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Item Detecting Attomolar DNA-Damaging Anticancer Drug Activity in Cell Lysates with Electrochemical DNA Devices(American Chemical Society, 2021-07-23) Wettasinghe, Ashan P.; Singh, Naveen; Starcher, Colton L.; DiTusa, Chloe C.; Ishak-Boushaki, Zakari; Kahanda, Dimithree; McMullen, Reema; Motea, Edward A.; Slinker, Jason D.; Biochemistry and Molecular Biology, School of MedicineHere, we utilize electrochemical DNA devices to quantify and understand the cancer-specific DNA-damaging activity of an emerging drug in cellular lysates at femtomolar and attomolar concentrations. Isobutyl-deoxynyboquinone (IB-DNQ), a potent and tumor-selective NAD(P)H quinone oxidoreductase 1 (NQO1) bioactivatable drug, was prepared and biochemically verified in cancer cells highly expressing NQO1 (NQO1+) and knockdowns with low NQO1 expression (NQO1−) by Western blot, NQO1 activity analysis, survival assays, oxygen consumption rate, extracellular acidification rate, and peroxide production. Lysates from these cells and the IB-DNQ drug were then introduced to a chip system bearing an array of DNA-modified electrodes, and their DNA-damaging activity was quantified by changes in DNA-mediated electrochemistry arising from base-excision repair. Device-level controls of NQO1 activity and kinetic analysis were used to verify and further understand the IB-DNQ activity. A 380 aM IB-DNQ limit of detection and a 1.3 fM midpoint of damage were observed in NQO1+ lysates, both metrics 2 orders of magnitude lower than NQO1− lysates, indicating the high IB-DNQ potency and selectivity for NQO1+ cancers. The device-level damage midpoint concentration in NQO1+ lysates was over 8 orders of magnitude lower than cell survival benchmarks, likely due to poor IB-DNQ cellular uptake, demonstrating that these devices can identify promising drugs requiring improved cell permeability. Ultimately, these results indicate the noteworthy potency and selectivity of IB-DNQ and the high sensitivity and precision of electrochemical DNA devices to analyze agents/drugs involved in DNA-damaging chemotherapies.Item Targeting Base Excision Repair in Cancer: NQO1-Bioactivatable Drugs Improve Tumor Selectivity and Reduce Treatment Toxicity Through Radiosensitization of Human Cancer(Frontiers, 2020-08-19) Starcher, Colton L.; Pay, S. Louise; Singh, Naveen; Yeh, I.-Ju; Bhandare, Snehal B.; Su, Xiaolin; Huang, Xiumei; Bey, Erik A.; Motea, Edward A.; Boothman, David A.; Biochemistry and Molecular Biology, School of MedicineIonizing radiation (IR) creates lethal DNA damage that can effectively kill tumor cells. However, the high dose required for a therapeutic outcome also damages healthy tissue. Thus, a therapeutic strategy with predictive biomarkers to enhance the beneficial effects of IR allowing a dose reduction without losing efficacy is highly desirable. NAD(P)H:quinone oxidoreductase 1 (NQO1) is overexpressed in the majority of recalcitrant solid tumors in comparison with normal tissue. Studies have shown that NQO1 can bioactivate certain quinone molecules (e.g., ortho-naphthoquinone and β-lapachone) to induce a futile redox cycle leading to the formation of oxidative DNA damage, hyperactivation of poly(ADP-ribose) polymerase 1 (PARP1), and catastrophic depletion of NAD+ and ATP, which culminates in cellular lethality via NAD+-Keresis. However, NQO1-bioactivatable drugs induce methemoglobinemia and hemolytic anemia at high doses. To circumvent this, NQO1-bioactivatable agents have been shown to synergize with PARP1 inhibitors, pyrimidine radiosensitizers, and IR. This therapeutic strategy allows for a reduction in the dose of the combined agents to decrease unwanted side effects by increasing tumor selectivity. In this review, we discuss the mechanisms of radiosensitization between NQO1-bioactivatable drugs and IR with a focus on the involvement of base excision repair (BER). This combination therapeutic strategy presents a unique tumor-selective and minimally toxic approach for targeting solid tumors that overexpress NQO1.