Directed Evolution of an Antitumor Drug (Arginine Deiminase PpADI) for Increased Activity at Physiological pH

Arginine deiminase (ADI; EC 3.5.3.6) has been studied as a potential antitumor drug for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. Studies with human lymphatic leukemia cell lines confirmed that ADI is an antiangiogenic agent for treating le...

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Published in:Chembiochem : a European journal of chemical biology 2010-03, Vol.11 (5), p.691-697
Main Authors: Zhu, Leilei, Tee, Kang Lan, Roccatano, Danilo, Sonmez, Burcu, Ni, Ye, Sun, Zhi-Hao, Schwaneberg, Ulrich
Format: Article
Language:English
Subjects:
Amino Acid Substitution
Angiogenesis Inhibitors - therapeutic use
Antineoplastic Agents - chemistry
Antineoplastic Agents - metabolism
Antineoplastic Agents - therapeutic use
antitumor agents
arginine deiminase
Citrulline - metabolism
directed evolution
Directed Molecular Evolution
Humans
Hydrogen-Ion Concentration
Hydrolases - chemistry
Hydrolases - genetics
Hydrolases - metabolism
Kinetics
leukemia
Leukemia - drug therapy
Mutagenesis, Site-Directed
pH optimum
Protein Engineering
Pseudomonas - enzymology
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Summary:Arginine deiminase (ADI; EC 3.5.3.6) has been studied as a potential antitumor drug for the treatment of arginine-auxotrophic tumors, such as hepatocellular carcinomas (HCCs) and melanomas. Studies with human lymphatic leukemia cell lines confirmed that ADI is an antiangiogenic agent for treating leukemia. The main limitation of ADI from Pseudomonas plecoglossicida (PpADI) lies in its pH-dependent activity profile, its pH optimum is at 6.5. A pH shift from 6.5 to 7.5 results in an approximately 80 % drop in activity. (The pH of human plasma is 7.35 to 7.45.) In order to shift the PpADI pH optimum, a directed-evolution protocol based on an adapted citrulline-screening protocol in microtiter-plate format was developed and validated. A proof of concept for ADI engineering resulted in a pH optimum of pH 7.0 and increased resistance under physiological and slightly alkaline conditions. At pH 7.4, variant M2 (K5T/D44E/H404R) is four times faster than the wild-type PpADI and retains ~50 % of its activity relative to its pH optimum, compared to ~10 % in the case of the wild-type PpADI.
ISSN:1439-4227
1439-7633
DOI:10.1002/cbic.200900717