Targeted mutagenesis of the Vibrio fischeri flavin reductase FRase I to improve activation of the anticancer prodrug CB1954.
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.
School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington, New Zealand. pearl.swe@qimr.edu.au
P M
PM
Swe
J N
JN
Copp
L K
LK
Green
C P
CP
Guise
A M
AM
Mowday
J B
JB
Smaill
A V
AV
Patterson
D F
DF
Ackerley
D048908:Aliivibrio fischeri / Q000201:enzymology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001388:Aziridines / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D038181:FMN Reductase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011355:Prodrugs / Q000494:pharmacology*; D013014:SOS Response, Genetics
D000970:Antineoplastic Agents; D001388:Aziridines; D001426:Bacterial Proteins; D011355:Prodrugs; C100099:tretazicar; D038181:FMN Reductase