title: Homologous recombination repair-dependent cytotoxicity of the benzotriazine di-N-oxide CEN-209: comparison with other hypoxia-activated prodrugs.
abstract: CEN-209 (SN30000) is a second-generation benzotriazine di-N-oxide currently in advanced preclinical development as a hypoxia-activated prodrug (HAP). Herein we describe the DNA repair-, hypoxia- and one-electron reductase-dependence of CEN-209 cytotoxicity. We deployed mutant CHO cell lines to generate DNA repair profiles for CEN-209, and compared the profiles with those for other HAPs. Hypoxic selectivity of CEN-209 was significantly greater than PR-104A and the nitro-chloromethylbenzindoline (nCBI/SN29428) and comparable to tirapazamine and TH-302. CEN-209 was selective for homologous recombination (HR) repair-deficient cells (Rad51d⁻/⁻), but less so than nitrogen mustard prodrugs TH-302 and PR-104A. Further, DNA repair profiles for CEN-209 differed under oxic and hypoxic conditions, with oxic cytotoxicity more dependent on HR. This feature was conserved across all three members of the benzotriazine di-N-oxide class examined (tirapazamine, CEN-209 and CEN-309/SN29751). Enhancing one-electron reduction of CEN-209 by forced expression of a soluble form of NADPH:cytochrome P450 oxidoreductase (sPOR) increased CEN-209 cytotoxicity more markedly under oxic than hypoxic conditions. Comparison of oxygen consumption, H₂O₂ production and metabolism of CEN-209 to the corresponding 1-oxide and nor-oxide reduced metabolites suggested that enhanced oxic cytotoxicity in cells with high one-electron reductase activity is due to futile redox cycling. This study supports the hypothesis that both oxic and hypoxic cell killing by CEN-209 is mechanistically analogous to tirapazamine and is dependent on oxidative DNA damage repaired via multiple pathways. However, HAPs that generate DNA interstrand cross-links, such as TH-302 and PR-104, may be more suitable than benzotriazine di-N-oxides for exploiting reported HR repair defects in hypoxic tumour cells.
authors.affiliation: Auckland Cancer Society Research Centre, University of Auckland, Private Bag 92019, Auckland, New Zealand.
authors.forename: Francis W
authors.initials: FW
authors.lastname: Hunter
authors.forename: Jingli
authors.initials: J
authors.lastname: Wang
authors.forename: Rita
authors.initials: R
authors.lastname: Patel
authors.forename: Huai-Ling
authors.initials: HL
authors.lastname: Hsu
authors.forename: Anthony J R
authors.initials: AJ
authors.lastname: Hickey
authors.forename: Michael P
authors.initials: MP
authors.lastname: Hay
authors.forename: William R
authors.initials: WR
authors.lastname: Wilson
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology / Q000633:toxicity*; D016466:CHO Cells; D006224:Cricetinae; D003497:Cyclic N-Oxides / Q000737:chemistry / Q000494:pharmacology / Q000633:toxicity*; D004249:DNA Damage / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects*; D005838:Genotype; D015394:Molecular Structure; D010100:Oxygen; D011355:Prodrugs / Q000737:chemistry / Q000494:pharmacology / Q000633:toxicity*; D014227:Triazines / Q000737:chemistry / Q000494:pharmacology / Q000633:toxicity*
chemical_list: D000970:Antineoplastic Agents; C570601:CEN-209; D003497:Cyclic N-Oxides; D011355:Prodrugs; D014227:Triazines; D010100:Oxygen