3-Amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine, TPZ) is a bioreductively-activated DNA-damaging agent that selectively kills the hypoxic cells found in virtually all solid tumors. This compound shows great clinical promise and is currently being examined in more than ten different clinical trials, including several phase III studies. Importantly, TPZ damages DNA via a completely novel sequence of chemical reactions in which the one-electron reduced drug initiates radical-mediated DNA damage and then the drug or its metabolites transfer oxygen atoms from their N-oxide functional groups to the resulting DNA radicals, thus, converting them to strand cleavage sites. While the medicinal properties of TPZ have been extensively investigated, many of its biologically relevant chemical properties remain poorly understood. The growing clinical interest in TPZ provides a practical incentive to characterize the relevant chemistry of this drug. In addition, characterization of this new chemical motif that efficiently delivers cytotoxic DNA-damaging radicals to the interior of hypoxic cells may be of fundamental interest in both medicinal chemistry and toxicology. The work described in this proposal is divided into three Specific Aims: 1. How does TPZ generate radical lesions on DNA? Does the drug serve to deliver the known DNA-damaging agent hydroxyl radical to hypoxic cells? How do the conditions of low oxygen and low pH found in tumor cells combine to facilitate DNA damage by the drug? 2. What is the chemical mechanism by which TPZ and its metabolites transfer oxygen atoms from their N-oxide functional groups to DNA radicals? This unprecedented reaction allows the drug to efficiently damage DNA under hypoxic conditions and may play a central role in defining the structural nature of the final DNA lesions caused by TPZ. 3. We propose studies to characterize the structural nature of the DNA strand breaks and base damage mediated by TPZ. These studies will explore the possibility that the unusual conditions under which TPZ operates, along with the unique chemical properties of the drug, lead to a unique spectrum of DNA lesions that confront cells with social challenges to their DNA-repair systems. ? ?
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