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Abstract
Phenylketonuria (PKU) is a genetic disease caused by deficient activity of human phenylalanine hydroxylase (hPAH) that, when untreated, can lead to severe psychomotor impairment. Protein misfolding is recognized as the main underlying pathogenic mechanism of PKU. Therefore, the use of stabilizers of protein structure and/or activity is an attractive therapeutic strategy for this condition. Here, we report that 3-hydroxyquinolin-2(1H)-one derivatives can act as protectors of hPAH enzyme activity. Electron paramagnetic resonance spectroscopy demonstrated that the 3-hydroxyquinolin-2(1H)-one compounds affect the coordination of the non-heme ferric center at the enzyme active-site. Moreover, surface plasmon resonance studies showed that these stabilizing compounds can be outcompeted by the natural substrate l-phenylalanine. Two of the designed compounds functionally stabilized hPAH by maintaining protein activity. This effect was observed on the recombinant purified protein and in a cellular model. Besides interacting with the catalytic iron, one of the compounds also binds to the N-terminal regulatory domain, although to a different location from the allosteric l-Phe binding site, as supported by the solution structures obtained by small-angle X-ray scattering.
Highlights
The truncated form of human phenylalanine hydroxylase (hPAH) corresponding to the N-terminal regulatory domain was expressed in E. coli TB1 cells in fusion with maltose binding protein (MBP) and a sequence recognized by Factor Xa (FXa) (MBP-FXa-hPAH-RD1–120 ) to allow further removal of the MBP tag, as described previously [17] (Supplementary Figure S2, peak 2)
The substituents in position 4 include L-Phe-like moieties predicted to enhance selectivity towards the catalytic site, while different electron withdrawing substituents at position 6 are expected to increase the acidity of the hydroxy group and the coordination with the iron center
The discovery of different classes of small molecules modulators of hPAH folding and/or activity will contribute to the development of pharmacological strategies to treat a larger cohort of PKU patients
Summary
Inherited metabolic disorders (IMDs) are genetic diseases causing the impaired function of enzymes or transporters involved in intermediary metabolism. The mutational spectrum of the majority of IMDs is dominated by missense mutations (50–80%), frequently leading to protein variants with deficient catalytic activity or impaired folding. The latter results in premature protein degradation and lower cellular protein steady-state levels. Attractive therapeutic approaches for these conditions are based on the use of small molecules acting as “pharmacological chaperones” rescuing variant proteins’
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