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: Chris P Guise, Anderson T Wang, Anke Theil, David J Bridewell, William R Wilson, Adam V Patterson
• Journal: Biochemical pharmacology (Biochem Pharmacol) Vol. 74 Issue 6 Pg. 810-20 (Sep 15 2007) ISSN: 0006-2952 [Print] England
• PMID: 17645874 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antineoplastic Agents
• Nitrogen Mustard Compounds
• PR-104A
• Prodrugs
• Oxidoreductases
• NADPH-Ferrihemoprotein Reductase
• NAD(P)H Dehydrogenase (Quinone)
• NQO1 protein, human

Topics

• Antineoplastic Agents (metabolism, pharmacokinetics)
• Cell Death (drug effects)
• Cell Hypoxia
• Cell Line, Tumor
• Humans
• NAD(P)H Dehydrogenase (Quinone) (metabolism)
• NADPH-Ferrihemoprotein Reductase (metabolism)
• Nitrogen Mustard Compounds (metabolism)
• Oxidoreductases (metabolism)
• Prodrugs (metabolism, pharmacokinetics)