Drug-DNA adducts as biomarkers for metabolic activation of the nitroaromatic nitrogen mustard prodrug PR-104A

作者：Stornetta Alessia; Deng Kai Cheng Kieren; Danielli Sara; Liyanage H D Sarath; Sturla Shana J^*; Wilson William R^*; Gu Yongchuan

来源：Biochemical Pharmacology, 2018, 154: 64-74.

DOI：10.1016/j.bcp.2018.04.004

• PR-104A
• DNA adducts
• Predictive biomarkers
• AKR1C3
• P450 oxidoreductase

PR-104A is a clinical-stage nitrogen mustard prodrug that is activated for DNA alkylation by reduction of a nitro group to the corresponding hydroxylamine (PR-104H) or amine (PR-104M). Metabolic reduction is catalysed by flavoreductases such as cytochrome P450 oxidoreductase (POR) under hypoxia, or by aldo-ketoreductase 1C3 (AKR1C3) independently of hypoxia. The unstable reduced metabolites are challenging to measure in biological samples, and biomarkers of the metabolic activation of PR-104A have not been used in the clinical evaluation of PR-104 to date. Here, we employ a selected reaction monitoring mass spectrometry assay for DNA crosslinks to assess the capacity of human cancer cells to bioactivate PR-104A. We also test whether the more abundant DNA monoadducts could be used for the same purpose. DNA monoadducts and crosslinks from PR-104A itself, and from its reduced metabolites, accumulated over 4h in AKRlC3-expressing TF1 erythroleukaemia cells under hypoxia, whereas intracellular concentrations of unstable PR-104H and PR-104M reached steady state within 1h. We then varied rates of PR-104A reduction by manipulating hypoxia or reductase expression in a panel of cell lines, in which AKR1C3 and POR were quantified by targeted proteomics. Hypoxia or reductase overe-expression induced large increases in PR-104A sensitivity (inhibition of proliferation), DNA damage response (gamma H2AX formation), steady-state concentrations of PR-104H/M and formation of reduced drug-DNA adducts but not DNA adducts retaining the dinitro groups of PR-104A. The fold-change in the sum of PR-104H and PR-104M correlated with the fold-change in reduced crosslinks or monoadducts (R-2 = 0.87 for both), demonstrating their potential for assessing the capacity of cancer cells to bioactivate PR-104A.

• 出版日期2018-8