Phenylalanine ammonia lyase (PAL): From discovery to enzyme substitution therapy for phenylketonuria.

Phenylketonuria (PKU) is a genetic inborn error in metabolism that impacts many people globally, with profound individual and societal consequences when left untreated. The journey of phenylalanine ammonia lyase (PAL) from plant enzyme to enzyme substitution therapy for PKU is a fascinating story that illustrates the importance of collaboration between basic scientists and industry in the drug development process. The story begins with the curiosity of plant physiologists about the origin of lignin, a polymer involved in maintaining the rigidity of plants. They learned that the critical element in this synthesis was an intermediary enzyme that deaminates phenylalanine to cinnamic acid and ammonia (later called phenylalanine ammonia lyase or PAL). Recognition of this ability to metabolize phenylalanine led to subsequent consideration of PAL as a treatment for PKU. This was initially attempted as enteral therapy with extracted enzyme, but that showed only minimal efficacy. Crucially, further development of PAL as a therapy for PKU required quantities of enzyme that could only be obtained after successfully cloning the gene, expressing the enzyme in vitro and modifying the protein via PEGylation to enable parenteral administration of this non-mammalian enzyme. Ultimately, PEGylated PAL was developed as an enzyme substitution therapy for PKU now approved under the name "Palynziq." The multidisciplinary academic-industrial partnership engaged throughout this process has been key to the successful pursuit of this therapeutic possibility and serves as a model for the development of future innovative therapies.

Chaperone-like therapy with tetrahydrobiopterin in clinical trials for phenylketonuria: is genotype a predictor of response?

Prospectively enrolled phenylketonuria patients (n=485) participated in an international Phase II clinical trial to identify the prevalence of a therapeutic response to daily doses of sapropterin dihydrochloride (sapropterin, KUVAN(®)). Responsive patients were then enrolled in two subsequent Phase III clinical trials to examine safety, ability to reduce blood Phenylalanine levels, dosage (5-20 mg/kg/day) and response, and bioavailability of sapropterin. We combined phenotypic findings in the Phase II and III clinical trials to classify study-related responsiveness associated with specific alleles and genotypes identified in the patients. We found that 17% of patients showed a response to sapropterin. The patients harbored 245 different genotypes derived from 122 different alleles, among which ten alleles were newly discovered. Only 16.3% of the genotypes clearly conferred a sapropterin-responsive phenotype. Among the different PAH alleles, only 5% conferred a responsive phenotype. The responsive alleles were largely but not solely missense mutations known to or likely to cause misfolding of the PAH subunit. However, the metabolic response was not robustly predictable from the PAH genotypes, based on the study design adopted for these clinical trials, and accordingly it seems prudent to test each person for this phenotype with a standardized protocol.

Evaluation of orally administered PEGylated phenylalanine ammonia lyase in mice for the treatment of Phenylketonuria.

Phenylketonuria (PKU), a Mendelian autosomal recessive phenotype (OMIM 261600), is an inborn error of metabolism causing impaired postnatal cognitive development in the absence of treatment. We used the Pah(enu2/enu2) PKU mouse model to study oral enzyme substitution therapy with various chemically modified formulations of phenylalanine ammonia lyase (Av-p.C503S/p.C565S/p.F18A PAL). In vivo studies with the most therapeutically effective formulation (5kDa PEG-Av-p.C503S/p.C565S/p.F18A PAL) revealed that this conjugate, given orally, yielded statistically significant (p=0.0029) and therapeutically relevant reduction (~40%) in plasma phenylalanine (Phe) levels. Phe reduction occurred in a dose- and loading-dependent manner; sustained clinically and statistically significant reduction of plasma Phe levels was observed with treatment ranging between 0.3 IU and 9 IU and with more frequent and smaller dosings. Oral PAL therapy could potentially serve as an adjunct therapy, perhaps with dietary treatment, and will work independently of phenylalanine hydroxylase (PAH), correcting such forms of hyperphenylalaninemias regardless of the PAH mutations carried by the patient.

Phenylalanine hydroxylase deficiency.

Phenylalanine hydroxylase deficiency is an autosomal recessive disorder that results in intolerance to the dietary intake of the essential amino acid phenylalanine. It occurs in approximately 1:15,000 individuals. Deficiency of this enzyme produces a spectrum of disorders including classic phenylketonuria, mild phenylketonuria, and mild hyperphenylalaninemia. Classic phenylketonuria is caused by a complete or near-complete deficiency of phenylalanine hydroxylase activity and without dietary restriction of phenylalanine most children will develop profound and irreversible intellectual disability. Mild phenylketonuria and mild hyperphenylalaninemia are associated with lower risk of impaired cognitive development in the absence of treatment. Phenylalanine hydroxylase deficiency can be diagnosed by newborn screening based on detection of the presence of hyperphenylalaninemia using the Guthrie microbial inhibition assay or other assays on a blood spot obtained from a heel prick. Since the introduction of newborn screening, the major neurologic consequences of hyperphenylalaninemia have been largely eradicated. Affected individuals can lead normal lives. However, recent data suggest that homeostasis is not fully restored with current therapy. Treated individuals have a higher incidence of neuropsychological problems. The mainstay of treatment for hyperphenylalaninemia involves a low-protein diet and use of a phenylalanine-free medical formula. This treatment must commence as soon as possible after birth and should continue for life. Regular monitoring of plasma phenylalanine and tyrosine concentrations is necessary. Targets of plasma phenylalanine of 120-360 µmol/L (2-6 mg/dL) in the first decade of life are essential for optimal outcome. Phenylalanine targets in adolescence and adulthood are less clear. A significant proportion of patients with phenylketonuria may benefit from adjuvant therapy with 6R-tetrahydrobiopterin stereoisomer. Special consideration must be given to adult women with hyperphenylalaninemia because of the teratogenic effects of phenylalanine. Women with phenylalanine hydroxylase deficiency considering pregnancy should follow special guidelines and assure adequate energy intake with the proper proportion of protein, fat, and carbohydrates to minimize risks to the developing fetus. Molecular genetic testing of the phenylalanine hydroxylase gene is available for genetic counseling purposes to determine carrier status of at-risk relatives and for prenatal testing.

Converting an injectable protein therapeutic into an oral form: phenylalanine ammonia lyase for phenylketonuria.

Phenylalanine ammonia lyase (PAL) has long been recognized as a potential enzyme replacement therapeutic for treatment of phenylketonuria. However, various strategies for the oral delivery of PAL have been complicated by the low intestinal pH, aggressive proteolytic digestion and circulation time in the GI tract. In this work, we report 3 strategies to address these challenges. First, we used site-directed mutagenesis of a chymotrypsin cleavage site to modestly improve protease resistance; second, we used silica sol-gel material as a matrix to demonstrate that a silica matrix can provide protection to entrapped PAL proteins against intestinal proteases, as well as a low pH of 3.5; finally, we demonstrated that PEGylation of AvPAL surface lysines can reduce the inactivation of the enzyme by trypsin.

Preclinical evaluation of multiple species of PEGylated recombinant phenylalanine ammonia lyase for the treatment of phenylketonuria.

Phenylketonuria (PKU) is a metabolic disorder, in which loss of phenylalanine hydroxylase activity results in neurotoxic levels of phenylalanine. We used the Pah(enu2/enu2) PKU mouse model in short- and long-term studies of enzyme substitution therapy with PEGylated phenylalanine ammonia lyase (PEG-PAL conjugates) from 4 different species. The most therapeutically effective PAL (Av, Anabaena variabilis) species was one without the highest specific activity, but with the highest stability; indicating the importance of protein stability in the development of effective protein therapeutics. A PEG-Av-p.C503S/p.C565S-PAL effectively lowered phenylalanine levels in both vascular space and brain tissue over a >90 day trial period, resulting in reduced manifestations associated with PKU, including reversal of PKU-associated hypopigmentation and enhanced animal health. Phenylalanine reduction occurred in a dose- and loading-dependent manner, and PEGylation reduced the neutralizing immune response to the enzyme. Human clinical trials with PEG-Av-p.C503S/p.C565S-PAL as a treatment for PKU are underway.

Garrod's Croonian Lectures (1908) and the charter 'Inborn Errors of Metabolism': albinism, alkaptonuria, cystinuria, and pentosuria at age 100 in 2008.

Garrod presented his concept of 'the inborn error of metabolism' in the 1908 Croonian Lectures to the Royal College of Physicians (London); he used albinism, alkaptonuria, cystinuria and pentosuria to illustrate. His lectures are perceived today as landmarks in the history of biochemistry, genetics and medicine. Garrod gave evidence for the dynamic nature of metabolism by showing involvement of normal metabolites in normal pathways made variant by Mendelian inheritance. His concepts and evidence were salient primarily among biochemists, controversial among geneticists because biometricians were dominant over Mendelists, and least salient among physicians who were not attracted to rare hereditary 'traits'. In 2008, at the centennial of Garrod's Croonian Lectures, each charter inborn error of metabolism has acquired its own genomic locus, a cloned gene, a repertoire of annotated phenotype-modifying alleles, a gene product with known structure and function, and altered function in the Mendelian variant.

A 25-year longitudinal analysis of treatment efficacy in inborn errors of metabolism.

We report here the results of treatment in a panel of 65 inborn errors of metabolism, obtained in the 25th year of a longitudinal project, first reported in 1983. The phenotypic impact of these 65 diseases was scored before and after treatment using a consistent set of parameters, which we have retained to measure change in clinical phenotype throughout the project. We observed significant improvements in the response to treatment for the disease set as a whole. The number of conditions for which there is no response to treatment has progressively decreased; from 31 in 1983, to 20 in 1993, to 17 in 2008. Concomitantly, there has been an increase in the number of conditions that fully respond to treatment (from 8 in 1983 and 1993, to 20 in 2008), and in those for which there is a partial response. Reasons for improved treatment responses include new small molecules, new enzyme replacement therapies, more conditions that can be treated by organ and cell transplantation, and new experimental approaches to substrate reduction and chaperone assisted therapy. However, the most important and new development was not found in one or other particular therapeutic modality but in the access to new knowledge surrounding the individual diseases via the Internet and related resources. Our longitudinal analysis of treatment efficacy for this subset of inborn errors of metabolism continues to constitute a robust and representative assessment of our ability to restore more normal homeostasis in the inborn errors of metabolism.

Structural and biochemical characterization of the therapeutic Anabaena variabilis phenylalanine ammonia lyase.

We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides a structural basis for further engineering of residues that could result in a better therapeutic molecule.

Structure-based epitope and PEGylation sites mapping of phenylalanine ammonia-lyase for enzyme substitution treatment of phenylketonuria.

Protein and peptide therapeutics are of growing importance as medical treatments but can frequently induce an immune response. This work describes the combination of complementary approaches to map the potential immunogenic regions of the yeast Rhodosporidium toruloides phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and to engineer the protein as a human therapeutic agent for the treatment of phenylketonuria (PKU), an inherited metabolic disorder. The identification of B and T cell epitopes on the PAL protein was performed by computational predictions based on the antigenicity and hydrophilicity of proteins, as well as by experimental epitope mapping using a PepSpots peptide array (Jerini AG). Human T cell epitope mapping was performed by applying the computational EpiMatrix algorithm (EpiVax, Inc.) for MHC Class I and Class II associated T cell epitopes on PAL, which predicts which sequences are associated with binding to several different HLA alleles, a requirement for antigen presentation and subsequent primary immune response. By chemical modification through PEGylation of surface lysine residues, it is possible to cover the immunogenic regions of a protein. To evaluate this strategy, we used mass spectrometry to determine which of the immunogenic epitopes are covered by the covalent PEGylation modification strategy. This approach has allowed us to determine whether additional lysines are needed in specific residue locations, or whether certain lysine residues can be removed in order to accomplish complete molecular coverage of the therapeutic enzyme.

Quantitation of phenylalanine and its trans-cinnamic, benzoic and hippuric acid metabolites in biological fluids in a single GC-MS analysis.

We describe a sensitive, simple and convenient stable isotope dilution assay developed to study endogenous metabolism of administered stable isotope-labeled phenylalanine (Phe) in phenylketonuric (PKU) mice treated experimentally with phenylalanine ammonia lyase (PAL). Mouse urine and plasma containing endogenous and administered labeled Phe together with internal standard Phe bearing a different pattern of labeling are converted by in situ diazotization to 2-chloro-3-phenylpropionic acid (CPP). A single solvent extraction is then used to isolate the isotopomers of CPP along with the trans-cinnamic acid (TCA) produced from Phe by PAL, as well as the TCA metabolites benzoic and hippuric acids. This procedure eliminates the need for a separate ion-exchange isolation step for Phe on a second sample aliquot and separate GC-MS analysis. Extracted CPP and the Phe metabolites are then measured by conversion to the pentafluorobenzyl esters and a single analysis by electron capture negative ion GC-MS. The estimated lower limit of quantitation is 0.1 microM.

The PAH gene, phenylketonuria, and a paradigm shift.

"Inborn errors of metabolism," first recognized 100 years ago by Garrod, were seen as transforming evidence for chemical and biological individuality. Phenylketonuria (PKU), a Mendelian autosomal recessive phenotype, was identified in 1934 by Asbjörn Fölling. It is a disease with impaired postnatal cognitive development resulting from a neurotoxic effect of hyperphenylalaninemia (HPA). Its metabolic phenotype is accountable to multifactorial origins both in nurture, where the normal nutritional experience introduces L-phenylalanine, and in nature, where mutations (>500 alleles) occur in the phenylalanine hydroxylase gene (PAH) on chromosome 12q23.2 encoding the L-phenylalanine hydroxylase enzyme (EC 1.14.16.1). The PAH enzyme converts phenylalanine to tyrosine in the presence of molecular oxygen and catalytic amounts of tetrahydrobiopterin (BH4), its nonprotein cofactor. PKU is among the first of the human genetic diseases to enter, through newborn screening, the domain of public health, and to show a treatment effect. This effect caused a paradigm shift in attitudes about genetic disease. The PKU story contains many messages, including: a framework on which to appreciate the complexity of PKU in which phenotype reflects both locus-specific and genomic components; what the human PAH gene tells us about human population genetics and evolution of modern humans; and how our interest in PKU is served by a locus-specific mutation database (http://www.pahdb.mcgill.ca; last accessed 20 March 2007). The individual Mendelian PKU phenotype has no "simple" or single explanation; every patient has her/his own complex PKU phenotype and will be treated accordingly. Knowledge about PKU reveals genomic components of both disease and health.

Recommendations of the 2006 Human Variome Project meeting.

Lists of variations in genomic DNA and their effects have been kept for some time and have been used in diagnostics and research. Although these lists have been carefully gathered and curated, there has been little standardization and coordination, complicating their use. Given the myriad possible variations in the estimated 24,000 genes in the human genome, it would be useful to have standard criteria for databases of variation. Incomplete collection and ascertainment of variants demonstrates a need for a universally accessible system. These and other problems led to the World Heath Organization-cosponsored meeting on June 20-23, 2006 in Melbourne, Australia, which launched the Human Variome Project. This meeting addressed all areas of human genetics relevant to collection of information on variation and its effects. Members of each of eight sessions (the clinic and phenotype, the diagnostic laboratory, the research laboratory, curation and collection, informatics, relevance to the emerging world, integration and federation and funding and sustainability) developed a number of recommendations that were then organized into a total of 96 recommendations to act as a foundation for future work worldwide. Here we summarize the background of the project, the meeting and its recommendations.

FINDbase: a relational database recording frequencies of genetic defects leading to inherited disorders worldwide.

Frequency of INherited Disorders database (FINDbase) (http://www.findbase.org) is a relational database, derived from the ETHNOS software, recording frequencies of causative mutations leading to inherited disorders worldwide. Database records include the population and ethnic group, the disorder name and the related gene, accompanied by links to any corresponding locus-specific mutation database, to the respective Online Mendelian Inheritance in Man entries and the mutation together with its frequency in that population. The initial information is derived from the published literature, locus-specific databases and genetic disease consortia. FINDbase offers a user-friendly query interface, providing instant access to the list and frequencies of the different mutations. Query outputs can be either in a table or graphical format, accompanied by reference(s) on the data source. Registered users from three different groups, namely administrator, national coordinator and curator, are responsible for database curation and/or data entry/correction online via a password-protected interface. Databaseaccess is free of charge and there are no registration requirements for data querying. FINDbase provides a simple, web-based system for population-based mutation data collection and retrieval and can serve not only as a valuable online tool for molecular genetic testing of inherited disorders but also as a non-profit model for sustainable database funding, in the form of a 'database-journal'.

Community genetics and dignity in diversity in the Quebec Network of Genetic Medicine.

The Quebec Network of Genetic Medicine (QNGM), implemented in 1971, has been an integrated program of community genetics serving the population (approximately 7.5 million) of Quebec province in Canada. QNGM reported to the Minister of Social Affairs and operated under an umbrella of universal health insurance in the province. The Network's programs have been run by members of the four university medical schools of the province under the direction of a central committee. A global annual budget was awarded to QNGM from its inception. Among its many programs, QNGM supported: (1) two newborn screening programs (using blood and urine samples) for early diagnosis, treatment and research in phenylketonuria, hereditary tyrosinemia, congenital hypothyroidism, and in a large number of other hereditary metabolic diseases; (2) follow-up of confirmatory diagnostic tests at regional centers, followed by supervision of ambulatory treatment modalities; (3) carrier screening and reproductive counseling for Tay-Sachs and beta-thalassemia diseases; (4) a spectrum of feasibility (research) studies (e.g., screening for biotinidase deficiency, neuroblastoma, hemoglobinopathies, and cystic fibrosis) to inform policy decisions. QNGM performed economic analyses of its major programs and followed prevailing ethical guidelines. Its global budget and integrated structure terminated in 1994, although some of its programs continue independently.

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.