Preclinical characterization of small molecule compounds as therapeutics for cancer and Chagas’ disease : tumor vascular disrupting agents and cysteine protease inhibitors.
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Odutola, Samuel O., 1986-
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The purpose of this study was the pre-clinical exploration of specific small molecule compounds as vascular disrupting agents and cysteine protease inhibitors, targeted towards the development of therapeutic agents for the treatment of cancer and Chaga's disease. Disruption of tumor vasculature is a selective approach to cancer therapy that results in tremendous tumor necrosis while leaving normal blood vessels relatively unaffected. The first part of this study describes an examination of the vascular disrupting ability and the mechanism of action of the indole-based tubulin-binding compound, OXi8006, and its water-soluble phosphate prodrug OXi8007. Treatment of rapidly proliferating human umbilical vein endothelial cells, used as a model for the tumor vasculature, with OXi8006 caused a dramatic disruption of the microtubule network and subsequent increase in cell contractility as observed from increased actin stress fiber and focal adhesion formation. The induced signaling cascade included increased phosphorylation of myosin light chain and focal adhesion kinase, and activation of the LIM kinase-cofilin pathway. It was demonstrated that these events were mediated by the intracellular G protein switch RhoA and its downstream target RhoA kinase, via the microtubule-binding guanine nucleotide exchange factor GEF-H1. A separate research project focused on the evaluation of synthetic thiosemicarbazone inhibitors of cysteine proteases as anticancer agents. Cathepsins L, K, and B are cysteine proteases that are implicated in tumor growth and metastasis. In addition, the cysteine protease cruzain, an essential enzyme in the life cycle of parasite Trypanosoma cruzi, is a validated therapeutic target for Chagas' disease. In this project, preliminary in vitro analysis of a group of synthetic thiosemicarbazones were carried out in order to characterize their potency and mode of inhibition against these enzymes. A number of compounds were found to be potent inhibitors of each enzyme. Results demonstrated that the most potent inhibitors in this library have slow binding, slowly reversible, competitive mechanisms of inhibition. Furthermore, results suggested that the best thiosemicarbazone inhibitors form a reversible covalent bond with each enzyme. Cathepsin L inhibitors were able to delay cell invasion of the MDA-MB-231 breast cancer cell line in a concentration dependent manner.