Phenylalanine hydroxylase misfolding and pharmacological chaperones

Abstract

Phenylketonuria (PKU) is a loss-of-function inborn error of metabolism. As many other inherited diseases the main pathologic mechanism in PKU is an enhanced tendency of the mutant phenylalanine hydroxylase (PAH) to misfold and undergo ubiquitin-dependent degradation. Recent alternative approaches with therapeutic potential for PKU aim at correcting the PAH misfolding, and in this respect pharmacological chaperones are the focus of increasing interest. These compounds, which often resemble the natural ligands and show mild competitive inhibition, can rescue the misfolded proteins by stimulating their renaturation in vivo. For PKU, a few studies have proven the stabilization of PKU-mutants in vitro, in cells, and in mice by pharmacological chaperones, which have been found either by using the tetrahydrobiopterin (BH(4)) cofactor as query structure for shape-focused virtual screening or by high-throughput screening of small compound libraries. Both approaches have revealed a number of compounds, most of which bind at the iron-binding site, competitively with respect to BH(4). Furthermore, PAH shares a number of ligands, such as BH(4), amino acid substrates and inhibitors, with the other aromatic amino acid hydroxylases: the neuronal/neuroendocrine enzymes tyrosine hydroxylase (TH) and the tryptophan hydroxylases (TPHs). Recent results indicate that the PAH-targeted pharmacological chaperones should also be tested on TH and the TPHs, and eventually be derivatized to avoid unwanted interactions with these other enzymes. After derivatization and validation in animal models, the PAH-chaperoning compounds represent novel possibilities in the treatment of PKU.

Fig. (1)

The biosynthetic and regeneration pathways of BH₄ and the reaction catalyzed by PAH. See main text for full names of the enzymes. The 3D structures of mammalian enzyme forms are also shown (for some only truncated conformations are available).

Fig. (2)

The structure of PAH. (A) The modeled structure of full-length tetrameric PAH (composite model prepared by combining the structures of tetrameric human PAH (residues 118-452; PDB 2PAH) and dimeric rat PAH (residues 19-427; PDB 2PHM)). (B) Detailed structure of PAH including thienylalanine and BH₄ (PDB 1KW0).

Fig. (3)

Compounds with potential pharmacological chaperone ability for PKU. (A) The hits from Pey et al. [50], where compounds III (3- amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one) and IV (5,6-dimethyl-3-(4-methyl-2-pyridinyl)-2-thioxo-2,3- dihydrothieno[2,3- d]pyrimidin-4(1H)-one) showed the best potential. (B) The hits from Santos-Sierra et al., [51], where compounds 1 (benzylhydantoin) and 2 (6-amino-5-(benzylamino)-uracil) showed the best potential.

Fig. (4)

Four alternative methodological approaches for virtual screening; these methods can be combined in a synergistic way. Partly based on [84].

Fig. (5)

The protocol for high-throughput screening through differential scanning fluorimetry. The enzyme is mixed with the compounds and a fluorescent dye. The temperature of the samples is gradually increased and fluorescence is measured. The resulting thermal unfolding curves are analyzed and hits are picked based on their ability to stabilize the enzyme thermally. DSF, differential scanning fluorimetry.

Fig. (6)

A vision for patient-tailored therapy in HPA/PKU with focus on pharmacological chaperones. A) The concept of genotypedependent therapy including some present (and future) alternatives in the treatment of HPA/PKU. ERT, enzyme replacement therapy; GT, gene therapy; AST, antisense therapy. B) Flow chart of translational approach in the process of discovery and validation of pharmacological chaperones. DSF, differential scanning fluorimetry; SPR, surface plasmon resonance.