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Published on: November 5, 2014
Directed evolution of an antitumor drug (arginine deiminase PpADI) for increased activity at physiological pH
Leilei Zhu1, Kang Lan Tee, Danilo Roccatano
1Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52056 Aachen, Germany.
Abstract:
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 approximately 50 % of its activity relative to its pH optimum, compared to approximately 10 % in the case of the wild-type PpADI.
Insights
Arginine deiminase (ADI) shows promise for treating certain cancers. Engineering improved its activity at physiological pH, making it a more viable therapeutic option for arginine-dependent tumors.
Area of Science:
- Biochemistry
- Enzymology
- Drug Development
Background:
- Arginine deiminase (ADI) is investigated as an antitumor agent for arginine-auxotrophic cancers like HCC and melanoma.
- ADI exhibits antiangiogenic properties beneficial for leukemia treatment.
- Pseudomonas plecoglossicida ADI (PpADI) has a suboptimal pH optimum of 6.5, limiting its efficacy in human plasma (pH 7.35–7.45).
Purpose of the Study:
- To engineer Pseudomonas plecoglossicida arginine deiminase (PpADI) for improved activity at physiological pH.
- To overcome the pH-dependent limitations of PpADI for enhanced therapeutic applications.
Main Methods:
- A directed-evolution strategy was employed.
- An adapted citrulline-screening protocol in microtiter plates was utilized for enzyme evolution.
- Variant screening focused on enhanced activity and stability under physiological and alkaline conditions.
Main Results:
- Engineered PpADI variants demonstrated a shifted pH optimum to 7.0.
- Variant M2 (K5T/D44E/H404R) exhibited increased resistance to physiological and alkaline conditions.
- At pH 7.4, variant M2 showed fourfold higher activity than wild-type PpADI, retaining 50% activity versus 10% for wild-type.
Conclusions:
- Directed evolution successfully enhanced PpADI's pH profile for therapeutic relevance.
- Engineered ADI variants offer improved efficacy for treating arginine-dependent tumors under physiological conditions.
- This engineered enzyme represents a promising advancement in cancer therapy development.
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