Abstract
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.
Highlights
Transthyretin-related hereditary amyloidosis is linked to more than 100 known heterozygous mutations in the gene coding for transthyretin (TTR) [1], a plasma protein responsible for transporting thyroxine (T4) and holo-retinol-binding protein [2]
The dissociation constant (Kd) for luteolin was determined to be 150 ± 70 nM, which is in accordance with the previously published value of 100 nM determined by isothermal titration calorimetry [37] (Fig 3A)
We have recently solved the structure of the TTR–luteolin complex, which shows how the aromatic A-ring of luteolin is inserted into the deep end of the hydrophobic T4-hormone binding site [21]
Summary
Transthyretin-related hereditary amyloidosis is linked to more than 100 known heterozygous mutations in the gene coding for transthyretin (TTR) [1], a plasma protein responsible for transporting thyroxine (T4) and holo-retinol-binding protein [2]. TTR-related hereditary amyloidosis is divided into two categories depending on the tissue that is predominantly affected: familial amyloid polyneuropathy (FAP), which mainly affects the peripheral nervous system, and familial amyloid cardiomyopathy (FAC), which predominantly affects the heart [4]. From a mechanistic point of view, the mutations in TTR frequently result in destabilization of the native tetrameric structure of TTR and increase its dissociation into monomers [6]. These monomeric species misfold and aggregate into amyloid fibrils within various tissues depending on the mutation [7,8]
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