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. As a test case, phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) was selected for enzyme replacement therapy in phenylketonuria [C.R. Scriver, S. Kaufman, Hyperphenylalaninemia:phenylalanine Hydroxylase Deficiency. The Metabolic and Molecular Bases of Inherited Disease, McGraw-Hill, New York, 2001, Chapter 77], an inherited metabolic disorder (OMIM 261600) causing mental retardation due to deficiency of the enzyme l-phenylalanine hydroxylase (EC 1.14.16.1). Previous in vivo studies of recombinant PAL demonstrated a lowering of blood l-phenylalanine levels; yet, the metabolic effect was not sustained due to protein degradation and immunogenicity [C.N. Sarkissian, Z. Shao, F. Blain, R. Peevers, H. Su, R. Heft, T.M. Chang, C.R. Scriver, A different approach to treatment of phenylketonuria:phenylalanine degradation with recombinant phenylalanine ammonia lyase, Proc. Natl. Acad. Sci. USA 96 (1999) 2339; J.A. Hoskins, G. Jack, H.E. Wade, R.J. Peiris, E.C. Wright, D.J. Starr, J. Stern, Enzymatic control of phenylalanine intake in phenylketonuria, Lancet 1 (1980) 392; C.M. Ambrus, S. Anthone, C. Horvath, K. Kalghatgi, A.S. Lele, G. Eapen, J.L. Ambrus, A.J. Ryan, P. Li, Extracorporeal enzyme reactors for depletion of phenylalanine in phenylketonuria, Ann. Intern. Med. 106 (1987) 531]. Here, we report the 1.6A three-dimensional structure of Rhodosporidium toruloides PAL, structure-based molecular engineering, pegylation of PAL, as well as in vitro and in vivo PKU mouse model studies on pegylated PAL formulations. Our results show that pegylation of R. toruloides PAL leads to promising therapeutic efficacy after subcutaneous injection by enhancing the in vivo activity, lowering plasma phenylalanine, and leading to reduced immunogenicity. The three-dimensional structure of PAL provides a basis for understanding the properties of pegylated forms of PAL and strategies for structure-based re-engineering of PAL for PKU treatment.

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. From a series of formulations of linear and branched polyethylene glycols chemically conjugated to PAL, we have created a parenteral therapeutic agent for PKU treatment. All the pegylated molecules were fully characterized in vitro and the most promising formulations were then tested in vivo in the PKU mouse model. The linear 20-kDa PEG-PAL combination abolished in vivo immunogenicity after repeated challenge while retaining full catabolic activity against phenylalanine, suggesting potential as a novel PKU therapeutic.

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. In this report, we have used polyethylene glycol derivatization (PEGylation) to produce protected forms of PAH for potential therapeutic use. Three recombinantly produced PAH enzymes were reacted with activated PEG species, with the aim of developing a stable and active PKU enzyme replacement. Tetrameric full-length human PAH, dimeric double-truncated (DeltaN102-DeltaC428) human PAH, and monomeric Chromobacterium violaceum PAH were PEGylated with succinimidyl succinate polyethylene glycol of molecular weight 5000 or 20,000 Da. Characterization of the PEGylated species was accomplished with MALDI-TOF mass spectrometry, SDS-PAGE, and specific activity measurements using ESI mass spectrometry. All PEG-derivatized PAH species retained catalytic activity, and, at low numbers of PEG molecules attached, these PEGylated PAH proteins were found to be more active and more stable than their non-derivatized PAH counterparts.

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. This structural information has helped formulate rules that might aid in predicting the likely effects of unclassified or newly discovered PAH mutations. Also, with the aid of recent crystal structure determinations of co-factor and substrate analogs bound at the PAH active site, the recently discovered tetrahydrobiopterin-responsive PKU/HPA genotypes can be mapped onto the PAH structure, providing a molecular basis for this tetrahydrobiopterin response.

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. Recent technological innovations such as submicroliter high-throughput crystallization, high-performance synchrotron beamlines and rapid binding-site analysis of de novo targets using virtual ligand screening and small molecule co-crystallization have resulted in a significant advance in structure-based drug discovery.

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. The enzyme phenylalanine hydroxylase (PheOH) catalyzes the hydroxylation of l-phenylalanine into l-tyrosine utilizing the cofactors (6R)-l-erythro-5,6,7,8 tetrahydrobiopterin (BH(4)) and molecular oxygen. Previously determined X-ray structures of human and rat PheOH, with a sequence identity of more than 93%, show that these two structures are practically identical. It is thus of interest to compare the structure of the divergent Chromobacterium violaceum phenylalanine hydroxylase (CvPheOH) ( approximately 24% sequence identity overall) to the related human and rat PheOH structures. We have determined crystal structures of CvPheOH to high resolution in the apo-form (no Fe-added), Fe(III)-bound form, and 7,8-dihydro-l-biopterin (7,8-BH(2)) plus Fe(III)-bound form. The bacterial enzyme displays higher activity and thermal melting temperature, and structurally, differences are observed in the N and C termini, and in a loop close to the active-site iron atom.