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 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.
Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52056 Aachen, Germany.
Leilei
L
Zhu
Kang Lan
KL
Tee
Danilo
D
Roccatano
Burcu
B
Sonmez
Ye
Y
Ni
Zhi-Hao
ZH
Sun
Ulrich
U
Schwaneberg
D019943:Amino Acid Substitution; D020533:Angiogenesis Inhibitors / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000378:metabolism / Q000627:therapeutic use; D002956:Citrulline / Q000378:metabolism; D019020:Directed Molecular Evolution; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D007938:Leukemia / Q000188:drug therapy; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D011549:Pseudomonas / Q000201:enzymology
D020533:Angiogenesis Inhibitors; D000970:Antineoplastic Agents; D002956:Citrulline; D006867:Hydrolases; C019723:arginine deiminase