Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2020 / Despite major advancements in the development of various chemotherapuetic agents, treatment for lung cancer remains costly, ineffective, toxic to neighbouring normal noncancerous cells and still hampered by high level of remissions (Wistuba et al., 2018; Tana et al., 2016; Schiller et al., 2002). Synthesis of novel quinoxalines with a wide spectrum of biological activities has recently received considerable attention with promising anticancer drug activity since most of them do not affect non-cancerous cells and are derived from readily available less costly raw materials (Srivastava et al., 2014). Since combination treatment has been shown to augment and improve single drug treatment, trace elements were employed in this study in combination with quinoxalines derivatives (Gomez et al., 2016; Kocdor et al., 2015; Ku et al., 2012; John et al., 2010; Killile and Killilea, 2007). Zinc is an essential element that is integral to many proteins and transcription factors which regulate key cellular functions such as the response to oxidative stress, DNA replication, DNA damage repair, cell cycle progression, and apoptosis (Dhawan and Chadha, 2010). Owing to the importance of these two approaches, the aim of this study was to provide in vitro preliminary anticancer activity data on A549 lung cancer cells using combination of zinc and quinoxaline derivatives. An assessment of the quinoxaline derivatives ferric reducing power and DPPH free radical scavenging activity was performed. The cytotoxic and anti-proliferation activity of these derivatives and zinc on cancer cell lines was determined using the MTT assay. The ability of the quinoxaline derivatives and zinc to modulate oxidative stress was evaluated using the H2DCFDA fluorescence assay. Cell cycle arrest stages were analysed by flow cytometry through propidium iodide cell cycle analyses. The ability of the quinoxaline derivatives to induce apoptosis in cancer cells was assessed using DAPI/PI, Acridine Orange/Ethidium Bromide (AO/EB) and Annexin V-FITC/Dead Cell assays. Western blot was used to investigate the Bcl/Bax expression ratios in A549 lung cancer cells after treatment with quinoxaline derivatives, zinc and in combination. Of the four quinoxaline derivatives tested, 3-(quinoxaline-3-yl) prop-2-ynyl methanosulphate (LA-39B) and 3-(quinoxaline-3-yl) prop-2-yn-1-ol (LA-55) produced significant anticancer properties against A549 lung cancer cells at minimal concentrations of 25μM. Both quinoxaline derivatives displayed antioxidant properties and did not induce cell death in non-cancerous Raw 267.4 macrophage cells. Cytotoxicity was observed in A549 lung cancer, HeLa cervical cancer and MCF-7 breast cancer cells albeit inhibition was more pronounced in A549 lung cancer cells. Treatment of cancer cells with zinc also resulted in pronounced cytotoxicity at a minimal concentration of 25μM. Although reduced oxidative stress was observed in Raw 264.7 macrophages, in A549 lung cancer cells both compounds were able to increase ROS production which was accompanied by high levels of apoptosis when treated with derivatives and zinc alone but when in combination an improved higher level of apoptosis is observed. The improved anti-cancer activity of this drug combination treatment was further accompanied by lower Bcl/Bax expression ratios with upregulation of Bax in A549 lung cancer cells. The results of the study suggest that 3-(quinoxaline-3-yl) prop-2-ynyl methanosulphate and 3-(quinoxaline-3-yl) prop- 2-yn-1-ol are potential candidates drug for treatment of lung cancer. The use of these quinoxaline derivatives in combination with zinc can offer alternative treatment options for lung cancer. / National Research Foundation (NRF)

Despite rapid advances in clinical oncology, acquired drug resistance still poses a significant threat to the long-term efficacy of current treatment regimens. Because most chemotherapy drugs aim to activate apoptosis in cancer cells, expansion of the pharmacopeia to include treatments targeting novel tumor cell death mechanisms is a promising anti-cancer strategy. Induction of ferroptosis, an iron-dependent form of regulated cell death, shows particular therapeutic potential as aggressive metastatic and drug-resistant cancer cell states have been demonstrated to possess an exquisite dependency on glutathione peroxidase 4 (GPX4), a key suppressor of the ferroptotic cell death pathway. However, current GPX4 inhibitors are limited by poor pharmacokinetic properties that preclude their clinical use. The development of novel drug-like GPX4 inhibitors would benefit from the discovery of new chemical scaffolds to both enhance our understanding of the structural basis of small molecule binding and inhibition as well as facilitate the rational design of future GPX4-targeted therapeutics. In this dissertation, we employed three high-throughput screening strategies to identify novel scaffolds of interest for GPX4 inhibitor development. First, a Lead-Optimized Compound (LOC) library was screened and we conducted further characterization and structure-activity relationship (SAR) studies on hit compound LOC880. Compared to the original hit, analogs QW-095 and QW-105 showed improved binding affinity and GPX4 inhibitory activity in vitro and also induced lipid peroxidation in cells suggestive of ferroptotic death. Further enhancement of the potency and ferroptosis specificity of this scaffold is still needed, but the potentially noncovalent and allosteric mechanism of action presents a novel approach for targeting GPX4. Second, we conducted extensive SAR analyses on another promising hit from the LOC library screen, LOC1886, which led to the identification of the lead compound QW-314. This analog showed significantly improved potency and ferroptosis specificity in multiple cancer cell contexts, including a drug-tolerant persister cell model of minimal residual disease. Characteristic markers of GPX4 inhibition and ferroptosis are also observed in cells treated with QW-314, including GPX4 protein degradation and induction of lipid peroxidation, and QW-314 exhibited excellent selectivity for GPX4 over another glutathione peroxidase family selenoprotein GPX1 in an in vitro assay using cell lysates. Moreover, we determined a baseline of pharmacokinetic measures including aqueous solubility and metabolic stability in human and mouse liver microsomes for further medicinal chemistry optimization. Lastly, we screened a DNA-encoded library (DEL) and an Enamine Diversity library, identifying 10 additional chemical starting points for future investigation.

Indiana University-Purdue University Indianapolis (IUPUI) / Breast cancer is the second leading cause of cancer death for women in the United States. Approximately, 1 in 5 women will recur with cancer within 10 years of completing treatment and recent publications have suggested that breast cancer stem cells confer resistance to therapy. These reports highlight aldehyde dehydrogenase 1A1 (ALDH1A1) and Yes-associated protein (YAP) as a biomarker and key mediator of the stem cell phenotype respectively. To further understand how YAP and ALDH1A1 facilitate chemoresistance, this study investigated how ALDH1A1 specific inhibition affected YAP activity and growth of basal-like breast cancer cells, which are enriched in cancer stem cells. Intriguingly, attenuation of growth by ALDH1A1 inhibition was observed when cells were plated on a reconstituted basement membrane. Further, the inhibition of cell growth correlated with cytosolic retention of YAP and a reduction in YAP signaling. In a complementary analysis, the overexpression of YAP correlated with an increased level of ALDH1A1 transcript. Results from this study indicate a novel mechanism by which basal-like breast cancer cells utilize YAP to maintain the stem cell phenotype and also suggest ALDH1A1 as a potential therapeutic target for breast cancer therapy.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy