Abstract
PR-104 is a dinitrobenzamide mustard currently in clinical trial as a hypoxia-activated prodrug. Its major metabolite, PR-104A, is metabolized to the corresponding hydroxylamine (PR-104H) and amine (PR-104M), resulting in activation of the nitrogen mustard moiety. We characterize DNA damage responsible for cytotoxicity of PR-104A by comparing sensitivity of repair-defective hamster Chinese hamster ovary cell lines with their repair-competent counterparts. PR-104H showed a repair profile similar to the reference DNA cross-linking agents chlorambucil and mitomycin C, with marked hypersensitivity of XPF(-/-), ERCC1(-/-), and Rad51D(-/-) cells but not of XPD(-/-) or DNA-PK(CS)(-/-) cells. This pattern confirmed the expected dependence on the ERCC1-XPF endonuclease, implicated in unhooking DNA interstrand cross-links at blocked replication forks, and homologous recombination repair (HRR) in restarting collapsed forks. However, even under anoxia, the hypersensitivity of XPF(-/-), ERCC1(-/-), and Rad51D(-/-) cells to PR-104A itself was lower than for chlorambucil. To test whether this reflects inefficient PR-104A reduction, a soluble form of human NADPH:cytochrome P450 oxidoreductase was stably expressed in Rad51D(-/-) cells and their HRR-restored counterpart. This expression increased hypoxic metabolism of PR-104A to PR-104H and PR-104M as well as hypoxia-selective cytotoxicity of PR-104A and its dependence on HRR. We conclude that PR-104A cytotoxicity is primarily due to DNA interstrand cross-linking by its reduced metabolites, although under conditions of inefficient PR-104A reduction (low reductase expression or aerobic cells), a second mechanism contributes to cell killing. This study shows that hypoxia, reductase activity, and DNA interstrand cross-link repair proficiency are key variables that interact to determine PR-104A sensitivity.
Highlights
Hypoxia is a ubiquitous feature of tumors and is a negative prognostic indicator in many contexts [1,2,3]
We examine the consequences of modulating the rate of nitroreduction of PR-104A by overexpressing a truncated form of human NADPH:cytochrome P450 oxidoreductase (CYPOR), known to be a major PR104A reductase in hypoxic cells [23] in homologous recombination repair (HRR)-defective and HRR-competent cell lines
This study shows the interaction between three determinants of cellular sensitivity to the prodrug PR-104A, each of which is likely to play a key role in its therapeutic activity in humans
Summary
Hypoxia is a ubiquitous feature of tumors and is a negative prognostic indicator in many contexts [1,2,3]. The importance of hypoxia as a therapeutic target has led to the development of hypoxia-activated prodrugs ( called bioreductive drugs or hypoxic cytotoxins) that are metabolized to active cytotoxins by pathways that are inhibited by oxygen [4, 10,11,12,13,14] One of these is PR-104 (see Fig. 1A for chemical structures), a phosphate ester pre-prodrug currently in clinical trial [15, 16], which is converted systemically to the corresponding alcohol PR-104A; the latter is metabolized selectively under hypoxia to the hydroxylamine PR104H and amine PR-104M by reduction of the nitro group para to the mustard moiety. This biotransformation of the electron withdrawing nitro group to electron-donating hydroxylamine or amine acts as a molecular switch to increase the alkylating reactivity of the latent nitrogen mustard moiety of dinitrobenzamide mustards such as PR-104A [15, 17, 18]
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