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.
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Research Article|
April 25 2013
FSL-61 is a 6-nitroquinolone fluorogenic probe for one-electron reductases in hypoxic cells
Jiechuang Su;
Jiechuang Su
1Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, 2013 Auckland, New Zealand
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Christopher P. Guise;
Christopher P. Guise
1Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, 2013 Auckland, New Zealand
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William R. Wilson
William R. Wilson
1
1Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, 2013 Auckland, New Zealand
1To whom correspondence should be addressed (email wr.wilson@auckland.ac.nz).
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Publisher: Portland Press Ltd
Received:
November 09 2012
Revision Received:
February 14 2013
Accepted:
March 12 2013
Accepted Manuscript online:
March 12 2013
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2013 Biochemical Society
2013
Biochem J (2013) 452 (1): 79–86.
Article history
Received:
November 09 2012
Revision Received:
February 14 2013
Accepted:
March 12 2013
Accepted Manuscript online:
March 12 2013
Citation
Jiechuang Su, Christopher P. Guise, William R. Wilson; FSL-61 is a 6-nitroquinolone fluorogenic probe for one-electron reductases in hypoxic cells. Biochem J 15 May 2013; 452 (1): 79–86. doi: https://doi.org/10.1042/BJ20121695
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