Cytotoxic natural products represent a class of cancer drug candidates that have remained largely untapped as payloads in the antibody-drug conjugate (ADC) therapeutic modality. The epothilones, a class of exquisitely cytotoxic natural products and their synthetic analogs, are a prime example, and our work has focused on the development of epothilones as ADC payloads. Strategies toward this goal have included total synthetic efforts toward four structurally distinct epothilone analogs equipped with linker functionality and structural modifications designed to improve metabolic and chemical stability. In addition, we have pursued the synthesis of octreotide- epothilone conjugates, structures designed to target epothilones into cells that overexpress somatostatin receptors 2 and 5. Biological evaluation via in vitro cellular assays revealed one of our epothilone analogs as a promising epothilone-inspired ADC payload. Synthetic efforts toward these goals will be discussed.

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Many experiments are being conducted to find compounds that offer radioprotection against radiation damage and that are also non-toxic. It is hopeful that in the future, research for this technology will benefit patients undergoing cancer treatment by reducing radiation damage to normal cells and therefore reducing short and long term side effects experienced from treatments. Hamster cells were irradiated at doses of 60 and 120 rad, with and without Sphingosine-1-Phosphate mixed in with their growth medium. Post irradiation, it was observed that the S1P molecule seemed to have a radioprotective effect by decreasing the amount of cell death compared to the amount of cell death that occurred with the absence of the molecule. The results of this experiment will sent to Dr. Jon Tilly at Massachusetts General Hospital. Dr. Tilly is currently researching S1P as a possible radioprotector. / Graduation date: 2003

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Bibliography: leaves 116-138 / [14], 138 leaves, 5 leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical & Experimental Pharmacology, 1988

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.

Mounting evidence implicates persister cancer cells as the key element of minimal residual disease (MRD) from which cancer relapse occurs. The observation that persister cells are differentially and specifically sensitive to ferroptosis, a unique form of metabolically-linked cell death, presents a critical weak point through which identification and targeting of persister cells in MRD may become possible. To identify biomarkers for targetable cells, the drivers of ferroptosis sensitivity in persister cells must be identified. Using three chemotherapeutics and cell lines, we derived persister models across diverse tissues of origin and found that: 1) activating transcription factor 4 (ATF4), previously demonstrated as central to lung cancer persister state formation, is differentially expressed in prostate and fibrosarcoma persisters vs parentals, 2) proteins key to ferroptosis are underexpressed in persisters, and revert expression upon persister to parental reversion, 3) the lung persister lipidome is significantly rewired to drive ferroptosis and 4) upon persister to parental reversion and re-acquisition of ferroptosis resistance, the lipid signature also reverts back to a parental-like state, and 5) although ATF4 elimination in persisters does not revert ferroptosis sensitivity, mitochondrial elimination in persisters does abrogate ferroptotic sensitivity. Collectively, these findings reveal the mechanism of persister ferroptosis sensitivity across multiple cancer types, opening up the possibility of leveraging ferroptosis for elimination of minimal residual disease.

Tong, Kit Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 91-104). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.x / List of Tables --- p.xii / List of Abbreviations --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cancer --- p.2 / Chapter 1.1.1 --- Tumor development --- p.2 / Chapter 1.1.2 --- Cell cycle --- p.4 / Chapter 1.1.3 --- Apoptosis --- p.9 / Chapter 1.1.3.1 --- The extrinsic pathway --- p.14 / Chapter 1.1.3.2 --- The intrinsic pathway --- p.16 / Chapter 1.1.3.3 --- The Bcl-2 family proteins --- p.17 / Chapter 1.1.3.4 --- Execution of apoptosis --- p.20 / Chapter 1.1.4 --- Melanoma --- p.22 / Chapter 1.2 --- Polyunsaturated fatty acids (PUFAs) --- p.24 / Chapter 1.2.1 --- "Chemistry, classification, metabolic conversion and sources …" --- p.24 / Chapter 1.2.2 --- Epidemiology studies --- p.27 / Chapter 1.2.3 --- Docosahexaenoic acid (DHA) --- p.28 / Chapter 1.2.3.1 --- Sources --- p.28 / Chapter 1.2.3.2 --- DHA and cancer --- p.29 / Chapter 1.3 --- Objectives --- p.33 / Chapter Chapter 2 --- Materials and Methods --- p.34 / Chapter 2.1 --- In vitro studies of DHA on growth and survival of human cancer cells --- p.34 / Chapter 2.1.1 --- Cell cultures --- p.34 / Chapter 2.1.2 --- Studies of growth inhibition of DHA on human cancer cells --- p.35 / Chapter 2.1.2.1 --- MTT assay --- p.35 / Chapter 2.1.2.2 --- Chemiluminescent-bromodeoxyuridine (Chemi-BrdU) immunoassay --- p.36 / Chapter 2.1.3 --- Studies of growth inhibitory mechanism of DHA on A375 cells. --- p.38 / Chapter 2.1.3.1 --- DNA -flow cytometry analysis --- p.38 / Chapter 2.1.3.2 --- Western blot analysis --- p.39 / Chapter 2.1.3.3 --- Caspase inhibitor studies --- p.42 / Chapter 2.1.3.4 --- Mitochondrial membrane potential analysis --- p.42 / Chapter 2.2 --- In vivo study of the anticancer effect of DHA on A375 cells --- p.44 / Chapter 2.2.1 --- Animals --- p.44 / Chapter 2.2.2 --- Cell inoculation and treatments --- p.44 / Chapter 2.2.3 --- Western blot analysis --- p.45 / Chapter 2.3 --- Statistical analysis --- p.46 / Chapter Chapter 3 --- Results --- p.47 / Chapter 3.1 --- In vitro studies of DHA on growth and survival of human canccr cells --- p.47 / Chapter 3.1.1 --- DHA reduced proliferation and survival of human cancer cells --- p.47 / Chapter 3.1.2 --- DHA modulated cell cycle of A375 cells --- p.52 / Chapter 3.1.3 --- DHA induced apoptosis in A375 cells --- p.55 / Chapter 3.1.4 --- Caspase activations were involved in the DHA-induced apoptosis in A375 cells --- p.59 / Chapter 3.1.5 --- "Caspase 3´ة 6, 8 and 9 were activated in DHA-induced apoptosis of A375 cells" --- p.62 / Chapter 3.1.6 --- DHA dissipated mitochondrial membrane potential in A375 cells --- p.66 / Chapter 3.1.7 --- DHA triggered the mitochondrial pathway of apoptosis --- p.68 / Chapter 3.1.8 --- DHA triggered the death receptor pathway of apoptosis --- p.71 / Chapter 3.2 --- In vivo study of the anticancer effect of DHA on A375 cells --- p.74 / Chapter 3.2.1 --- Effect of DHA on the growth ofA375 xenograft in athymic Bαlb/c mice --- p.74 / Chapter 3.2.2 --- DR4 and TRAIL were upregulated by DHA treatment in A375 solid tumor --- p.77 / Chapter Chapter 4 --- Discussion --- p.79 / References --- p.91

Ho Yi-Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 120-131). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Table of Contents --- p.v / List of Figures --- p.ix / List of Tables --- p.xi / Abbreviations --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Corticosteroids in ophthalmology --- p.1 / Chapter 1.1.1 --- History of the clinical use of corticosteroids --- p.1 / Chapter 1.1.2 --- Administration --- p.1 / Chapter 1.1.3 --- General biological effects of corticosteroids --- p.4 / Chapter 1.1.4 --- Application of corticosteroids in ocular diseases --- p.5 / Chapter 1.1.5 --- Side effects of ocular corticosteroid treatment --- p.6 / Chapter 1.1.6 --- General introduction to commonly used corticosteroids in ophthalmology --- p.6 / Chapter 1.1.6.1 --- Hydrocortisone --- p.6 / Chapter 1.1.6.2 --- Dexamethasone --- p.7 / Chapter 1.1.6.3 --- Triamcinolone --- p.7 / Chapter 1.1.6.4. --- Chemical structures and relative anti-inflammatory potencies --- p.8 / Chapter 1.1.7 --- Cytotoxicity of triamcinolone --- p.12 / Chapter 1.2 --- Retinal ganglion cells --- p.13 / Chapter 1.2.1 --- Basic structures of the eye --- p.13 / Chapter 1.2.2 --- Anatomical structure of retina --- p.13 / Chapter 1.2.3 --- Functions of retinal ganglion cells --- p.18 / Chapter 1.2.4 --- Culture models to study RGCs --- p.20 / Chapter 1.3 --- Aim of study --- p.25 / Chapter Chapter 2 --- Methodology --- p.26 / Chapter 2.1 --- Intravitreal injection of TA (IVTA) --- p.26 / Chapter 2.1.1 --- Materials --- p.26 / Chapter 2.1.1.1 --- Animals --- p.26 / Chapter 2.1.1.2 --- Chemicals --- p.26 / Chapter 2.1.1.3 --- Instruments --- p.26 / Chapter 2.1.2 --- Procedures --- p.26 / Chapter 2.2 --- Peripheral Nerve - Optic Nerve Grafting (PN-ON) Procedure --- p.27 / Chapter 2.3 --- Retrograde Labeling of regenerating RGCs --- p.27 / Chapter 2.3.1 --- Materials --- p.27 / Chapter 2.3.2 --- Procedures --- p.27 / Chapter 2.3.3 --- Statistical analysis --- p.28 / Chapter 2.4 --- Immunohistochemistry --- p.28 / Chapter 2.4.1 --- Materials --- p.28 / Chapter 2.4.2 --- Procedures --- p.29 / Chapter 2.4.3 --- Statistical analysis --- p.29 / Chapter 2.5 --- Histology --- p.29 / Chapter 2.5.1 --- Materials --- p.29 / Chapter 2.5.2 --- Procedures --- p.29 / Chapter 2.6 --- Primary rat retinal ganglion cell culture --- p.30 / Chapter 2.6.1 --- Materials --- p.30 / Chapter 2.6.1.1 --- Animals --- p.30 / Chapter 2.6.1.2 --- Chemicals --- p.30 / Chapter 2.6.1.3 --- Solutions and buffers --- p.30 / Chapter 2.6.1.4 --- Instruments --- p.31 / Chapter 2.6.2 --- Preparations --- p.31 / Chapter 2.6.2.1 --- Working media --- p.31 / Chapter 2.6.2.2 --- Plate coating --- p.32 / Chapter 2.6.3 --- Cell culture process --- p.32 / Chapter 2.6.3.1 --- Dissection of retinal tissues --- p.32 / Chapter 2.6.3.2 --- Purification of RGCs --- p.33 / Chapter 2.6.3.3 --- Culture condition and cell seeding --- p.34 / Chapter 2.7 --- Corticosteroid treatment --- p.34 / Chapter 2.7.1 --- Materials --- p.34 / Chapter 2.7.2 --- Preparations --- p.34 / Chapter 2.7.3 --- Treatment --- p.35 / Chapter 2.8 --- Cell viability assay and morphometric study --- p.36 / Chapter 2.8.1 --- Materials --- p.36 / Chapter 2.8.2 --- Calcein-AM staining --- p.36 / Chapter 2.8.3 --- Cell viability --- p.37 / Chapter 2.8.4 --- Morphometry study --- p.37 / Chapter 2.9 --- TUNEL Assay --- p.38 / Chapter 2.9.1 --- Materials --- p.38 / Chapter 2.9.2 --- Procedure --- p.38 / Chapter 2.9.3 --- Statistical analysis --- p.39 / Chapter 2.10 --- Quantitative Reverse transcription - Polymerase Chain Reaction (qRT-PCR) --- p.39 / Chapter 2.10.1 --- Materials --- p.39 / Chapter 2.10.1.1 --- "Chemicals, reagents, and kits" --- p.39 / Chapter 2.10.1.2 --- Primers --- p.40 / Chapter 2.10.1.3 --- Equipment --- p.41 / Chapter 2.10.1.4 --- Software --- p.41 / Chapter 2.10.2 --- Procedures --- p.41 / Chapter 2.10.2.1 --- Cell collection and RNA isolation --- p.41 / Chapter 2.10.2.2 --- Reverse Transcription --- p.42 / Chapter 2.10.2.3 --- Real-time PCR --- p.43 / Chapter 2.10.3 --- Statistical analysis --- p.43 / Chapter 2.11 --- Western blotting --- p.44 / Chapter 2.11.1 --- Sample preparation --- p.44 / Chapter 2.11.1.1 --- Materials --- p.44 / Chapter 2.11.1.1.1 --- Chemicals and materials --- p.44 / Chapter 2.11.1.1.2 --- Equipment --- p.44 / Chapter 2.11.1.2 --- Procedures --- p.44 / Chapter 2.11.2 --- Protein assay --- p.45 / Chapter 2.11.2.1 --- Materials --- p.45 / Chapter 2.11.2.1.1 --- Chemicals and materials --- p.45 / Chapter 2.11.2.1.2 --- Equipment and software --- p.46 / Chapter 2.11.2.2 --- Procedures --- p.46 / Chapter 2.11.3 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.46 / Chapter 2.11.3.1 --- Materials --- p.46 / Chapter 2.11.3.1.1 --- Chemicals and reagents --- p.46 / Chapter 2.11.3.1.2 --- Equipment --- p.46 / Chapter 2.11.3.1.3 --- Solutions and buffers --- p.47 / Chapter 2.11.3.2 --- Gel preparation --- p.48 / Chapter 2.11.3.3 --- Electrophoresis --- p.49 / Chapter 2.11.3.4 --- Transblotting (semi-dry transfer) --- p.49 / Chapter 2.11.3.5 --- Band visualization --- p.49 / Chapter 2.11.4 --- Immunostaining --- p.50 / Chapter 2.11.4.1 --- Materials --- p.50 / Chapter 2.11.4.1.1 --- Antibodies --- p.50 / Chapter 2.11.4.1.2 --- Chemicals and reagents --- p.50 / Chapter 2.11.4.1.3 --- Equipment --- p.50 / Chapter 2.11.4.2 --- Procedures --- p.50 / Chapter 2.12 --- Gas-chromatography-electron-capture negative-ion mass spectrometry (GC-NCI-MS) --- p.51 / Chapter 2.12.1 --- Standard and reagents --- p.51 / Chapter 2.12.2 --- Chromatography and mass spectrometry --- p.52 / Chapter 2.12.3 --- Sample collection --- p.52 / Chapter 2.12.3 --- Standard and sample preparation --- p.53 / Chapter 2.12.4 --- Validation --- p.54 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Effect of triamcinolone on RGCs in vivo --- p.55 / Chapter 3.2 --- Cell viability of RGCs after IVTA plus PN-ON grafting --- p.55 / Chapter 3.3 --- Abnormal retinal morphology under different IVTA conditions --- p.59 / Chapter 3.4 --- Cell viability assay --- p.66 / Chapter 3.4.1 --- Effects of triamcinolone on RGC viability --- p.66 / Chapter 3.4.2 --- Effects of dexamethasone on RGC viability --- p.68 / Chapter 3.4.3 --- Effects of hydrocortisone on RGC viability --- p.70 / Chapter 3.4.4 --- Effects of filtered fraction of triamcinolone on RGC viability --- p.72 / Chapter 3.5 --- Morphometric study analysis of RGCs --- p.74 / Chapter 3.5.1 --- Percentage of RGCs showing neurite outgrowth --- p.74 / Chapter 3.5.2 --- Average neurite length --- p.77 / Chapter 3.5.3 --- Neurite spanning area --- p.77 / Chapter 3.5.4 --- Neurite count --- p.80 / Chapter 3.5.5 --- Neurite branching --- p.83 / Chapter 3.6 --- Determination of concentration of TA in culture media by GC-NCI-MS --- p.85 / Chapter 3.7 --- TUNEL assay --- p.90 / Chapter 3.8 --- Real-time quantitative Reverse transcription - Polymerase Chain Reaction --- p.93 / Chapter 3.9 --- Western blot --- p.99 / Chapter Chapter 4 --- Discussion --- p.102 / Chapter Chapter 5 --- References --- p.120

[Truncated abstract] In the last ten years regulator T (Tr) cells have re-emerged as an integral part of the immune system. Research in this field has rapidly demonstrated the role of these cells in the maintenance of immune homeostasis and their involvement in disease. Tr cells are generated in the thymus as a normal part of the developing immune system. Furthermore, antigen-specific Tr cells are induced in the periphery by a mechanism which is yet to be completely elucidated, but is likely to involve dendritic cells. Tr cells play an important role in autoimmune disease, transplantation tolerance, cancer. Most recently Tr cell involvement has been demonstrated in a growing number of infectious diseases. Tr cell induction was reported in Friend Virus infection at the commencement of this study, and subsequent to publication of our findings have also been identified in FIV and HIV. Murine AIDS (MAIDS) is a fatal chronic retroviral infection induced in susceptible strains of mice by infection with BM5d, a replication defective virus, in a viral mixture which is designated LP-BM5. The manipulation of Tr cells detailed in this thesis and the related publication represent the first reported therapy utilising targeted removal of Tr cells. Chapter 1 summarises the literature relevant to this study up to November 2004. Chapter 2 details the materials and methodologies used in this work. Chapter 3 investigates whether Tr cells are involved in the development of murine AIDS, particularly in the early stages of infection. The data presented in this chapter provides evidence of a population of CD4+ Tr cells which express CD25 on their cell surface and secrete TGF-β, some IL-10 and low levels of IL-4 are induced following infection with LP-BM5. These cells were found to arise by day 12 post infection (pi) by flow cytometry and immunosuppressive cytokine expression was found to peak at day 16 pi indicating a role in the early stages of disease progression. Chapter 4 investigates the effect of therapeutically targeting these induced Tr cells using the antimitotic agent Vinblastine during their induction period. The efficacy of treatment was found to be time dependent and was shown to abrogate disease progression maximally when given at day 14 pi. Treatment with anti-CD4 monoclonal antibody was also found to be efficacious at day 14 pi and confirmed the identity of the Tr cells as being CD4+ T cells. Adoptive transfer studies demonstrated that the return of these cells to a successfully treated host results in renewed MAIDS progression, confirming their role in disease progression