title: New enzyme for reductive cancer chemotherapy, YieF, and its improvement by directed evolution.
abstract: Reductive prodrugs, mitomycin C and 5-aziridinyl-2,4-dinitrobenzamide (CB 1954), are nontoxic in their native form but become highly toxic upon reduction. Their effectiveness in cancer chemotherapy can be enhanced by delivering to tumors enzymes with improved prodrug reduction kinetics. We report the discovery of a new prodrug-reducing enzyme, YieF, from Escherichia coli, and the improvement of its kinetics for reducing mitomycin C and CB 1954. A YieF-derived enzyme, Y6, killed HeLa spinner cells with >or=5-fold efficiency than the wild-type enzymes, YieF and NfsA, at a variety of drug and enzyme concentrations and incubation times. With adhered HeLa cells and Salmonella typhimurium SL 7838 bacteria as enzyme delivery vehicle, at least an order of magnitude less of Y6-producing bacteria were required to kill >90% of tumor cells compared with bacteria expressing the wild-type enzymes, which at a comparable level killed < 5% of the cells. Thus, Y6 is a promising enzyme for use in cancer chemotherapy, and Salmonella strain SL 7838, which specifically targets tumors, may be used to deliver the prodrug-activating enzymes to tumors.
authors.affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Sherman Fairchild Science Building, 299 Campus Drive, Stanford, CA 94305, USA.
authors.forename: Yoram
authors.initials: Y
authors.lastname: Barak
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Thorne
authors.forename: David F
authors.initials: DF
authors.lastname: Ackerley
authors.forename: Susan V
authors.initials: SV
authors.lastname: Lynch
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Contag
authors.forename: A
authors.initials: A
authors.lastname: Matin
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism* / Q000494:pharmacology; D001388:Aziridines / Q000378:metabolism / Q000494:pharmacology; D016503:Drug Delivery Systems / Q000379:methods; D029968:Escherichia coli Proteins / Q000008:administration & dosage / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D006367:HeLa Cells; D006801:Humans; D016685:Mitomycin / Q000378:metabolism / Q000494:pharmacology; D009369:Neoplasms / Q000188:drug therapy; D010088:Oxidoreductases / Q000187:drug effects / Q000378:metabolism*; D011355:Prodrugs / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein
chemical_list: D000970:Antineoplastic Agents; D001388:Aziridines; D029968:Escherichia coli Proteins; D011355:Prodrugs; D016685:Mitomycin; C100099:tretazicar; D010088:Oxidoreductases; C508489:YieF protein, E coli