title: Pseudomonas aeruginosa NfsB and nitro-CBI-DEI--a promising enzyme/prodrug combination for gene directed enzyme prodrug therapy.
abstract: BACKGROUND
  The nitro-chloromethylbenzindoline prodrug nitro-CBI-DEI appears a promising candidate for the anti-cancer strategy gene-directed enzyme prodrug therapy, based on its ability to be converted to a highly cytotoxic cell-permeable derivative by the nitroreductase NfsB from Escherichia coli. However, relative to some other nitroaromatic prodrugs, nitro-CBI-DEI is a poor substrate for E. coli NfsB. To address this limitation we evaluated other nitroreductase candidates from E. coli and Pseudomonas aeruginosa.
  
  
  RESULTS
  Initial screens of candidate genes in the E. coli reporter strain SOS-R2 identified two additional nitroreductases, E. coli NfsA and P. aeruginosa NfsB, as being more effective activators of nitro-CBI-DEI than E. coli NfsB. In monolayer cytotoxicity assays, human colon carcinoma (HCT-116) cells transfected with P. aeruginosa NfsB were >4.5-fold more sensitive to nitro-CBI-DEI than cells expressing either E. coli enzyme, and 23.5-fold more sensitive than untransfected HCT-116. In three dimensional mixed cell cultures, not only were the P. aeruginosa NfsB expressing cells 540-fold more sensitive to nitro-CBI-DEI than pure cultures of untransfected HCT-116, the activated drug that they generated also displayed an unprecedented local bystander effect.
  
  
  CONCLUSIONS
  We posit that the discrepancy in the fold-sensitivity to nitro-CBI-DEI between the two and three dimensional cytotoxicity assays stems from loss of activated drug into the media in the monolayer cultures. This emphasises the importance of evaluating high-bystander GDEPT prodrugs in three dimensional models. The high cytotoxicity and bystander effect exhibited by the NfsB_Pa/nitro-CBI-DEI combination suggest that further preclinical development of this GDEPT pairing is warranted.
authors.affiliation: School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington, New Zealand.
authors.forename: Laura K
authors.initials: LK
authors.lastname: Green
authors.forename: Sophie P
authors.initials: SP
authors.lastname: Syddall
authors.forename: Kendall M
authors.initials: KM
authors.lastname: Carlin
authors.forename: Glenn D
authors.initials: GD
authors.lastname: Bell
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Guise
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Mowday
authors.forename: Michael P
authors.initials: MP
authors.lastname: Hay
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Smaill
authors.forename: Adam V
authors.initials: AV
authors.lastname: Patterson
authors.forename: David F
authors.initials: DF
authors.lastname: Ackerley
mesh_terms: D024201:Bystander Effect; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D057487:Enzyme Therapy; D015870:Gene Expression; D015316:Genetic Therapy; D045325:HCT116 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D009601:Nitroreductases / Q000235:genetics / Q000378:metabolism*; D011355:Prodrugs / Q000378:metabolism* / Q000494:pharmacology / Q000633:toxicity; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D014410:Tumor Stem Cell Assay
chemical_list: D011355:Prodrugs; D009601:Nitroreductases