2025 Undergraduate Research Showcase

Investigating the Mechanism of DNA-Protein Crosslink Formation Facilitated by Synthetic Aziridinomitosenes

Document Type

Student Presentation

Presentation Date

4-15-2025

Faculty Sponsor

Dr. Don Warner

Abstract

Aziridinomitosenes (AZMs) are quinone-containing DNA alkylating agents, discovered as metabolites of the chemotherapeutic drug mitomycin C (MC). Through the formation of DNA-interstrand crosslinks (ICLs), MC is able to inhibit DNA replication and ultimately cause cell death in tumors. However, prior to DNA binding, MC must undergo a bioreductive activation sequence that has been well studied and characterized. Historically known to produce DNA-monoalkylated adducts in the absence of reductive activation, it has also been shown that synthetic AZMs are able to generate not only ICLs, but also DNA-protein crosslinks (DPCs) in the absence of any reducing species. Given that these were previously unreported adducts for AZMs, a nucleophilic activation/DNA modification sequence involving either of the C-6 or C-7 positions on the quinone ring (mitomycin numbering) was proposed. To test the proposed mechanism, analogs where the C-6 and/or C-7 positions have been blocked have been synthesized and then subjected to a variety of DNA-binding experiments, including in vitro K-SDS assays to quantify the impact of AZM substitution on DPC formation. C6-AZM and H-AZM formed significant amounts of DPCs compared to untreated (41.3 ± 1.9% and 10.2 ± 0.7% respectively). MC and dimethyl-AZM, both having a fully substituted quinone ring, formed equivalent amounts of DPCs based on a two-way ANOVA (2.8 ± 0.4% and 5.6 ± 1.2% respectively). Currently we aim to repeat these DNA-binding experiments with untested AZM analogs as well as treat the various analogs with nucleophilic amines and characterize the products. Upon characterization of these products we aim to further determine whether or not incorporation of a nucleophile at the C-6 and/or C-7 sites occurs, thus supporting or disproving the hypothesized mechanism.

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