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

Biochem Pharmacol. 2012 Sep 15;84(6):775-83. doi: 10.1016/j.bcp.2012.07.002. Epub 2012 Jul 14.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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


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