DNA alkylating agents, including nitrogen mustards (NM) such as cyclophosphamide, are mainstays in the treatment of a variety of cancers, including breast cancers. A central theme of our project is that initially formed DNA alkylation products undergo chemical transformations into complex DNA damage, involving formamidopyrimidine (Fapy-dG) chemistry, interstrand cross-linking (ICL) and apurinic (AP) site formation, and this unmasks chemical functionalities that modulate repair or replication or serve as obligate intermediates to facilitate secondary reactions. These secondary reactions can be exploited to design and implement therapeutic modalities designed to enhance treatment outcomes. Specifically, the mechanistic basis of combination anthracycline-cyclophosphamide and anthracycline-cyclophosphamide-taxane (AC or AC-T) treatments, used clinically in breast cancer chemotherapy is of interest. Two secondary products arise following N7-dG alkylation and are key players in forming complex damage sites: formamidopyrimidine (alkyl-Fapy-dG) adducts resulting from the imidazole ring-opening of guanine, and AP sites resulting from depurination of N7-dG alkylation products. We will utilize both NMR and crystallographic approaches to rigorously characterize the structural biology of these complex damage sites, including the potential for anthracyclines and other small molecules to form conjugates at AP sites that are generated following nitrogen mustard alkylation in DNA; these sites may be complex and clustered in the DNA. Project 1 and the DNA Synthesis Resource Core will prepare samples for structural analyses. A fundamental knowledge of the structural biology of complex DNA damage that is formed during AC or AC-T treatments is essential to explaining the cellular processing of this damage by genome surveillance, replication, repair and tolerance proteins, which is the goal of Project 2. Indeed, Project 3 lies at the crucial intersection between complex DNA damage induced by specific agents or combination chemotherapies, and the cellular processing of this damage, which influences the ultimate fate of the cell. We will utilize NMR to determine the structures of these lesions in DNA, which are related to their recognition by repair enzymes, and we will utilize crystallography to determine there structures in complex with repair and replication proteins involved in their processing, in an effort to understand the molecular origins of genotoxicity and cytotoxicity. With Project 1, we will use NMR and crystallography to delineate the structural biology of AP-site conjugates involving anthracyline antibiotics, new analogs of these antibiotics prepared by Project 1, and small molecule AP-lyase inhibitors identified by Project 2. Ultimately our goal is to leverage a more complete understanding of the chemistry and biology of DNA alkylating agents to discover new therapeutic strategies in clinical oncology.! !
The alkylation of DNA by clinically used chemotherapeutic agents such as nitrogen mustards (NM) and thioTEPA results in complex forms of DNA damage. These may be genotoxic or cytotoxic, and in addition, may react further with other chemotherapeutic agents, such as the anthracyline antibiotics. We will utilize NMR and crystallography to probe the molecular mechanisms underlying this chemistry and biology.The rigorous characterization of structural biology associated with complex DNA damage generated following specific combination chemotherapy regimens is essential to explaining the cellular processing of this damage by genome surveillance, replication, repair and tolerance proteins.
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