Bacterial Cytosine Deaminase Mutants Created by Molecular Engineering Demonstrate Improved 5FC-Mediated Cell Killing In Vitro and In Vivo

• Published in: Cancer research (Chicago, Ill.) Vol. 69; no. 11; pp. 4791 - 4799
• Main Authors: Fuchita, Michi, Ardiani, Andressa, Zhao, Lei, Serve, Kinta, Stoddard, Barry L., Black, Margaret E.
• Format: Journal Article
• Language: English
• Published: 01-06-2009
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/0008-5472.CAN-09-0615

Cytosine deaminase is used in combination with 5-fluorocytosine as an enzyme-prodrug combination for targeted genetic cancer treatment. This approach is limited by inefficient gene delivery and poor prodrug conversion activities. Previously, we reported individual point mutations within the substrate binding pocket of bacterial cytosine deaminase (bCD) that result in marginal improvements in the ability to sensitize cells to 5FC. Here we describe an expanded random mutagenesis and selection experiment that yielded enzyme variants which provide significant improvement in prodrug sensitization. Three of these mutants were evaluated using enzyme kinetic analyses and then assayed in three cancer cell lines for 5FC sensitization, bystander effects and formation of 5FU metabolites. All variants displayed 18 to 19-fold shifts in substrate preference toward 5FC, a significant reduction in IC
50
values and improved bystander effect compared to wild-type bCD. In a xenograft tumor model the best enzyme mutant was shown to prevent tumor growth at much lower doses of 5FC than is observed when tumor cells express wild-type bCD. Crystallographic analyses of this construct demonstrates the basis for improved activity towards 5FC, and also how two different mutagenesis strategies yield closely related, but mutually exclusive mutations that each result in a significant alteration of enzyme specificity.