title: Directed arginine deiminase evolution for efficient inhibition of arginine-auxotrophic melanomas.
abstract: Arginine deiminase (ADI) is a therapeutic protein for cancer therapy of arginine-auxotrophic tumors. However, ADI's application as anticancer drug is hampered by its low activity for arginine under physiological conditions mainly due to its high "K M" (S₀.₅) values which are often 1 magnitude higher than the arginine concentration in blood (0.10-0.12 mM arginine in human plasma). Previous evolution campaigns were directed by us with the aim of boosting activity of PpADI (ADI from Pseudomonas plecoglossicida, k cat = 0.18 s(-1); S₀.₅ = 1.30 mM), and yielded variant M6 with slightly reduced S₀.₅ values and enhanced k cat (S₀.₅ = 0.81 mM; k cat = 11.64 s(-1)). In order to further reduce the S₀.₅ value and to increase the activity of PpADI at physiological arginine concentration, a more sensitive screening system based on ammonia detection in 96-well microtiter plate to reliably detect ≥0.005 mM ammonia was developed. After screening ~5,500 clones with the ammonia detection system (ADS) in two rounds of random mutagenesis and site-directed mutagenesis, variant M19 with increased k cat value (21.1 s(-1); 105.5-fold higher compared to WT) and reduced S₀.₅ value (0.35 mM compared to 0.81 mM (M6) and 1.30 mM (WT)) was identified. Improved performance of M19 was validated by determining IC₅₀ values for two melanoma cell lines. The IC₅₀ value for SK-MEL-28 dropped from 8.67 (WT) to 0.10 (M6) to 0.04 μg/mL (M19); the IC₅₀ values for G361 dropped from 4.85 (WT) to 0.12 (M6) to 0.05 μg/mL (M19).
authors.affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.
authors.forename: Feng
authors.initials: F
authors.lastname: Cheng
authors.forename: Leilei
authors.initials: L
authors.lastname: Zhu
authors.forename: Hongqi
authors.initials: H
authors.lastname: Lue
authors.forename: Jürgen
authors.initials: J
authors.lastname: Bernhagen
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schwaneberg
mesh_terms: D000641:Ammonia / Q000032:analysis; D000970:Antineoplastic Agents / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D045744:Cell Line, Tumor; D019020:Directed Molecular Evolution / Q000379:methods*; D004353:Drug Evaluation, Preclinical; D057166:High-Throughput Screening Assays; D006801:Humans; D006867:Hydrolases / Q000235:genetics* / Q000378:metabolism*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008545:Melanoma / Q000188:drug therapy; D016296:Mutagenesis; D011549:Pseudomonas / Q000201:enzymology*
chemical_list: D000970:Antineoplastic Agents; D000641:Ammonia; D001120:Arginine; D006867:Hydrolases; C019723:arginine deiminase