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Kinetic stabilization of transthyretin and its role as an inhibitor of Aβ amyloid formation
Umeå University, Faculty of Science and Technology, Department of Chemistry.ORCID iD: 0000-0003-2074-3584
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Amyloid formation occurs when normally soluble proteins and peptides misfold and aggregate into intractable threadlike structures called fibrils. There are currently more than 30 proteins associated with this aberrant structure, including the Aβ peptide in Alzheimer’s disease (AD) and transthyretin (TTR) in TTR amyloidosis. TTR is a homotetrameric transporter protein present in both cerebrospinal fluid and plasma. Dissociation of its tetrameric structure is required for the formation of amyloid fibrils. Small molecule ligands able to bind and stabilize the tetrameric structure of TTR thus represent a potential therapeutic intervention. Interestingly, apart from TTR’s role as a toxic agent in TTR amyloidosis, it also has a role as an inhibitor of the Aβ toxicity associated with AD. The work presented in this thesis focused on small molecules that have the potential ability to prevent TTR amyloidosis. We also sought to gain a greater understanding of the interaction between TTR and the Aβ peptide with respect to Aβ fibril formation.

The ability of a drug to stabilize TTR is directly correlated to its binding affinity. However, since TTR is a plasma protein, it is of great importance that the drug binds selectively to TTR. In paper I, we used a newly developed urea denaturation assay, in combination with isothermal titration calorimetry, to show that, in a complex environment such as plasma, the enthalpy of binding correlates better with a drug’s ability to stabilize TTR than the binding affinity. In paper II, we modified the highly selective but rapidly degraded TTR ligand luteolin in order to increase its resistance against biotransformation. Using a liver-based microsome assay, in combination with HPLC, we show how the luteolin analogues have gained increased stability. However, using the urea assay, we also show that the analogues have lost much of luteolin’s selectivity. In paper III, we show that tetrabromobisphenol A is a highly selective binder of TTR in plasma and is able to rescue cells from TTR-induced toxicity. In paper IV, we studied the interaction of TTR with Aβ and its effect on Aβ fibril formation. We used a ThT fluorescence-based assay and dot blotting to show that TTR inhibits Aβ amyloid formation and promotes the formation of high molecular weight assemblies with an open N-terminus. Using surface plasmon resonance, we further show how TTR is unable to inhibit fibril elongation and instead targets the nucleation processes, both primary and fibril-catalyzed secondary nucleation. To conclude, we present new molecules with the ability to selectively stabilize TTR that can serve as scaffolds in drug design. We also elucidate TTR’s inhibiting effects on toxic Aβ amyloid formation.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2017. , 55 p.
Keyword [en]
Amyloid, transthyretin, amyloid beta, kinetic stabilizer
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-131776ISBN: 978-91-7601-676-3 (print)OAI: oai:DiVA.org:umu-131776DiVA: diva2:1076042
Public defence
2017-03-17, Lilla hörsalen, KBC-huset, Linnaeusväg 6, 90756 Umeå, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2017-02-23 Created: 2017-02-21 Last updated: 2017-02-22Bibliographically approved
List of papers
1. Enthalpic Forces Correlate with Selectivity of Transthyretin-Stabilizing Ligands in Human Plasma
Open this publication in new window or tab >>Enthalpic Forces Correlate with Selectivity of Transthyretin-Stabilizing Ligands in Human Plasma
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2015 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 58, no 16, 6507-6515 p.Article in journal (Refereed) Published
Abstract [en]

The plasma protein transthyretin (TTR) is linked to human amyloidosis. Dissociation of its native tetrameric assembly is a rate-limiting step in the conversion from a native structure into a pathological amyloidogenic fold. Binding of small molecule ligands within the thyroxine binding site of TTR can stabilize the tetrameric integrity and is a potential therapeutic approach. However, through the characterization of nine different tetramer-stabilizing ligands we found that unspecific binding to plasma components might significantly compromise ligand efficacy. Surprisingly the binding strength between a particular ligand and TTR does not correlate well with its selectivity in plasma. However, through analysis of the thermodynamic signature using isothermal titration calorimetry we discovered a better correlation between selectivity and the enthalpic component of the interaction. This is of specific interest in the quest for more efficient TTR stabilizers, but a high selectivity is an almost universally desired feature within drug design and the finding might have wide-ranging implications for drug design.

Keyword
transthyretin, entalpic, enthropic
National Category
Medicinal Chemistry
Research subject
biological chemistry
Identifiers
urn:nbn:se:umu:diva-106724 (URN)10.1021/acs.jmedchem.5b00544 (DOI)000360415800015 ()26214366 (PubMedID)
Available from: 2015-08-04 Created: 2015-08-04 Last updated: 2017-05-10Bibliographically approved
2. Modifications of the 7-Hydroxyl Group of the Transthyretin Ligand Luteolin Provide Mechanistic Insights into Its Binding Properties and High Plasma Specificity
Open this publication in new window or tab >>Modifications of the 7-Hydroxyl Group of the Transthyretin Ligand Luteolin Provide Mechanistic Insights into Its Binding Properties and High Plasma Specificity
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2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 4, e0153112Article in journal (Refereed) Published
Abstract [en]

Amyloid formation of the plasma protein transthyretin (TTR) has been linked to familial amyloid polyneuropathy and senile systemic amyloidosis. Binding of ligands within its natural hormone binding site can stabilize the tetrameric structure and impair amyloid formation. We have recently shown that the flavonoid luteolin stabilizes TTR in human plasma with a very high selectivity. Luteolin, however, is inactivated in vivo via glucuronidation for which the preferred site is the hydroxy group at position 7 on its aromatic A-ring. We have evaluated the properties of two luteolin variants in which the 7-hydroxy group has been exchanged for a chlorine (7-Cl-Lut) or a methoxy group (7-MeO-Lut). Using an in vitro model, based on human liver microsomes, we verified that these modifications increase the persistence of the drug. Crystal structure determinations show that 7-Cl-Lut binds similarly to luteolin. The larger MeO substituent cannot be accommodated within the same space as the chlorine or hydroxy group and as a result 7-MeO-Lut binds in the opposite direction with the methoxy group in position 7 facing the solvent. Both 7-Cl-Lut and 7-MeO-Lut qualify as high-affinity binders, but in contrast to luteolin, they display a highly non-specific binding to other plasma components. The binding of the two conformations and the key-interactions to TTR are discussed in detail. Taken together, these results show a proof-of-concept that the persistence of luteolin towards enzymatic modification can be increased. We reveal two alternative high-affinity binding modes of luteolin to TTR and that modification in position 7 is restricted only to small substituents if the original orientation of luteolin should be preserved. In addition, the present work provides a general and convenient method to evaluate the efficacy of TTR-stabilizing drugs under conditions similar to an in vivo environment.

Place, publisher, year, edition, pages
Public Library Science, 2016
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-119997 (URN)10.1371/journal.pone.0153112 (DOI)000373603500101 ()27050398 (PubMedID)
Available from: 2016-05-04 Created: 2016-05-04 Last updated: 2017-02-22Bibliographically approved
3. Tetrabromobisphenol A Is an Efficient Stabilizer of the Transthyretin Tetramer
Open this publication in new window or tab >>Tetrabromobisphenol A Is an Efficient Stabilizer of the Transthyretin Tetramer
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2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 4, e0153529Article in journal (Refereed) Published
Abstract [en]

Amyloid formation of the human plasma protein transthyretin (TTR) is associated with several human disorders, including familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis. Dissociation of TTR’s native tetrameric assembly is the rate-limiting step in the conversion into amyloid, and this feature presents an avenue for intervention because binding of an appropriate ligand to the thyroxin hormone binding sites of TTR stabilizes the native tetrameric assembly and impairs conversion into amyloid. The desired features for an effective TTR stabilizer include high affinity for TTR, high selectivity in the presence of other proteins, no adverse side effects at the effective concentrations, and a long half-life in the body. In this study we show that the commonly used flame retardant tetrabromobisphenol A (TBBPA) efficiently stabilizes the tetrameric structure of TTR. The X-ray crystal structure shows TBBPA binding in the thyroxine binding pocket with bromines occupying two of the three halogen binding sites. Interestingly, TBBPA binds TTR with an extremely high selectivity in human plasma, and the effect is equal to the recently approved drug tafamidis and better than diflunisal, both of which have shown therapeutic effects against FAP. TBBPA consequently present an interesting scaffold for drug design. Its absorption, metabolism, and potential side-effects are discussed.

Place, publisher, year, edition, pages
Public Library Science, 2016
National Category
Chemical Sciences
Identifiers
urn:nbn:se:umu:diva-119999 (URN)10.1371/journal.pone.0153529 (DOI)000374541200025 ()27093678 (PubMedID)
Available from: 2016-05-04 Created: 2016-05-04 Last updated: 2017-02-22Bibliographically approved
4. Transthyretin Impairs Nucleation in the Path of Amyloid β Fibril formation
Open this publication in new window or tab >>Transthyretin Impairs Nucleation in the Path of Amyloid β Fibril formation
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(English)Manuscript (preprint) (Other academic)
National Category
Organic Chemistry
Identifiers
urn:nbn:se:umu:diva-131805 (URN)
Available from: 2017-02-22 Created: 2017-02-22 Last updated: 2017-02-22

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