PT  - JOURNAL ARTICLE
AU  - Christopher P Guise
AU  - Maria R Abbattista
AU  - Smitha R Tipparaju
AU  - Neil K Lambie
AU  - Jiechuang Su
AU  - Dan Li
AU  - William R Wilson
AU  - Gabi U Dachs
AU  - Adam V Patterson
TI  - Diflavin Oxidoreductases Activate the Bioreductive Prodrug PR104A Under Hypoxia
AID  - 10.1124/mol.111.073759
DP  - 2011 Oct 07
TA  - Molecular Pharmacology
PG  - mol.111.073759
4099  - http://molpharm.aspetjournals.org/content/early/2011/10/07/mol.111.073759.short
4100  - http://molpharm.aspetjournals.org/content/early/2011/10/07/mol.111.073759.full
AB  - The clinical agent PR-104 is converted systemically to PR-104A, a nitrogen mustard prodrug designed to target tumour 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 PR104A in an anoxia-specific manner. Diflavin oxidoreductase-dependent PR104A 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 tumour 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 POR as a useful biomarker for PR-104A activation. Immunostaining for carbonic anhydrase 9 (CAIX), reportedly an endogenous marker of hypoxia, revealed only moderate co-expression (9.6%) of both CAIX and POR across a subset of five cancer types.