Targeted mutagenesis of the Vibrio fischeri flavin reductase FRase I to improve activation of the anticancer prodrug CB1954.

Abstract

Phase I/II cancer gene therapy trials of the Escherichia coli nitroreductase NfsB in partnership with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] have indicated that CB1954 toxicity is dose-limiting at concentrations far below the enzyme K(M). Here we report that the flavin reductase FRase I from Vibrio fischeri is also a CB1954 nitroreductase, which has a substantially lower apparent K(M) than E. coli NfsB. To enhance the activity of FRase I with CB1954 we used targeted mutagenesis and an E. coli SOS reporter strain to engineer single- and multi-residue variants that possess a substantially reduced apparent K(M) and an increased k(cat)/K(M) relative to the wild type enzyme. In a bacteria-delivered model for enzyme prodrug therapy, the engineered FRase I variants were able to kill human colon carcinoma (HCT-116) cells at significantly lower CB1954 concentrations than wild type FRase I or E. coli NfsB.

• Authors: P M Swe, J N Copp, L K Green, C P Guise, A M Mowday, J B Smaill, A V Patterson, D F Ackerley
• Journal: Biochemical pharmacology (Biochem Pharmacol) Vol. 84 Issue 6 Pg. 775-83 (Sep 15 2012) ISSN: 1873-2968 [Electronic] England
• PMID: 22796568 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2012 Elsevier Inc. All rights reserved.

Chemical References

• Antineoplastic Agents
• Aziridines
• Bacterial Proteins
• Prodrugs
• tretazicar
• FMN Reductase

Topics

• Aliivibrio fischeri (enzymology)
• Antineoplastic Agents (pharmacology)
• Aziridines (pharmacology)
• Bacterial Proteins (genetics, metabolism)
• Cell Line, Tumor
• Cell Proliferation (drug effects)
• Escherichia coli (genetics)
• FMN Reductase (genetics, metabolism)
• Humans
• Kinetics
• Models, Molecular
• Mutagenesis, Site-Directed
• Point Mutation
• Prodrugs (pharmacology)
• SOS Response, Genetics