Phenylketonuria (PKU) is an inborn error of metabolism caused by mutations in phenylalanine hydroxylase (PAH). Over 500 disease-causing mutations have been identified in humans, most of which result in PAH protein misfolding and increased turnover in vivo. The use of pharmacological chaperones to stabilize or promote correct folding of mutant proteins represents a promising new direction in the treatment of misfolding diseases. We performed a high-throughput ligand screen of over 1,000 pharmacological agents and identified 4 compounds (I–IV) that enhanced the thermal stability of PAH and did not show substantial inhibition of PAH activity. In further studies, 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) stabilized the functional tetrameric conformation of recombinant WT-PAH and PKU mutants. These compounds also significantly increased activity and steady-state PAH protein levels in cells transiently transfected with either WT-PAH or PKU mutants. Furthermore, PAH activity in mouse liver increased after a 12-day oral administration of low doses of compounds III and IV. Thus, we have identified 2 small molecules that may represent promising alternatives in the treatment of PKU.

Phenylketonuria patients harboring a subset of phenylalanine hydroxylase (PAH) mutations have recently shown normalization of blood phenylalanine levels upon oral administration of the PAH cofactor tetrahydrobiopterin [(6 R )- l - erythro -5,6,7,8-tetrahydrobiopterin (BH ₄ )]. Several hypotheses have been put forward to explain BH ₄ responsiveness, but the molecular basis for the corrective effect(s) of BH ₄ has not been understood. We have investigated the biochemical, kinetic, and structural changes associated with BH ₄ -responsive mutations (F39L, I65T, R68S, H170D, E178G, V190A, R261Q, A300S, L308F, A313T, A373T, V388M, E390G, P407S, and Y414C). The biochemical and kinetic characterization of the 15 mutants studied points toward a multifactorial basis for the BH ₄ responsiveness; the mutants show residual activity (>30% of WT) and display various kinetic defects, including increased K _m (BH ₄ ) and reduced cooperativity of substrate binding, but no decoupling of cofactor (BH ₄ ) oxidation. For some, BH ₄ seems to function through stabilization and protection of the enzyme from inactivation and proteolytic degradation. In the crystal structures of a phenylketonuria mutant, A313T, minor changes were seen when compared with the WT PAH structures, consistent with the mild effects the mutant has upon activity of the enzyme both in vitro and in vivo . Truncations made in the A313T mutant PAH form revealed that the N and C termini of the enzyme influence active site binding. Of fundamental importance is the observation that BH ₄ appears to increase Phe catabolism if at least one of the two heterozygous mutations has any residual activity remaining.

When analyzed in the context of the phenylalanine hydroxylase (PAH) three‐dimensional structure, only a minority of the PKU mutations described world‐wide affect catalytic residues. Consistent with these observations, recent data point to defective folding and subsequent aggregation/degradation as a predominant disease mechanism for several mutations. In this work, we use a combined approach of expression in eukaryotic cells at different temperatures and a prokaryotic system with co‐expression of chaperonins to elucidate and confirm structural consequences for 18 PKU mutations. Three mutations are located in the amino terminal regulatory domain and 15 in the catalytic domain. Four mutations were found to abolish the specific activity in all conditions. Two are catalytic mutations (Y277D and E280K) and two are severe structural defects (IVS10–11G>A and L311P). All the remaining mutations (D59Y, I65T, E76G, P122Q, R158Q, G218V, R243Q, P244L, R252W, R261Q, A309V, R408Q, R408W, and Y414C) are folding defects causing reduced stability and accelerated degradation, although some of them probably affect residues involved in regulation. In these cases, we have demonstrated that the amount of mutant PAH protein and residual activity could be modulated by in vitro experimental conditions, and therefore the observed in vivo metabolic variation may be explained by interindividual variation in the quality control systems. The results derived provide an experimental framework to define the mutation severity relating genotype to phenotype. They also explain the observed inconsistencies for some mutations in patients with similar genotype and different phenotypes. Hum Mutat 21:370–378, 2003.