Abstract
The clinical agent PR-104 is converted systemically to PR-104A, a nitrogen mustard prodrug designed to target tumor hypoxia. Reductive activation of PR-104A is initiated by one-electron oxidoreductases in a process reversed by oxygen. The identity of these oxidoreductases is unknown, with the exception of cytochrome P450 reductase (POR). To identify other hypoxia-selective PR-104A reductases, nine candidate oxidoreductases were expressed in HCT116 cells. Increased PR-104A-cytotoxicity was observed in cells expressing methionine synthase reductase (MTRR), novel diflavin oxidoreductase 1 (NDOR1), and inducible nitric-oxide synthase (NOS2A), in addition to POR. Plasmid-based expression of these diflavin oxidoreductases also enhanced bioreductive metabolism of PR-104A in an anoxia-specific manner. Diflavin oxidoreductase-dependent PR-104A metabolism was suppressed >90% by pan-flavoenzyme inhibition with diphenyliodonium chloride. Antibodies were used to quantify endogenous POR, MTRR, NDOR1, and NOS2A expression in 23 human tumor cell lines; however, only POR protein was detectable and its expression correlated with anoxic PR-104A reduction (r2 = 0.712). An anti-POR monoclonal antibody was used to probe expression using human tissue microarrays; 13 of 19 cancer types expressed detectable POR with 21% of cores (185 of 874) staining positive; this heterogeneity suggests that POR is a useful biomarker for PR-104A activation. Immunostaining for carbonic anhydrase 9 (CAIX), reportedly an endogenous marker of hypoxia, revealed only moderate coexpression (9.6%) of both CAIX and POR across a subset of five cancer types.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This research was supported by the Health Research Council of New Zealand [Grant 08/103].
W.R.W. is a founding scientist, stockholder, and consultant to Proacta Inc., which is undertaking the clinical development of PR-104. A.V.P. is a consultant to and has stock options in Proacta Inc.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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ABBREVIATIONS:
- PR-104
- 2-((2-bromoethyl)(2,4-dinitro-6-((2-(phosphonooxy)ethyl)carbamoyl)phenyl)amino)ethyl methanesulfonate
- PR-104A
- 2-((2-bromoethyl)-2-{[(2-hydroxyethyl) amino]carbonyl}-4,6-dinitroanilino)ethyl methanesulfonate
- TH-302
- N,N′-bis(2-bromoethyl)-(1-methyl-2-nitro-1H-imidazol-5-yl)phosphorodiamidic acid methyl ester
- RH1
- 2,5-di(aziridin-1-yl)-3-(hydroxymethyl)-6-methylcyclohexa-2,5-diene-1,4-dione
- AKR1C3
- aldo-keto reductase 1C3
- POR
- cytochrome P450 reductase
- DPI
- diphenyliodonium
- FDXR
- adrenodoxin oxidoreductase
- CYB5R
- cytochrome B5 reductase
- NOS
- nitric-oxide synthase
- NOS2A
- inducible nitric-oxide synthase
- NQO1
- NAD(P)H quinone oxidoreductase 1
- NQO2
- NAD(P)H quinone oxidoreductase 1
- XD
- xanthine dehydrogenase
- MTRR
- methionine synthase reductase
- NDOR1
- novel diflavin oxidoreductase 1
- FFPE
- formalin fixed paraffin embedded
- LC
- liquid chromatography
- MS/MS
- tandem mass spectrometry
- mAb
- monoclonal antibody
- WT
- wild type
- IHC
- immunohistochemistry
- TMA
- tissue microarray
- CAIX
- carbonic anhydrase 9
- H score
- Histo score
- HIF-1
- hypoxia-inducible factor 1
- UPR
- unfolded protein response
- SN30000/CEN-209
- 3-(3-morpholinopropyl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine-1,4-dioxide
- CB1954
- 5-(aziridin-1-yl)-2,4-dinitrobenzamide.
- Received May 24, 2011.
- Accepted October 7, 2011.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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