Engineering and Characterization of an Enzyme Replacement Therapy for Classical Homocystinuria.

Homocystinuria due to loss of cystathionine beta-synthase (CBS) causes accumulation of homocysteine and depletion of cysteine. Current treatments are suboptimal, and thus the development of an enzyme replacement therapy based on PEGylated human truncated CBS (PEG-CBS) has been initiated. Attenuation of potency was observed, which necessitated a screen of several PEG-CBS conjugates for their efficacy to correct and maintain the plasma metabolite profile of murine homocystinuria after repeated administrations interrupted with washouts. We found that CBS coupling with maleimide PEG inconsistently modified the enzyme. In contrast, the PEG-CBS conjugate with 20 kDa N-hydroxysuccinimide-PEG showed very little loss of potency likely due to a reproducible PEGylation resulting in species modified with five PEGs per subunit on average. We developed assays suitable for monitoring the extent of CBS PEGylation and demonstrated a sustainable partial normalization of homocystinuria upon continuous PEG-CBS administration via osmotic pumps. Taken together, we identified the PEG-CBS conjugate suitable for manufacturing and clinical development.

Human cystathionine ß-synthase (CBS) contains two classes of binding sites for S-adenosylmethionine (SAM): complex regulation of CBS activity and stability by SAM.

CBS (cystathionine ß-synthase) is a multidomain tetrameric enzyme essential in the regulation of homocysteine metabolism, whose activity is enhanced by the allosteric regulator SAM (S-adenosylmethionine). Missense mutations in CBS are the major cause of inherited HCU (homocystinuria). In the present study we apply a novel approach based on a combination of calorimetric methods, functional assays and kinetic modelling to provide structural and energetic insight into the effects of SAM on the stability and activity of WT (wild-type) CBS and seven HCU-causing mutants. We found two sets of SAM-binding sites in the C-terminal regulatory domain with different structural and energetic features: a high affinity set of two sites, probably involved in kinetic stabilization of the regulatory domain, and a low affinity set of four sites, which are involved in the enzyme activation. We show that the regulatory domain displays a low kinetic stability in WT CBS, which is further decreased in many HCU-causing mutants. We propose that the SAM-induced stabilization may play a key role in modulating steady-state levels of WT and mutant CBS in vivo. Our strategy may be valuable for understanding ligand effects on proteins with a complex architecture and their role in human genetic diseases and for the development of novel pharmacological strategies.

Pharmacokinetics and pharmacodynamics of PEGylated truncated human cystathionine beta-synthase for treatment of homocystinuria.

AIMS: PEGylated human truncated cystathionine beta-synthase, lacking the C-terminal regulatory domain (PEG-CBS), is a promising preclinical candidate for enzyme replacement therapy in homocystinuria (HCU). It was designed to function as a metabolic sink to decrease the severely elevated plasma and tissue homocysteine concentrations. In this communication, we evaluated pharmacokinetics (PK), pharmacodynamics (PD) and sub-chronic toxicity of PEG-CBS in homocystinuric mice, wild type rats and monkeys to estimate the minimum human efficacious dose for clinical trials. MAIN METHODS: Animal models received single or multiple doses of PEG-CBS. Activity of PEG-CBS and sulfur amino acid metabolites were determined in plasma and used to determine PK and PD. KEY FINDINGS: The plasma half-lives of PEG-CBS after a single subcutaneous (SC) injection were approximately 20, 44 and 73 h in mouse, rat and monkey, respectively. The SC administration of PEG-CBS resulted in a significant improvement or full correction of metabolic imbalance in both blood and tissues of homocystinuric mice. The PD of PEG-CBS in mouse was dose-dependent, but less than dose-proportional, with the maximal efficacy achieved at 8 mg/kg. PEG-CBS was well-tolerated in mice and monkeys, but resulted in dose-dependent minimal-to-moderate inflammation at the injection sites and vacuolated macrophages in rats. Allometric scaling of animal data was linear and the estimated human efficacious dose was determined as 0.66 mg/kg administered once a week. SIGNIFICANCE: These results provide critical preclinical data for the design of first-in-human PEG-CBS clinical trial.