title: The flavoprotein FOXRED2 reductively activates nitro-chloromethylbenzindolines and other hypoxia-targeting prodrugs.
abstract: 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.
authors.affiliation: Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Francis W
authors.initials: FW
authors.lastname: Hunter
authors.affiliation: Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Jagdish K
authors.initials: JK
authors.lastname: Jaiswal
authors.affiliation: Department of Molecular Medicine and Pathology and Bioinformatics Institute, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Hurley
authors.affiliation: Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: H D Sarath
authors.initials: HD
authors.lastname: Liyanage
authors.affiliation: Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Sarah P
authors.initials: SP
authors.lastname: McManaway
authors.affiliation: Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Yongchuan
authors.initials: Y
authors.lastname: Gu
authors.affiliation: Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Susan
authors.initials: S
authors.lastname: Richter
authors.affiliation: Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Jingli
authors.initials: J
authors.lastname: Wang
authors.affiliation: Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Moana
authors.initials: M
authors.lastname: Tercel
authors.affiliation: Department of Molecular Medicine and Pathology and Bioinformatics Institute, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: Cristin G
authors.initials: CG
authors.lastname: Print
authors.affiliation: Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors.forename: William R
authors.initials: WR
authors.lastname: Wilson
authors.affiliation: Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand. Electronic address: f.pruijn@auckland.ac.nz.
authors.forename: Frederik B
authors.initials: FB
authors.lastname: Pruijn
mesh_terms: D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology; D003497:Cyclic N-Oxides / Q000737:chemistry / Q000494:pharmacology; D005420:Flavoproteins / Q000096:biosynthesis*; D045325:HCT116 Cells; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D007211:Indoles / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000096:biosynthesis / Q000378:metabolism*; D011355:Prodrugs / Q000008:administration & dosage / Q000737:chemistry* / Q000378:metabolism*; D014227:Triazines / Q000737:chemistry / Q000494:pharmacology
chemical_list: C570601:CEN-209; D003497:Cyclic N-Oxides; D005420:Flavoproteins; D007211:Indoles; D011355:Prodrugs; D014227:Triazines; C560801:Foxred2 protein, rat; D010088:Oxidoreductases
keywords: FOXRED2; Hypoxia-targeting prodrug; NitroCBI; PR-104; TH-302; Tirapazamine