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Modifications of the 7-Hydroxyl Group of the Transthyretin Ligand Luteolin Provide Mechanistic Insights into Its Binding Properties and High Plasma Specificity
Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Science and Technology, Department of Chemistry. Swedish Defence Research Agency, CBRN Defence and Security.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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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. Vol. 11, no 4, e0153112
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:umu:diva-119997DOI: 10.1371/journal.pone.0153112ISI: 000373603500101PubMedID: 27050398OAI: oai:DiVA.org:umu-119997DiVA: diva2:926120
Available from: 2016-05-04 Created: 2016-05-04 Last updated: 2017-11-30Bibliographically approved
In thesis
1. Selection of transthyretin amyloid inhibitors
Open this publication in new window or tab >>Selection of transthyretin amyloid inhibitors
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
Transthyretin, TTR, ATTR, TTR-stabilizing drugs, selectivity
National Category
Other Basic Medicine
Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-123939 (URN)978-91-7601-528-5 (ISBN)
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
2. Kinetic stabilization of transthyretin and its role as an inhibitor of Aβ amyloid formation
Open this publication in new window or tab >>Kinetic stabilization of transthyretin and its role as an inhibitor of Aβ amyloid formation
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
Amyloid, transthyretin, amyloid beta, kinetic stabilizer
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-131776 (URN)978-91-7601-676-3 (ISBN)
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

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