Elucidation of thioredoxin reductase 1 as an anticancer drug target

Abstract: Cancer constantly finds ways to survive, so we must find new ways to stop it. A major attribute of cancer cells is increased oxidative stress, occurring in the form of reactive oxygen species (ROS). Basal ROS generation commonly occurs in all types of cells and is essential for normal cellular growth and function. However, in contrast to its beneficial attributes when generated at low concentrations, excessive production of ROS is harmful to the cell. High levels of ROS can damage cellular function to the point of cell senescence or cell death. Certain cells are able to effectively adapt to increased ROS levels, activating endogenous antioxidant pathways as a way to survive the aberrant onslaught of oxidative stress. One antioxidant pathway that is often found to be upregulated in cancer cells is the thioredoxin pathway, and within the thioredoxin pathway exists a highly reactive selenocysteine-containing enzyme called thioredoxin reductase 1 (TrxR1). The observed overexpression of the antioxidant enzyme TrxR1 in cancer cells suggests that the enzyme serves as an integral combatant to increased oxidative stress levels, allowing cancer cells to survive and even thrive in the nocuous environment of elevated ROS. The studies comprising this thesis further examine the ability to inhibit TrxR1 function with small molecule drug candidates, the role such inhibition has on modulating ROS levels, and whether such inhibition is sufficient to elicit anticancer therapeutic effects. Paper I established a novel recombinant TrxR1 assay designed for high-throughput screening capabilities. The assay was designed to be dual-purpose, with the ability to detect TrxR1 substrate or inhibitory activity of the test compound within a single test sample. Using the library of pharmacologically active compounds (LOPAC1280), known substrates and inhibitors of TrxR1 in the library validated the assay. Protoporphyrin IX (PpIX), a previously unknown inhibitor of TrxR1, was discovered to inhibit the enzyme in the screen. PpIX and two of its analogs displayed irreversible inhibition to the enzyme, with the capacity to inhibit cellular TrxR1 activity and inhibit cancer cell viability. The three porphyrin compounds illustrated how slight chemical modifications to the porphyrin ring core of PpIX could alter the inhibitory activity of TrxR1. Paper II examined various pharmacodymics and activities of the proteasome inhibitor b-AP15. b- AP15 was found to be rapidly taken up in cancer cells and quickly induce cell death irrespective of brief exposure times. The reactive site of b-AP15 was determined to exist at the α,β-unsaturated carbonyl Michael acceptor moiety of the compound. The half-life of b-AP15 in plasma was determined to be short, but coincided with the observed rapid uptake of the compound into cells. In human hepatocytes, over 17 different metabolites were observed after compound treatment. b-AP15 and many of its analogs, as opposed to bortezomib, were also found to be potent inhibitors of TrxR1. b-AP15 was also successfully able to inhibit TrxR1 in a cellular context. Paper III describes the effects of MJ25, a novel p53 transactivator and TrxR1 inhibitor, and Auranofin against malignant melanoma. Both compounds were found to be effective inhibitors of malignant melanoma cell growth and viability. In redox profiling, both compounds irreversibly inhibited of TrxR1, displayed selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) activity, and caused increased cellular ROS production. Paper IV screened for novel TrxR1 inhibitors on a large scale and tested whether the newly discovered inhibitors would elicit anticancer effects. A structure activity relationship analysis of the two top TrxR1 inhibitors (TRi-1 and TRi-2) correlated enzyme inhibition to inhibition of cell viability. Both compounds exhibited potency across multiple cancer cell types in the NCI60 cell panel and individual cell line testing. Differential SecTRAP forming capabilities of the two compounds, compared with Auranofin, correlated a SecTRAP dependent cellular induction of H2O2 while lacking effects on mitochondrial function. TRi-1 effectively inhibited tumor growth, decreased tumor metabolic activity, and was well tolerated in mouse models. TRi-1 and Auranofin effectively inhibited tumor growth in syngenic mouse models. These studies reinforce the candidacy of TrxR1 as an anticancer drug target through the introduction of novel inhibitors of the enzyme displaying anticancer effects in vitro and in vivo, and through the exposition of anticancer drug candidates as inhibitors of the enzyme.