Date of Award

12-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Track

Bioanalysis

Research Advisor

Wei Li, Ph.D.

Committee

Subhash Chauhan, Ph.D. Bernd Meibohm, Ph.D. Duane D. Miller, Ph.D. Tiffany N. Seagroves, Ph.D.

Abstract

Tubulin inhibitors are widely used as chemotherapeutic agents, and their successis attributed to their ability to target microtubule dynamics and disrupt critical cellular functions including cell signaling, motility, intracellular trafficking, and mitosis. Interference with microtubule dynamics consequently disrupts mitotic progression and ultimately leads to apoptosis, validating microtubule dynamics as an excellent target for anticancer agents. While this class of drug has proven to be effective against many cancer types, the clinical efficacy of current tubulin inhibitors is often limited by the development of multidrug resistance. The most common form of resistance to these agents arises from the overexpression of drug efflux transporters. Extensive research efforts have attempted to develop colchicine binding site inhibitors, which are reported to be significantly less susceptible to multidrug resistance and have therapeutic advantages over agents that target the taxane and vinca alkaloid site. Herein, we evaluated the anticancer activity of novel small-molecules that target the colchicine binding site, focusing on the most promising compounds from several structural scaffolds including indolyl-imidazopyridines (DJ95 and DJ101), VERU-111 analogs with a modified indole moiety (10ab and 10bb) or 3,4,5-trimethoxyphenyl moiety (13f), and heterocyclic pyrimidines (4a, 6a, 5a, and 5b). We demonstrated the cytotoxic potency of these compounds against a variety of cancer cell lines, including malignant melanomas, taxaneresistant prostate cancer cells, and drug efflux pump-overexpressing cell lines. Their mechanism of action was revealed through tubulin polymerization inhibition, disruption of microtubule networks and mitotic spindle formation, and confirmed through X-ray crystallography, which detailed their specific molecular interactions with tubulin in the colchicine binding pocket. Furthermore, these compounds exhibited hallmark characteristics of colchicine binding site agents, such as arresting cells in the G2/M phase of the cell cycle, inducing apoptosis in a concentration-dependent manner, and impeding cancer cell proliferation and migration. Finally, the compounds were efficacious in vivo against melanoma and taxane-resistant prostate cancer xenograft tumors. Several agents were evaluated for ability to prevent melanoma metastases to the lungs in experimental mouse models, and they potently inhibited the development metastatic foci. Safety assessment by pharmacological profiling demonstrated minimal interactions to physiologically important targets and pathophysiological analysis of major organs from the in vivo treatment groups did not expose apparent drug-related injury. Several of the investigated compounds also demonstrated vascular disrupting properties by targeting tumor vasculature and inhibiting capillary-like network formation of endothelial cells. Ultimately, these compounds exhibit strong anticancer efficacy, specifically target the colchicine binding site, and have great potential as cancer therapeutics, particularly for multidrug resistance phenotypes.

Another target we explored for anticancer intervention was survivin. Survivin is the smallest member the inhibitor of apoptosis protein family and its overexpression in tumor cells is been positively correlated with the development of multidrug resistance and radiation resistance. Because it is differentially expressed in healthy tissues and tumors, it is an attractive therapeutic target. Using the scaffold of UC-112, which was previously identified through virtual screening, we evaluated a series of analogs designed to optimize potency and improve selectivity to survivin over other inhibitor of apoptosis proteins. We identified compound 10f, which was highly cytotoxic to melanoma and Pglycoprotein overexpressing cell lines, induced apoptotic cascades in a concentrationdependent manner, specifically downregulated survivin protein levels, and significantly inhibited tumor growth in vivo. Ultimately, these results validated our in-depth biological investigation of novel scaffolds of survivin inhibitors and verified the anticancer efficacy of 10f.

ORCID

http://orcid.org/0000-0002-4429-590X

DOI

10.21007/etd.cghs.2018.0469

Available for download on Saturday, February 13, 2021

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