title: Identification of human reductases that activate the dinitrobenzamide mustard prodrug PR-104A: a role for NADPH:cytochrome P450 oxidoreductase under hypoxia.
abstract: Hypoxia is a common trait found in many solid tumours and thus represents a therapeutic target with considerable potential. PR-104, a hypoxia-activated prodrug currently in clinical trial, is a water-soluble phosphate ester which is converted in vivo to the corresponding alcohol, PR-104A. This 3,5-dinitrobenzamide-2-nitrogen mustard is activated by reduction to the corresponding 5-hydroxylamine (PR-104H) and 5-amine (PR-104M) in hypoxic cells. The clinical effectiveness of PR-104 will depend in part on the expression of reductases within tumours that can effect this reduction. Here, we evaluate the roles of NADPH:cytochrome P450 oxidoreductase (CYPOR; E.C.1.6.2.4) and NAD(P)H:quinone oxidoreductase (NQO1; E.C.1.6.99.2) as candidate PR-104A reductases. A weak correlation was observed between NQO1 activity and aerobic cytotoxicity in a panel of eight tumour cell lines. However, overexpression of human NQO1 did not increase cytotoxicity of PR-104A or the formation of PR-104H/M, showing that PR-104A is not a substrate for NQO1. Overexpression of human CYPOR did, however, increase the hypoxic cytotoxicity of PR-104A, and its metabolism to PR-104H and PR-104M, demonstrating it to be a PR-104A reductase. To assess the contribution of CYPOR to overall activation of PR-104A in hypoxic SiHa cells, a combination of siRNA transfection and antisense expression were used to suppress CYPOR protein by 91% (+/-3%), a phenotype which conferred 45% (+/-7%) decrease in cytotoxic potency of PR-104A. Regression analysis of all CYPOR depletion data was found to correlate with cytoprotection and metabolism (p<0.001). Residual PR-104A reductase activity could be inhibited by the flavoprotein inhibitor diphenyliodonium. We conclude that CYPOR is an important PR-104A reductase, but that other flavoenzymes also contribute to its activation in hypoxic SiHa cells.
authors.affiliation: Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors.forename: Chris P
authors.initials: CP
authors.lastname: Guise
authors.forename: Anderson T
authors.initials: AT
authors.lastname: Wang
authors.forename: Anke
authors.initials: A
authors.lastname: Theil
authors.forename: David J
authors.initials: DJ
authors.lastname: Bridewell
authors.forename: William R
authors.initials: WR
authors.lastname: Wilson
authors.forename: Adam V
authors.initials: AV
authors.lastname: Patterson
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism / Q000493:pharmacokinetics; D016923:Cell Death / Q000187:drug effects; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D006801:Humans; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D009588:Nitrogen Mustard Compounds / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism; D011355:Prodrugs / Q000378:metabolism* / Q000493:pharmacokinetics
chemical_list: D000970:Antineoplastic Agents; D009588:Nitrogen Mustard Compounds; C524170:PR-104A; D011355:Prodrugs; D010088:Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human