Document Type
Thesis - University Access Only
Award Date
2007
Degree Name
Doctor of Philosophy (PhD)
Department / School
Pharmaceutical Sciences
Abstract
Glutathione is a nucleophile and antioxidant that serves a protective and detoxifying function in the body. It is involved in phase II drug metabolism of reactive electrophiles and quenches free radicals and reactive oxygen species produced from endogenous and exogenous sources. Glutathione levels are maintained in two ways: the de novo biosynthesis of glutathione from the constituent amino acids and the reduction of oxidized glutathione back to reduced glutathione by glutathione reductase. Tumor drug resistance occurs when cancer cells fail to respond to chemotherapy either intrinsically or acquired through previous exposure to a chemotherapeutic agent. The mechanisms by which tumor drug resistance can develop are numerous and complex. Glutathione contributes to drug resistance in three ways: increased chemotherapy drug metabolism, quenching of free radicals and reactive oxygen species, and association with the drug efflux pump MRP. Because of the important role of glutathione in drug resistance, research efforts have focused on mechanisms to alter the glutathione pathway and increase the sensitivity of cancer to chemotherapy. One method of achieving this is through inhibition of glutathione reductase. Inhibition of this enzyme should alter the ratio of reduced to oxidized glutathione and shift the cell toward a state of oxidative stress. The use of glutathione reductase inhibitors for this purpose has been hampered by the lack of inhibitors that are potent, stable, selective, and easily synthesized. The objectives of this research project were to design and synthesize a series of novel glutathione reductase inhibitors and determine their effectiveness in increasing the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin. 2-Acetylarnino-3-[ 4-(2-acetylarnino-2 carboxyethylsulfanylcarbonylarnino )phenylcarbarnoylsulfanyl ]propionic acid and its derivatives were designed and synthesized as irreversible glutathione reductase inhibitors. The K; and kinact values of 2- acetylarnino-3-[ 4-(2-acetylarnino-2-carboxyethylsulfanylcarbonylarnino) phenylcarbarnoylsulfanyl ]propi onic acid, the most potent derivative of the series, are 88 μMand 0.1 min"1 respectively with yeast glutathione reductase. Although the K; value of the inhibitor is in the micromolar range, it is more potent than N,N-bis(2-chloroethyl)-Nnitrosourea (BCNU), which is currently the most commonly used irreversible glutathione reductase inhibitor with a literature reported ICso of 646 μM. Despite demonstrating effective glutathione reductase inhibition with yeast enzyme, the compound failed to pass through biological membranes to inhibit intracellular glutathione reductase. Because of the cell permeability problems with the originally designed inhibitor, a cell permeable structural analog was synthesized. 2-Acetylamino-3-[4-(2-acetylarnino-2- carboxyethylsulfanylthiocarbonylarnino)phenylthiocarbarnoylsulfanyl]propionic acid is also an irreversible glutathione reductase inhibitor. The K and kinact values of this compound against yeast glutathione reductase were determined to be 56 μMand 0.1 min-1 respectively. By employing CV -1 cells, a monkey kidney cell line, the compound produced 97% glutathione reductase inhibition, 25% glutathione reduction, and five-fold increase in oxidized glutathione at 0.1 mM concentration in 20 minutes. To determine the effect of glutathione reductase inhibition in increasing the sensitivity to doxorubicin, OVCAR-3 cells, a drug resistant ovarian cancer cell line were treated with the two drugs, and the synergistic effect was calculated. An 18% synergistic effect was observed indicating a significant increase in doxorubicin cytotoxicity from this combination. The glutathione reductase inhibitor at a concentration of 25 μM was able to inhibit the intracellular enzyme activity by 51 % with a concomitant increase in the disulfide to free thiol ratio indicating that oxidative stress is the mechanism behind the synergistic effect. While glutathione reductase inhibition did increase the sensitivity of ovarian cancer cells to doxorubicin, it did not sensitize cardiomyocytes to the chemotherapeutic agent; this is important as cardiotoxicity is the dose-limiting adverse effect of doxorubicin. In conclusion, a novel series of glutathione reductase inhibitors was designed, synthesized, and evaluated. One inhibitor, 2-acetylamino-3-[ 4-(2-acetylamino-2- carboxyethylsulfanylthiocarbonylamino )phenylthiocarbamoylsulfanyl]propionic acid was found to inhibit intracellular glutathione reductase and produce an alteration in thiol redox state. Use of this GR inhibitor increased the sensitivity of an ovarian cancer cell line to doxorubicin.
Library of Congress Subject Headings
Cancer -- Chemotherapy
Glutathione
Enzyme inhibitors
Doxorubicin
Format
application/pdf
Number of Pages
163
Publisher
South Dakota State University
Recommended Citation
Seefeldt, Teresa M., "Design, Synthesis, and Evaluation of Novel Glutathione Reductase Inhibitors and Their Effect on Cancer Sensitivity to Doxorubicin" (2007). Electronic Theses and Dissertations. 1404.
https://openprairie.sdstate.edu/etd2/1404
