| NF-kappaB transcription factors control a variety of cellular processes, such as immune responses, cell growth and apoptosis, and are chronically activated in many human diseases, including ones associated with inflammation and cancer. In collaboration with the laboratory of Dr. John A. Porco Jr. (Department of Chemistry, Boston University), I have characterized various natural and synthetic epoxyquinoids for their ability to inhibit activation of NF-kappaB. In this thesis, it is shown that jesterone dimer (JD) and epoxyquinone A monomer (EqM) block activation of NF-kappaB by inhibiting multiple molecular targets: IkappaB kinase beta (IKKbeta) and the NF-kappaB subunits p65 and REL. JD and EqM inhibit tumor necrosis factor-alpha-induced IkappaBalpha phosphorylation and degradation by targeting IKKbeta. Cysteine residue 179 (Cys-179) in the activation loop of IKKbeta is critical for this inhibition in that substitution of Cys-179 with Ala abolishes JD- and EqM-mediated inhibition of IKKbeta. JD and EqM also directly inhibit p65 and REL, but not p50, DNA-binding activity; moreover, replacement of a conserved Cys residue with Serin p65 and REL reduces JD- and EqM-mediated inhibition of DNA binding. Pre-incubation of cells with the reducing agent dithiothreitol dose-dependently reduces EqM-mediated inhibition of NF-kappaB, further suggesting that these epoxyquinoids directly modify the thiol groups of Cys residues in their protein targets. It is proposed that the reactive functional moieties (i.e., alpha,beta-unsaturated ketones and/or epoxides) found on JD and EqM covalently bind to the thiols of Cys residues in target proteins.; Consistent with their ability to inhibit multiple steps in the NF-kappaB pathway, JD and EqM induce apoptosis in human lymphoma SUDHL-4 (with IkappaBalpha) and RC-K8 (without IkappaBalpha) cell lines that have constitutively active REL DNA-binding complexes due to different cellular alterations. In addition, low doses of EqM selectively inhibit the growth of leukemia, kidney, and colon cancer cells in a panel of 60 human tumor cell lines.; The results presented in this thesis suggest that two synthetic epoxyquinoids, JD and EqM, block activation of NF-kappaB by inhibiting two steps in its activation (i.e., (IKKbeta) and DNA binding). These results also demonstrate the utility of assessing the bioactivity of synthetic derivatives of natural products. |
