Structure-based chemical modification strategy for enzyme replacement treatment of phenylketonuria.

Structure-based protein engineering coupled with chemical modifications (e.g., pegylation) is a powerful combination to significantly improve the development of proteins as therapeutic agents.

Development of pegylated forms of recombinant Rhodosporidium toruloides phenylalanine ammonia-lyase for the treatment of classical phenylketonuria.

Phenylketonuria (PKU) is a metabolic disorder due primarily to mutations in the PAH gene that impair both phenylalanine hydroxylase activity and disposal of l-phenylalanine from the normal diet. Excess phenylalanine is toxic to cognitive development and a low-phenylalanine diet prevents mental retardation, but it is a difficult therapeutic option. Previous studies with recombinant phenylalanine ammonia-lyase, PAL, demonstrated pharmacologic and physiologic proofs of principle for PAL as an alternative therapy for PKU but its immunogenicity was problematic.

Toward PKU enzyme replacement therapy: PEGylation with activity retention for three forms of recombinant phenylalanine hydroxylase.

Phenylketonuria (PKU) is a disease in which phenylalanine and phenylalanine-derived metabolites build up to neurotoxic levels due to mutations in the phenylalanine hydroxylase gene (PAH). Enzyme replacement therapy is a viable option to supply active PAH. However, the inherent protease sensitivity and potential immunogenicity of PAH have precluded adoption of this approach.

Structural studies on phenylalanine hydroxylase and implications toward understanding and treating phenylketonuria.

Mutations in the gene encoding for phenylalanine hydroxylase (PAH) result in phenylketonuria (PKU) or hyperphenylalaninemia (HPA). Several 3-dimensional structures of truncated forms of PAH have been determined in our laboratory and by others, using x-ray crystallographic techniques. These structures have allowed for a detailed mapping of the >250 missense mutations known to cause PKU or HPA found throughout the 3 domains of PAH.

The genesis of high-throughput structure-based drug discovery using protein crystallography.

Over the past 12 years, drugs have been developed using structure-based drug design relying upon traditional crystallographic methods. Established successes, such as the drugs designed against HIV-1 protease and neuraminidase, demonstrate the utility of a structure-based approach in the drug-discovery process. However, the approach has historically lacked throughput and reliability capabilities; these bottlenecks are being overcome by breakthroughs in high-throughput structural biology.

Structural comparison of bacterial and human iron-dependent phenylalanine hydroxylases: similar fold, different stability and reaction rates.

Structure determination of bacterial homologues of human disease-related proteins provides an efficient path to understanding the three-dimensional fold of proteins that are associated with human diseases. However, the precise locations of active-site residues are often quite different between bacterial and human versions of an enzyme, creating significant differences in the biological understanding of enzyme homologs. To study this hypothesis, phenylalanine hydroxylase from a bacterial source has been structurally characterized at high resolution and comparison is made to the human analog.