Abstract Background: SN30000 is a 2nd generation benzotriazine-N-oxide hypoxia-activated prodrug (tirapazamine analogue) and is currently in preclinical development. We have previously shown that reductive activation of the hypoxia marker EF5 and SN30000 are correlated across a panel of cancer cells under hypoxia (Wang et al, Clin Can Res 18:1684, 2012), suggesting that they are activated by the same reductases. The identities of these reductases are not known, except NADPH:cytochrome p450 oxidoreductase (POR). Further, knockout of POR in HCT116 and SiHa cell lines had little or no effect on reduction of either compound. Here, we utilise a previously reported panel of HCT116 isogenic cell lines (Guise et al, Can Res 70:1573, 2010; Mol Pharmacol 81:31, 2012) over-expressing nine oxidoreductases with known or suspected roles in bioreductive prodrug metabolism (AKR1C3, CYB5R, FDXR, MTRR, NDOR1, NOS2A, NQO1, NQO2 and POR) to identify reductases capable of reducing SN30000 and EF5. Methods: Drug metabolism rates under oxic and anoxic conditions were determined in each cell line. The metabolism of SN 30000 to its corresponding 1-oxide and nor-oxide metabolites were measured by LC/MS/MS and the reduction of EF5 by quantifying covalent protein binding of 14C-EF5. SN30000 anoxic cytotoxicity was determined by clonogenic assay. Oxidoreductase expression profiling in cancer patient samples was conducted using Oncomine Premium edition. Results: There was minimal reductive metabolism under oxic conditions (0.1 to 4.8% of anoxic values) for both compounds in all cell lines. Under anoxic conditions, 4 to 5 fold differences were obtained for EF5 and SN30000 across the 10 isogenic cell lines. Our results confirmed our previous observations that POR is one of the reductases capable for SN30000 and EF5 metabolism. In addition to POR, three other members of the diflavin oxidoreductase family (MTRR, NOS2A and NDOR1) had increased anoxic metabolism rates compared to the parental line for both SN30000 and EF5. There was a good correlation between EF5 binding and SN30000 anoxic metabolism across the cell lines (R2=0.78, p=0.0015). SN30000 cytotoxicity under anoxic conditions was also increased in the cells over-expressing POR, NDOR1, MTRR and NOS2A. Expression of these four reductases showed considerable variation between individual human tumour biopsies. In general, the ranking of transcript abundance between the four reductases was POR > MTRR > NOS2A ≈ NDOR1 in most cancer types. In breast cancer, expression of these reductases was similar in tumours with high and low expression of the hypoxia marker gene CA9. Conclusion: we have identified 4 flavoreductases (POR, MTRR, NOS2A and NDOR1) that are capable of activating both SN30000 and EF5 under anoxia when overexpressed in HCT116 cells. However it is not yet clear whether they play a significant role at basal levels of expression in cell lines or tumours. Citation Format: Jingli Wang, Chris P. Guise, Huai-Ling Hsu, Daniel Hurley, William R. Wilson, Adam V. Patterson. Identification of reductases capable of metabolic activation of hypoxia targeting prodrug SN30000 and hypoxia marker EF5. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2111. doi:10.1158/1538-7445.AM2013-2111