amyloidogenic TTR by stabilization of native tetrameric conformation, using binding ligands to prevent TTR dissociation.

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. However, regardless of a noticeable decrease of the TTR’s amyloidogenic potential [4], prolonged NSAIDs administration could provoke renal failure, cardiac side effects, and gastrointestinal ulcers. [7] 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. [4]









Dibenzofuran-4,6-dicarboxylic acid

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)

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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 [8]. In the outer cavity are positioned the HBP1 and HBP1' pockets, in the inner cavity are placed the HBP3 and HBP3’ pockets, and the HBP2 and HBP2’ pockets are at the interface between the inner and outer cavity

DDBF is bounded according two symmetric equivalent modes [9]. 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 [10][11]. Thanks to the complementarity of shape and hydrophobicity, DDBF enters nicely the outer portion of HBPs pockets [9]. Besides, the tricyclic ring system interacts with Lys15, Leu17 and Ala108 from two adjacent TTR subunits [9]. Additionally, carboxylates at the position 4 and 6 of DDBF make electrostatic interactions at the entrance of HBP1 and HBP1’ with Lys15 on the ε-NH3+ groups [9].


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 [11]. There is not conformational change of Ser117 and Thr119 of TTR, contrary to other inhibitor, such as FLU. Consequently, DDBF creates a bridge between two adjacent subunits stabilized by ionic and hydrophobic interactions.






Improvements

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)

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To exploit the TTR inner cavity, DDBR can be ameliorate [11]. An additional substituent like an aryl ring could be link at DDBR thought a heteroatom or directly via a covalent bond [9]. 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 [11]. 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. [11]




Advantages

The substituted DDBR possess numerous advantages. In vivo, at a molar ratio of 1, there is more of 50% of fibril inhibition activity [8] [10]. 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.[11] Additionally, these drugs don’t affect the cyclooxygenase activities [8]. Consequently, DDBR and its derivatives are potent drug for human transthyretin amyloid disease.


References

  1. ↑ Seibert FB, Nelson JW. Electrophoretic study of the blood protein response in tuberculosis. J Biol Chem 1942; 143: 29–38.
  2. ↑ 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
  3. ↑ ARTICLE Human brain amyloidoses
  4. ↑ 4.0 4.1 4.2 Article RATIONAL DESIGN
  5. ↑ 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: https://dx.doi.org/10.1161/JAHA.113.000098
  6. ↑ Amyloid fibril composition and transthyretin gene structure in senile systemic amyloidosis. Gustavsson A1, Jahr H, Tobiassen R, Jacobson DR, Sletten K, Westermark P., of Pathology I, Linköping University, Sweden
  7. ↑ 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: https://dx.doi.org/10.1136/bmj.j1909
  8. ↑ 8.0 8.1 8.2 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
  9. ↑ 9.0 9.1 9.2 9.3 9.4 Petrassi HM, Johnson SM, Purkey HE, Chiang KP, Walkup T, Jiang X, Powers ET, Kelly JW. Potent and selective structure-based dibenzofuran inhibitors of transthyretin amyloidogenesis: kinetic stabilization of the native state. J Am Chem Soc. 2005 May 11;127(18):6662-71. doi: 10.1021/ja044351f. PMID:15869287 doi:https://dx.doi.org/10.1021/ja044351f
  10. ↑ 10.0 10.1 Link text, PubChem Database. CID:3022(accessed on Dec. 26, 2019). Cite error: Invalid <ref> tag; name "National Center for Biotechnology Information" defined multiple times with different content
  11. ↑ 11.0 11.1 11.2 11.3 11.4 11.5 Klabunde T, Petrassi HM, Oza VB, Raman P, Kelly JW, Sacchettini JC. Rational design of potent human transthyretin amyloid disease inhibitors. Nat Struct Biol. 2000 Apr;7(4):312-21. PMID:10742177 doi:https://dx.doi.org/10.1038/74082
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