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  • uvrB gene deletion enhances SOS chromotest sensitivity for nitroreductases that preferentially generate the 4-hydroxylamine metabolite of the anti-cancer prodrug CB1954.

uvrB gene deletion enhances SOS chromotest sensitivity for nitroreductases that preferentially generate the 4-hydroxylamine metabolite of the anti-cancer prodrug CB1954.

Journal of biotechnology (2010-08-24)
G A Prosser, A V Patterson, D F Ackerley
ABSTRACT

CB1954 is an anti-cancer prodrug that can be reduced at either of two nitro groups to form cytotoxic metabolites. We describe here two efficient and previously uncharacterized nitroreductases, YfkO from Bacillus subtilis which reduces CB1954 exclusively at the 4-NO(2) position, and NfsA from Klebsiella pneumoniae which preferentially reduces the 2-NO(2) group. Utilizing these novel enzymes, together with three previously characterized nitroreductases, we demonstrate that the Escherichia coli SOS-chromotest assay can differentially detect the 4-nitro versus 2-nitro reduction products of CB1954 following deletion of the nucleotide excision repair gene uvrB, but not mismatch repair (mutS) or methyltransferase (ada/ogt) genes. These findings may hold significance for identification and selection of nitroreductases for CB1954-mediated gene therapy, particularly when targeting tumors that are deficient in nucleotide excision repair. Moreover, we demonstrate that comparative SOS chromotest analysis in wild type and uvrB mutant strains can be used to determine whether or not nucleotide excision repair plays a significant role in processing DNA damage resulting from activation of different nitroaromatic prodrugs.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
CB 1954, solid
Supelco
Nitrofurazone solution, VETRANAL®, 100 μg/mL in acetonitrile, analytical standard
Supelco
Nitrofurazone, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
5-Nitro-2-furaldehyde semicarbazone, ≥97.0% (HPLC)