Paul A. Fitzpatrick

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.

Delivery of an enzyme-IGFII fusion protein to the mouse brain is therapeutic for mucopolysaccharidosis type IIIB

Significance Mucopolysaccharidosis type IIIB (MPS IIIB) is a devastating and currently untreatable disease affecting mainly the brain. The cause is lack of the lysosomal enzyme, α–N-acetylglucosaminidase (NAGLU), and storage of heparan sulfate. Using a mouse model of MPS IIIB, we administered a modified NAGLU by injection into the left ventricle of the brain, bypassing the blood–brain barrier. The modification consisted of a fragment of IGFII, which allows receptor-mediated uptake and delivery to lysosomes. The modified enzyme was taken up avidly by cells in both brain and liver, where it reduced pathological accumulation of heparan sulfate and other metabolites to normal or near-normal levels. The results suggest the possibility of treatment for MPS IIIB. Mucopolysaccharidosis type IIIB (MPS IIIB, Sanfilippo syndrome type B) is a lysosomal storage disease characterized by profound intellectual disability, dementia, and a lifespan of about two decades. The cause is mutation in the gene encoding α–N-acetylglucosaminidase (NAGLU), deficiency of NAGLU, and accumulation of heparan sulfate. Impediments to enzyme replacement therapy are the absence of mannose 6-phosphate on recombinant human NAGLU and the blood–brain barrier. To overcome the first impediment, a fusion protein of recombinant NAGLU and a fragment of insulin-like growth factor II (IGFII) was prepared for endocytosis by the mannose 6-phosphate/IGFII receptor. To bypass the blood–brain barrier, the fusion protein (“enzyme”) in artificial cerebrospinal fluid (“vehicle”) was administered intracerebroventricularly to the brain of adult MPS IIIB mice, four times over 2 wk. The brains were analyzed 1–28 d later and compared with brains of MPS IIIB mice that received vehicle alone or control (heterozygous) mice that received vehicle. There was marked uptake of the administered enzyme in many parts of the brain, where it persisted with a half-life of approximately 10 d. Heparan sulfate, and especially disease-specific heparan sulfate, was reduced to control level. A number of secondary accumulations in neurons [β-hexosaminidase, LAMP1(lysosome-associated membrane protein 1), SCMAS (subunit c of mitochondrial ATP synthase), glypican 5, β-amyloid, P-tau] were reduced almost to control level. CD68, a microglial protein, was reduced halfway. A large amount of enzyme also appeared in liver cells, where it reduced heparan sulfate and β-hexosaminidase accumulation to control levels. These results suggest the feasibility of enzyme replacement therapy for MPS IIIB.

Structural basis for the inhibition of poly(ADP-ribose) polymerases 1 and 2 by BMN 673, a potent inhibitor derived from dihydropyridophthalazinone.

BMN 673, a novel PARP1/2 inhibitor in clinical development with substantial tumor cytotoxicity, forms extensive hydrogen-bonding and π-stacking in the nicotinamide pocket, with its unique disubstituted scaffold extending towards the less conserved edges of the pocket. These interactions might provide structural insight into the ability of BMN 673 to both inhibit catalysis and affect DNA-binding activity.

Erythrocyte-mediated delivery of phenylalanine ammonia lyase for the treatment of phenylketonuria in BTBR-Pahenu2 mice

Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by defects in the phenylalanine hydroxylase gene. Preclinical and clinical investigations suggest that phenylalanine ammonia lyase (PAL) could be an effective alternative for the treatment of PKU. The aim of this study is to investigate if erythrocytes loaded with PAL may act as a safe delivery system able to overcome bioavailability issues and to provide, in vivo, a therapeutically relevant concentration of enzyme. Murine erythrocytes were loaded with recombinant PAL from Anabaena variabilis (rAvPAL) and their ability to perform as bioreactors was assessed in vivo in adult BTBR-Pah(enu2) mice, the genetic murine model of PKU. Three groups of mice were treated with a single i.v. injection of rAvPAL-RBCs at three different doses to select the most appropriate one for assessment of efficacy. Repeated administrations at 9-10 day-intervals of the selected dose for 10 weeks showed that the therapeutic effect was persistent and not affected by the generation of antibodies induced by the recombinant enzyme. This therapeutic approach deserves further in vivo evaluation either as a potential option for the treatment of PKU patients or as a possible model for the substitutive enzymatic treatment of other inherited metabolic disorders.

Formulation and PEGylation optimization of the therapeutic PEGylated phenylalanine ammonia lyase for the treatment of phenylketonuria

Phenylketonuria (PKU) is a genetic metabolic disease in which the decrease or loss of phenylalanine hydroxylase (PAH) activity results in elevated, neurotoxic levels of phenylalanine (Phe). Due to many obstacles, PAH enzyme replacement therapy is not currently an option. Treatment of PKU with an alternative enzyme, phenylalanine ammonia lyase (PAL), was first proposed in the 1970s. However, issues regarding immunogenicity, enzyme production and mode of delivery needed to be overcome. Through the evaluation of PAL enzymes from multiple species, three potential PAL enzymes from yeast and cyanobacteria were chosen for evaluation of their therapeutic potential. The addition of polyethylene glycol (PEG, MW = 20,000), at a particular ratio to modify the protein surface, attenuated immunogenicity in an animal model of PKU. All three PEGylated PAL candidates showed efficacy in a mouse model of PKU (BTBR Pahenu2) upon subcutaneous injection. However, only PEGylated Anabaena variabilis (Av) PAL-treated mice demonstrated sustained low Phe levels with weekly injection and was the only PAL evaluated that maintained full enzymatic activity upon PEGylation. A PEGylated recombinant double mutant version of AvPAL (Cys503Ser/Cys565Ser), rAvPAL-PEG, was selected for drug development based on its positive pharmacodynamic profile and favorable expression titers. PEGylation was shown to be critical for rAvPAL-PEG efficacy as under PEGylated rAvPAL had a lower pharmacodynamic effect. rAvPAL and rAvPAL-PEG had poor stability at 4°C. L-Phe and trans-cinnamate were identified as activity stabilizing excipients. rAvPAL-PEG is currently in Phase 3 clinical trials to assess efficacy in PKU patients.

Clearance of Heparan Sulfate and Attenuation of CNS Pathology by Intracerebroventricular BMN 250 in Sanfilippo Type B Mice

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB), caused by inherited deficiency of α-N-acetylglucosaminidase (NAGLU), required for lysosomal degradation of heparan sulfate (HS), is a pediatric neurodegenerative disorder with no approved treatment. Intracerebroventricular (ICV) delivery of a modified recombinant NAGLU, consisting of human NAGLU fused with insulin-like growth factor 2 (IGF2) for enhanced lysosomal targeting, was previously shown to result in marked enzyme uptake and clearance of HS storage in the Naglu−/− mouse brain. To further evaluate regional, cell type-specific, and dose-dependent biodistribution of NAGLU-IGF2 (BMN 250) and its effects on biochemical and histological pathology, Naglu−/− mice were treated with 1–100 μg ICV doses (four times over 2 weeks). 1 day after the last dose, BMN 250 (100 μg doses) resulted in above-normal NAGLU activity levels, broad biodistribution, and uptake in all cell types, with NAGLU predominantly localized to neurons in the Naglu−/− mouse brain. This led to complete clearance of disease-specific HS and reduction of secondary lysosomal defects and neuropathology across various brain regions lasting for at least 28 days after the last dose. The substantial brain uptake of NAGLU attainable by this highest ICV dosage was required for nearly complete attenuation of disease-driven storage accumulations and neuropathology throughout the Naglu−/− mouse brain.