title: Identification of one-electron reductases that activate both the hypoxia prodrug SN30000 and diagnostic probe EF5.
abstract: SN30000 is a second-generation benzotriazine-N-oxide hypoxia-activated prodrug scheduled for clinical trial. Previously we showed that covalent binding of the hypoxia probe EF5 predicts metabolic activation of SN30000 in a panel of cancer cell lines under anoxia, suggesting that they are activated by the same reductases. However the identity of these reductases is unknown. Here, we test whether forced expression of nine oxidoreductases with known or suspected roles in bioreductive prodrug metabolism (AKR1C3, CYB5R3, FDXR, MTRR, NDOR1, NOS2A, NQO1, NQO2 and POR) enhances oxic or anoxic reduction of SN30000 and EF5 by HCT116 cells. Covalent binding of (14)C-EF5 and reduction of SN30000 to its 1-oxide and nor-oxide metabolites was highly selective for anoxia in all lines, with significantly elevated anoxic metabolism of both compounds in lines over-expressing POR, MTRR, NOS2A or NDOR1. There was a strong correlation between EF5 binding and SN30000 metabolism under anoxia across the cell lines (R(2)=0.84, p=0.0001). Antiproliferative potency of SN30000 under anoxia was increased most strongly by overexpression of MTRR and POR. Transcript abundance in human tumours, evaluated using public domain mRNA expression data, was highest for MTRR, followed by POR, NOS2A and NDOR1, with little variation between tumour types. Immunostaining of tissue microarrays demonstrated variable MTRR protein expression across 517 human cancers with most displaying low expression. In conclusion, we have identified four diflavin reductases (POR, MTRR, NOS2A and NDOR1) capable of reducing both SN30000 and EF5, further supporting use of 2-nitroimidazole probes to predict the ability of hypoxic cells to activate SN30000.
authors.affiliation: Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors.forename: Jingli
authors.initials: J
authors.lastname: Wang
authors.affiliation: Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, New Zealand.
authors.forename: Chris P
authors.initials: CP
authors.lastname: Guise
authors.affiliation: Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, New Zealand; Department of Pathology, University of Otago Christchurch, PO Box 4345, Christchurch 8140, New Zealand.
authors.forename: Gabi U
authors.initials: GU
authors.lastname: Dachs
authors.affiliation: Department of Pathology, University of Otago Christchurch, PO Box 4345, Christchurch 8140, New Zealand.
authors.forename: Yen
authors.initials: Y
authors.lastname: Phung
authors.affiliation: Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors.forename: Annie Huai-Ling
authors.initials: AH
authors.lastname: Hsu
authors.affiliation: Department of Anatomical Pathology, Prince of Wales Hospital, Barker Street, Randwick, NSW 2031, Australia.
authors.forename: Neil K
authors.initials: NK
authors.lastname: Lambie
authors.affiliation: Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, New Zealand.
authors.forename: Adam V
authors.initials: AV
authors.lastname: Patterson
authors.affiliation: Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, New Zealand. Electronic address: wr.wilson@auckland.ac.nz.
authors.forename: William R
authors.initials: WR
authors.lastname: Wilson
mesh_terms: D015687:Cell Hypoxia; D003497:Cyclic N-Oxides / Q000494:pharmacology*; D004583:Electrons; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D005434:Flow Cytometry; D045325:HCT116 Cells; D006801:Humans; D010088:Oxidoreductases / Q000378:metabolism*; D011355:Prodrugs / Q000494:pharmacology*; D014227:Triazines / Q000494:pharmacology*
chemical_list: C570601:CEN-209; D003497:Cyclic N-Oxides; D011355:Prodrugs; D014227:Triazines; D010088:Oxidoreductases; C111241:methionine synthase reductase; D005287:Ferredoxin-NADP Reductase
keywords: EF5; Hypoxia; Hypoxia-activated prodrugs; Methionine synthase reductase; SN30000