Production of "biobetter" variants of glucarpidase with enhanced enzyme activity.

Abstract

Glucarpidase, also known as carboxypeptidase G2, is a Food and Drug Administration-approved enzyme used in targeted cancer strategies such as antibody-directed enzyme prodrug therapy (ADEPT). It is also used in drug detoxification when cancer patients have excessive levels of the anti-cancer agent methotrexate. The application of glucarpidase is limited by its potential immunogenicity and limited catalytic efficiency. To overcome these pitfalls, mutagenesis was applied to the glucarpidase gene of Pseudomonas sp. strain RS-16 to isolate three novels "biobetter" variants with higher specific enzyme activity. DNA sequence analysis of the genes for the variants showed that each had a single point mutation, resulting in the amino acid substitutions: I100 T, G123S and T239 A. Km, Vmax and Kcat measurements confirmed that each variant had increased catalytic efficiency relative to wild type glucarpidase. Additionally, circular dichroism studies indicated that they had a higher alpha-helical content relative to the wild type enzyme. However, three different software packages predicted that they had reduced protein stability, which is consistent with having higher activities as a tradeoff. The novel glucarpidase variants presented in this work could pave the way for more efficient drug detoxification and might allow dose escalation during chemotherapy. They also have the potential to increase the efficiency of ADEPT and to reduce the number of treatment cycles, thereby reducing the risk that patients will develop antibodies to glucarpidase.

• Authors: Alanod D Al-Qahtani, Sara S Bashraheel, Fatma B Rashidi, C David O'Connor, Atilio Reyes Romero, Alexander Domling, Sayed K Goda
• Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie (Biomed Pharmacother) Vol. 112 Pg. 108725 (Apr 2019) ISSN: 1950-6007 [Electronic] France
• PMID: 30970523 (Publication Type: Journal Article)
• Copyright: Copyright © 2019. Published by Elsevier Masson SAS.

Chemical References

• Antimetabolites, Antineoplastic
• Prodrugs
• Recombinant Proteins
• glucarpidase
• gamma-Glutamyl Hydrolase
• Methotrexate

Topics

• Antimetabolites, Antineoplastic (adverse effects, pharmacokinetics)
• Cloning, Molecular
• Drug Design
• Enzyme Stability
• Enzyme Therapy (methods)
• Methotrexate (adverse effects, pharmacokinetics)
• Models, Molecular
• Neoplasms (drug therapy, immunology)
• Point Mutation
• Prodrugs (administration & dosage, therapeutic use)
• Pseudomonas putida (genetics)
• Recombinant Proteins (genetics, immunology, therapeutic use)
• gamma-Glutamyl Hydrolase (genetics, immunology, therapeutic use)