One-electron
reductases that reduce
nitro compounds in hypoxic human tumour cells are poorly characterized, but are important for targeting
hypoxia with nitroaromatic
prodrugs. Fluorogenic probes with defined
reductase profiles are needed to interrogate the activity of these
enzymes in intact cells. In the present paper, we report a 6-nitroquinolone
ester (FSL-61) as a fluorogenic probe for POR (
NADPH:cytochrome P450 oxidoreductase) activity under
hypoxia, and demonstrate its suitability of monitoring POR by flow cytometry. Reduction of FSL-61 by purified recombinant human POR generated the corresponding
hydroxylamine, which was non-fluorescent, but was reduced further to the fluorescent
amine in cells. Hydrolysis of the
ester side chain facilitated cellular entrapment, enabling detection of heterogeneous POR expression in mixed populations of cells. In addition to POR, forced expression of three other diflavin
reductases [MTRR (
methionine synthase reductase), NDOR1 (
NADPH-dependent diflavin
oxidoreductase 1) and NOS2A (
nitric oxide synthase 2A)] or
NADPH:adrenoredoxin
oxidoreductase in HCT116 cells significantly increased hypoxic activation of FSL-61. This
reductase profile is similar to that for the dinitrobenzamide
prodrug PR-104A under
hypoxia, and fluorogenic metabolism of FSL-61 correlated significantly with
PR-104A activation in a panel of 22 human tumour cell lines. The present study thus demonstrates the utility of FSL-61 for rapid and non-destructive interrogation of the activity of one-electron
reductases in hypoxic cells at the single-cell level.