The nitro-chloromethylbenzindoline
prodrug SN29428 has been rationally designed to target tumour
hypoxia. SN29428 is metabolised to
a DNA minor groove
alkylator via
oxygen-sensitive reductive activation initiated by unknown one-electron
reductases. The present study sought to identify
reductases capable of activating SN29428 in tumours. Expression of candidate
reductases in cell lines was modulated using forced expression and, for P450 (
cytochrome)
oxidoreductase (POR), by
zinc finger nuclease-mediated gene knockout. Affymetrix microarray
mRNA expression of flavoreductases was correlated with SN29428 activation in a panel of 23
cancer cell lines. Reductive activation and cytotoxicity of
prodrugs were measured using mass spectrometry and antiproliferative assays, respectively. SN29428 activation under
hypoxia was strongly attenuated by the pan-
flavoprotein inhibitor
diphenyliodonium, but less so by knockout of POR suggesting other flavoreductases contribute. Forced expression of 5-methyltetrahydrofolate-homocysteine
methyltransferase reductase (MTRR), as well as POR, increased activation of SN29428 in hypoxic HCT 116 cells. SN29428 activation strongly correlated with expression of POR and also
FAD-dependent
oxidoreductase domain containing 2 (FOXRED2), in
cancer cell lines. This association persisted after removing the effect of POR
enzyme activity using first-order partial correlation. Forced expression of FOXRED2 increased SN29428 activation and cytotoxicity in hypoxic HEK293 cells and also increased activation of
hypoxia-targeted
prodrugs PR-104A,
tirapazamine and
SN30000, and increased cytotoxicity of the clinical-stage
prodrug TH-302. Thus this study has identified three flavoreductases capable of enzymatically activating SN29428, one of which (FOXRED2) has not previously been implicated in
xenobiotic metabolism. These results will inform future development of
biomarkers predictive of SN29428 sensitivity.