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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].

Structure TTR structure with natural ligand : T4 and retinol

Disease

Type of disease

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)^[3]. 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. ^[4]

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. ^[5] 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 ^[6][7]

Drug development

First drugs developed: Non-steroidal anti-inflammatory drugs

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 ^[5], prolonged NSAIDs administration could provoke renal failure, cardiac side effects, and gastrointestinal ulcers. ^[8] 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. ^[5]

Dibenzofuran-4,6-dicarboxylic acid

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 ^[9]. 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 ^[10]. 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 ^[11][12]. Thanks to the complementarity of shape and hydrophobicity, DDBF enters nicely the outer portion of HBPs pockets ^[10]. Besides, the tricyclic ring system interacts with from two adjacent TTR subunits ^[10]. Additionally, carboxylates at the position 4 and 6 of DDBF make electrostatic interactions at the entrance of HBP1 and HBP1’ with on the ε-NH3+ groups ^[10].

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 ^[12]. There is not conformational change of and 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

To exploit the TTR inner cavity, DDBR can be ameliorate ^[12]. An additional substituent like an aryl ring could be link at DDBR thought a heteroatom or directly via a covalent bond ^[10]. 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 ^[12]. 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. ^[12]

Advantages

The substituted DDBR possess numerous advantages. In vivo, at a molar ratio of 1, there is more of 50% of fibril inhibition activity ^{[9] [11]}. 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.^[12] Additionally, these drugs don’t affect the cyclooxygenase activities ^[9]. 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. ↑ 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
4. ↑ ARTICLE Human brain amyloidoses
5. ↑ ^{5.0 5.1 5.2} Article RATIONAL DESIGN
6. ↑ 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
7. ↑ 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
8. ↑ 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
9. ↑ ^{9.0 9.1 9.2} 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
10. ↑ ^{10.0 10.1 10.2 10.3 10.4} 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:http://dx.doi.org/10.1021/ja044351f
11. ↑ ^{11.0 11.1} Link text, PubChem Database. CID:3022(accessed on Dec. 26, 2019).
12. ↑ ^{12.0 12.1 12.2 12.3 12.4 12.5} 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:http://dx.doi.org/10.1038/74082