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Selection of transthyretin amyloid inhibitors
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.ORCID iD: 0000-0003-2910-4850
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Amyloidosis is a group of clinical disorders caused by the aggregation of specific proteins into abnormal extracellular deposits. Today, 31 different proteins have been linked to amyloid diseases including transthyretin-related amyloidosis (ATTR). ATTR occurs through the aggregation of either wild-type plasma protein transthyretin (TTR) or a mutated form. TTR is a homotetramer that under normal circumstances functions as a carrier of thyroxine and retinol binding protein. The aggregation cascade requires dissociation of the tetramer into monomers, and preventing this dissociation represents a potential mode of intervention. Interestingly, small molecules, referred as kinetic stabilizers, can bind to TTR’s thyroxine-binding site (TBS) and such molecules are currently being used as a therapeutic approach to impair tetramer dissociation.

The efficacy of TTR stabilization is directly correlated to the binding affinity of the ligand to TBS. However, the binding of the ligand to TTR in vivo can be affected by other plasma components resulting in poor efficacy. Thus, the selectivity of ligands is an important parameter. We have designed an assay where the ability to stabilize TTR can be directly evaluated in plasma and we have investigated the stabilizing effect of nine potential TTR binders (Paper I). The results, surprisingly, revealed that the binding affinity of molecules has a poor correlation to its selectivity. However, the nature of protein-ligand complex formation can also be described by enthalpic (∆H) and entropic (∆S) energy contributions. ∆H represents the change in chemical bonds and frequently requires a higher order of orientation compared to the ∆S component, which mainly represents the hydrophobic effect via the exclusion of water. We hypothesized that ligands possessing high ΔH in binding to their co-partner would also be more specific in a complex environment such as plasma. By applying a thermodynamic analysis using isothermal titration calorimetry, we found that the selectivity in plasma correlates well with the ∆H contribution and might, therefore, be a better predictor for selectivity.

Luteolin was found to be a highly selective stabilizer of TTR and was investigated further (Paper II). The ligand displayed a significant rescuing effect in both cell culture and animal models. However, luteolin undergoes rapid enzymatic degradation in the liver and this impairs its use as a potential therapeutic drug. To attempt to circumvent this issue, we modified the most exposed hydroxyl group thus rendering the molecule inert towards glucuronidation (Paper III). The substitutions resulted in higher stability in the face of hepatic degradation molecules, but they also affected the selectivity in a negative manner.

The screening for new TTR stabilizers resulted in the discovery of tetrabromobisphenol A, which displayed a very high selectivity (Paper IV). This study also included a comparison with the drug Vyndaqel™ which currently is in clinically use, and showed how the dosage could be altered to acquire a better level of saturation and possibly also a better clinical effect.

Taken together we present new molecules with the ability to stabilize TTR, and these can serve as scaffolds for the design of new drugs. We present a method to measure the efficacy of a TTR-stabilizing drugs in a complex matrix and as well as a way to adjust the dosage of existing drugs. We also show that the selectivity of a drug is affected by the relative proportion of ∆H and ∆S, and this is of interest for drug design in general.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2016. , 72 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1826
Keyword [en]
Transthyretin, TTR, ATTR, TTR-stabilizing drugs, selectivity
National Category
Other Basic Medicine
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-123939ISBN: 978-91-7601-528-5OAI: oai:DiVA.org:umu-123939DiVA: diva2:947035
Public defence
2016-09-09, KB3A9, byggnad KBC, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2016-08-18 Created: 2016-07-06 Last updated: 2016-08-16Bibliographically 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: 2016-08-16Bibliographically approved
2. The flavonoid luteolin, but not luteolin-7-o-glucoside, prevents a transthyretin mediated toxic response
Open this publication in new window or tab >>The flavonoid luteolin, but not luteolin-7-o-glucoside, prevents a transthyretin mediated toxic response
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 5, e0128222Article in journal (Refereed) Published
Abstract [en]

Transthyretin (TTR) is a homotetrameric plasma protein with amyloidogenic properties that has been linked to the development of familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The in vivo role of TTR is associated with transport of thyroxine hormone T4 and retinol-binding protein. Loss of the tetrameric integrity of TTR is a rate-limiting step in the process of TTR amyloid formation, and ligands with the ability to bind within the thyroxin binding site (TBS) can stabilize the tetramer, a feature that is currently used as a therapeutic approach for FAP. Several different flavonoids have recently been identified that impair amyloid formation. The flavonoid luteolin shows therapeutic potential with low incidence of unwanted side effects. In this work, we show that luteolin effectively attenuates the cytotoxic response to TTR in cultured neuronal cells and rescues the phenotype of a Drosophila melanogaster model of FAP. The plant-derived luteolin analogue cynaroside has a glucoside group in position 7 of the flavone A-ring and as opposed to luteolin is unable to stabilize TTR tetramers and thus prevents a cytotoxic effect. We generated high-resolution crystal-structures of both TTR wild type and the amyloidogenic mutant V30M in complex with luteolin. The results show that the A-ring of luteolin, in contrast to what was previously suggested, is buried within the TBS, consequently explaining the lack of activity from cynaroside. The flavonoids represent an interesting group of drug candidates for TTR amyloidosis. The present investigation shows the potential of luteolin as a stabilizer of TTR in vivo. We also show an alternative orientation of luteolin within the TBS which could represent a general mode of binding of flavonoids to TTR and is of importance concerning the future design of tetramer stabilizing drugs.

Place, publisher, year, edition, pages
Public Library Science, 2015
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-103893 (URN)10.1371/journal.pone.0128222 (DOI)000355187300089 ()26020516 (PubMedID)
Funder
Swedish Research Council
Available from: 2015-06-02 Created: 2015-06-02 Last updated: 2016-08-16Bibliographically approved
3. 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: 2016-08-16Bibliographically approved
4. 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: 2016-08-16Bibliographically approved

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