Modulation of Human Phenylalanine Hydroxylase by 3-Hydroxyquinolin-2(1H)-One Derivatives

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.

Impact of Fluorinated Ionic Liquids on Human Phenylalanine Hydroxylase—A Potential Drug Delivery System

“…These results are in agreement with the blue native electrophoresis described above, confirming that the addition of FIL at the tested concentrations does not influence the oligomerization of hPAH + L-Phe, while for the non-activated protein, a different oligomeric profile was only observed with 0.6% [N 1112(OH) ][C 4 F 9 SO 3 ]. Structural parameters such as radius of gyration (R g ), Porod volume (V P ), and approximated average molecular mass (MM), described in Table 5, are in agreement with published hPAH batch (i.e., non-SEC) measurements describing a mixture of oligomeric states [28]. These parameters tend to decrease with increasing FIL concentrations, indicating higher protein compactness on average or the dissociation of larger assemblies into tetrameric/dimeric and monomeric forms.…”

“…Furthermore, an oligomerization analysis (see Figure 7b) of the scattering data, using the high-resolution domain structures of hPAH (PDB ID: 5FII, 2PAH) to define association/dissociation components, also suggests the presence of larger species, even before the addition of FIL. This effect has been reported in previous SAXS data collection of hPAH, where the protein tends to equilibrate between different oligomeric states [28,36]. SEC coupled to SAXS (SEC-SAXS) can usually overcome this; however, the interaction between ionic liquids and column matrices precluded the use of the SEC-SAXS mode in this case [39].…”

Systematic Modification and Evaluation of Enzyme-Loaded Chitosan Nanoparticles

“…The hPAH activity was measured as previously described [ 42 ]. Briefly, the reaction mixture in a 200 μL final volume contained 1 mM l- Phe, 0.1 M Na-Hepes, pH 7.0, 0.1 mg/mL catalase, 5 μg of recombinant hPAH tetramers, 5 mM dithiothreitol DTT and 100 μM ferrous ammonium sulphate.…”

Role of misfolding in rare enzymatic deficits and use of pharmacological chaperones as therapeutic approach

“…Recently, Lopes R.R. et al [142] applied a different strategy to target the whole PAH active site, including the binding sites for the substrate, the cofactor, and the non-heme ferric center. Through a combination of studies using purified PAH and cellular models, aiming at investigating both the direct binding of the molecules and their protective effect against enzyme denaturation/degradation, the authors were able to identify putative PCs that stabilize PAH in the active conformation.…”

“…Through a combination of studies using purified PAH and cellular models, aiming at investigating both the direct binding of the molecules and their protective effect against enzyme denaturation/degradation, the authors were able to identify putative PCs that stabilize PAH in the active conformation. Interestingly, one of them binds a region remote from the catalytic and the allosteric sites, thus possibly representing a "second-generation" non-inhibitory PC [142]. In this regard, a strategy based on the use of small-molecules that bind PAH in a site remote from the active site has been also considered promising for mutations affecting the equilibrium among alternative PAH structures.…”