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The development of hypoxia activated DNA repair inhibitors Lindquist, Kirstin Elizabeth 2009

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    THE DEVELOPMENT OF HYPOXIA ACTIVATED DNA REPAIR INHIBITORS  by    Kirstin Elizabeth Lindquist BSc. Simon Fraser University, 2007     A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Pathology and Laboratory Medicine)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)    November 2009     © Kirstin Lindquist 2009   ii Abstract The human body’s vast network of blood vessels provides oxygen and nutrients throughout the body, however hypoxic cells (cells with lower than normal physiological oxygen levels) are found in human tumours. Hypoxic cells are resistant to ionizing radiation and therefore are an impediment to the effectiveness of radiotherapy. Ionizing radiation kills cells by causing DNA damage; the principal lethal lesion formed is the DNA double strand break (DSB).  This work concerns the development of a hypoxia activated DNA repair inhibitor prodrug. The specific target of the prodrug is a critical DSB repair enzyme of the non-homologous end joining pathway (NHEJ), called DNA dependent protein kinase (DNA-PK). This work concerns the development of pharmaceutical screening tools for agents that act as radiosensitizers of oxic and hypoxic cells. These tools were then used to demonstrate that DNA-PK deficiency can sensitize hypoxic mammalian cells to the effects of ionizing radiation, and that 2-nitroimidazole derivatives of the DNA-PK inhibitor IC86621 cell- based screening results from hypoxia activated DNA repair inhibitors that display hypoxia selective radiosensitization of CB.17 murine embryonic fibroblast and HeLa human cervical carcinoma cells.     iii Table of contents  Abstract ................................................................................................................ ii! Table of contents................................................................................................ iii! List of tables........................................................................................................vi! List of figures.....................................................................................................vii! Acknowledgements............................................................................................ ix! Dedication ............................................................................................................x! Glossary ..............................................................................................................xi! Chapter 1: Introduction.......................................................................................1! 1.1! Hypoxia and radiation therapy ...................................................................1!  The oxygen effect ............................................................................1! 1.2! DNA double strand break repair ................................................................2!  Homologous recombination repair ...................................................2!  Non-homologous end joining ...........................................................3! 1.3! Hypoxic cell radiation sensitizers and hypoxia activated prodrugs............4! 1.4! Current status of DNA-PK inhibitors for use in cancer treatment ..............6! Chapter 2: Materials and methods.....................................................................9! 2.1! Hypoxia and radiation treatments ..............................................................9! 2.2! Inhibitors, drugs and prodrugs .................................................................10! 2.3! Cell culture...............................................................................................10! 2.4! Resazurin reduction assay ......................................................................11! 2.5! Clonogenic survival assay .......................................................................12! 2.6! Flow cytometry of !H2AX and cell cycle analysis ....................................13! 2.7! Mouse liver microsome preparation.........................................................13! 2.8! Microsomal stability assay .......................................................................14! 2.9! High pressure liquid chromatography (HPLC) .........................................14! 2.10! Cell lysis and western blotting ...............................................................15! Chapter 3: Development of prodrug screening assays .................................17! 3.1! Hypoxia chamber development: the effect of cyclic evacuation and pressurization on cell viability............................................................17!  Introduction ....................................................................................17!  Approach .......................................................................................18!  Results ...........................................................................................19! 3.2 ! Hypoxia chamber development: the number of gas exchange cycles necessary to achieve maximum radiobiological hypoxia........22!   iv  Introduction ....................................................................................22!  Approach .......................................................................................22!  Results ...........................................................................................22! 3.3 ! Hypoxia chamber development: differentiation between pO2 tensions achieved using the hypoxia chambers................................25!  Introduction ....................................................................................25!  Approach .......................................................................................25!  Results ...........................................................................................25! 3.4! Testing of a multi-attenuator insert to achieve multiple radiation doses with a single administration.....................................................28!  Introduction ....................................................................................28!  Approach .......................................................................................28!  Results ...........................................................................................29! Chapter 4: Establishment of proof of principle ..............................................31! 4.1 DNA-PKcs, Ku70, and Ku80 protein expression is stable in HeLa human cervical cancer cells under hypoxic conditions......................31!  Introduction ....................................................................................31!  Approach .......................................................................................32!  Results ...........................................................................................32! 4.2! Genetic DNA-PK deficiency causes sensitivity to ionizing radiation independent of hypoxia .....................................................................35!  Introduction ....................................................................................35!  Approach .......................................................................................36!  Results ...........................................................................................36! 4.3! Chemical inhibition of DNA-PK can sensitize both oxic and hypoxic cells to the effects of ionizing radiation..............................................39!  Introduction ....................................................................................39!  Approach .......................................................................................40!  Results ...........................................................................................40! 4.4! Residual DNA damage is increased in cells when treated with the DNA-PK inhibitor IC86621 in combination with ionizing radiation.............................................................................................44!  Introduction ....................................................................................44!  Approach .......................................................................................44!  Results ...........................................................................................45! 4.5! IC86621 loses DNA-PK inhibitory activity when modified at the 2- hydroxyl site ......................................................................................49!  Introduction ....................................................................................49!  Approach .......................................................................................50!  Results ...........................................................................................53! Chapter 5: Screening and development..........................................................55! 5.1! Prodrug HAPI2 displays hypoxia selective radiosensitization and dose dependent toxicity under both oxic and hypoxic conditions ..........................................................................................55!  Introduction ....................................................................................55!   v  Approach .......................................................................................56!  Results ...........................................................................................57! 5.2!  Prodrug HAPI3 decreases cell viability selectively in hypoxic cells treated with ionizing radiation............................................................59!  Introduction ....................................................................................59!  Approach .......................................................................................59!  Results ...........................................................................................60! 5.3! Prodrug HAPI3 is reduced by an NADPH dependent enzyme to the DNA-PK inhibitor IC86621 selectively under hypoxic conditions ......63!  Introduction ....................................................................................63!  Approach .......................................................................................63!  Results ...........................................................................................63! References .........................................................................................................66! Appendices ........................................................................................................72! Appendix A: Chemical structures of various DNA-PK inhibitors.......................73! Appendix B: Published IC50 values of various DNA-PK inhibitors against PIKK and PI3K family members ........................................................74! Appendix C: Chemical structures and table of common and International Union of Pure and Applied Chemistry names for compounds synthesised as part of the HADRI project .........................................75!    vi List of tables  Table 3-1! A list of the attenuating agents used to make the multi- attenuator and corresponding percent attenuation...............................29!    vii List of figures  Figure 1.1 ! Mechanism and components of hypoxia activated DNA repair inhibitor prodrugs..........................................................................8! Figure 3.1! Photograph and diagram of an aluminium hypoxia chamber ........20! Figure 3.2 Cell viability following gassing protocol with and without 5 Gy irradiation..............................................................................................21! Figure 3.3! HeLa cell viability following 2-10 cycles of gas exchange in hypoxia chambers and 5 Gy ionizing radiation.....................................24! Figure 3.4! Percent viability of CB.17 cells equilibrated to various levels of hypoxia in response to IR. ................................................................27! Figure 3.5! Photograph and X-ray film of the multi-attenuator insert. ..............30! Figure 4.1 HeLa cell protein expression of DNA dependent protein kinase subunits following 4-72 hours of hypoxia at 1% oxygen.......................34! Figure 4.2 ! The survival of oxic and hypoxic CB.17 and SCID/st cells following exposure to ionizing radiation................................................38! Figure 4.3 ! Radiosensitization of oxic and hypoxic CB.17 cells by IC86621 ................................................................................................42! Figure 4.4 ! Radiosensitization of oxic and hypoxic CB.17 cells by NU7026 ................................................................................................43! Figure 4.5 ! DNA-PK inhibitor IC86621 increases residual DNA damage in HeLa cells 24h following treatment  combination with ionizing radiation................................................................................................47! Figure 4.6 DNA-PK inhibitor IC86621 in combination with ionizing radiation causes an IC86621 does dependent increase in residual DNA damage and elicits a G2 block .............................................................48! Figure 4.7 ! Reduction scheme of HAPI 3 in hypoxia and structural comparison of IC86621 to LY294002 ...................................................52! Figure 4.8 ! Modification of the 2'-hydroxyl site on IC86621 to an O- methyl eliminates radiosensitization activity .........................................54! Figure 5.1 ! HAPI2 displays dose-dependent hypoxia selective radiosensitization ability........................................................................58! Figure 5.2 ! HAPI3 displays no off-target toxicity in the 25-100µM range.........61! Figure 5.3 ! HAPI3 has activity as a hypoxia selective radiosensitizer .............62!   viii Figure 5.4 ! Bioreduction of HAPI3 by liver microsomes requires NADPH and selectively produces IC86621 under hypoxic conditions ...............65!    ix Acknowledgements  Foremost I would like to acknowledge my supervisor throughout this project, Dr. Andrew Minchinton, whose innovation, mentorship, and encouragement made this work possible.  I would like to thank Dr. Alastair Kyle whose brilliance in the laboratory and contribution to this work is difficult to overstate. Dr. Kyle’s dedication to his work, creativity, skilfulness and willingness to help others constantly inspire me.  A special thanks to Dr. Robyn Seipp who carried out the flow cytometry experiments and was a joy to work with on this project.  Also a thank-you to my former and present lab mates Dr. Lynsey Huxham and Jennifer Baker. Lynsey who mentored me patiently when I was a young naïve co-op student and Jenn who has been an understanding friend and lab mate over the years.  Lastly I would like to thank the many co-op students whose hard work contributed to the work presented in this thesis, particularly Misa Noda and Jordan Cran.    x  Dedication  This work is dedicated to my parents Gordon and Elizabeth Lindquist for their unwavering commitment and support of my education AND to my best friend and partner Scott Yuzwa, whose laughter, love & encouragement made this work possible on even the darkest rainiest Vancouver days.    xi Glossary !H2AX Variant of histone H2A phosphorylated at serine residue 139. " Light wavelength µM Micromolar ARCON Accelerated radiotherapy, carbogen and nicotinamide ATM Ataxia telangiectasia mutated ATR Ataxia telangiectasia and Rad3 related DNA Deoxyribonucleic acid dsDNA Double stranded deoxyribonucleic acid DNA-PK  Deoxyribonucleic acid dependent protein kinase DNA-PKcs Deoxyribonucleic acid dependent protein kinase catalytic subunit D-PBS Dulbecco’s modified phosphate buffered saline DSB DNA double strand break ECL Enhanced chemiluminescence Gy Gray: one gray is the absorption of one joule of energy, in the form of ionizing radiation, by one kilogram of matter. h hours H2A.X The X variant of histone H2A HIF1-# Hypoxia inducible factor 1 alpha HRP Horseradish peroxidase HRR Homologous recombination repair   xii IC50 Half maximal inhibitory concentration: The concentration of a substance necessary to inhibit enzymatic activity by 50% IR Ionizing radiation kDa Kilodaltons min Minutes mM Millimolar mm Millimeter mTOR Mammalian target of rapamycin NADPH Nicotinamide adenine dinucleotide phosphate NHEJ Non-homologous end joining OER Oxygen enhancement ratio p110# Phosphotidylinositol-3-Kinase p110 alpha p110$ Phosphotidylinositol-3-Kinase p110 beta p110! Phosphotidylinositol-3-Kinase p110 gamma p110% Phosphotidylinositol-3-Kinase p110 delta PBST Dulbecco’s modified phosphate buffered saline containing 0.01% polysorbate 20 (commercially known as Tween-20) pCO2 Partial pressure of carbon dioxide PHD Prolyl hydroxylase PIKK Phosphotidylinositol-3-kinase like kinase PI3-K Phosphotidylinositol-3-kinase pO2 Partial pressure of oxygen prkdc Protein kinase, DNA-activated, catalytic polypeptide. This gene encodes the DNA-PKcs transcript. s Seconds   xiii SCID Severe combined immunodeficiency SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis TLD Thermoluminescent dosimetry XLF XRCC4-like factor XRCC4 X-ray repair cross complementing protein 4    1 Chapter 1:  Introduction 1.1 Hypoxia and radiation therapy Radiotherapy is a mainstay in the treatment of many types of cancer, however the efficacy of radiotherapy is limited by the damage caused to normal tissues surrounding the tumour volume. At doses given clinically, radiation resistant populations of hypoxic cells that exist within the tumour microenvironment reduce the ability of radiotherapy to cause the formation of highly reactive radical species within cells. These radical species result in multiple forms of cellular damage including protein-protein and protein-DNA cross-links, as well as various forms of DNA damage (1). The most lethal lesions caused by radiotherapy are DNA double strand breaks (DSB), in which both strands of a DNA double helix are severed within 15-20 base pairs (2). The importance of DSB is evident in the close correlation between the number of DSB generated by ionizing radiation and the resulting cell death, and additionally by the severe radiosensitivity resulting from loss-of-function mutations in genes which code for proteins involved in DSB repair pathways (3, 4). The oxygen effect The presence of molecular oxygen is thought to potentiate the effect of ionizing radiation by a mechanism described in the oxygen fixation hypothesis (5). The oxygen fixation hypothesis maintains that the critical product of radiation, DNA radicals, can be oxidized by cellular molecular oxygen to form enzymatically irreparable peroxyl-DNA adducts (5). According to this hypothesis, hypoxia renders cells resistant to the effects of ionizing radiation owing to the low concentration of molecular oxygen available to oxidize   2 the DNA radicals and the presence of a cellular environment that is more conducive to the reduction of DNA radicals by thiol containing compounds, such as glutathione. When molecular oxygen is scarce, thiol containing compounds are thought to effectively compete for reduction, rather than oxidation of the DNA radicals, resulting either directly in restitution of the DNA structure or in a form of damage amenable to repair cellular DNA repair enzymes (2). The cell survival difference observed between cells irradiated under well-oxygenated and hypoxic conditions is commonly quantified by a term called the oxygen enhancement ratio (OER). OER is defined as the ratio of the hypoxic and oxic doses of radiation required to produce a given surviving fraction of cells. The OER varies modestly between cell lines and reaches a maximum value close to 3.0 (6). 1.2 DNA double strand break repair Homologous recombination repair The mammalian cell has two primary pathways used to repair DNA double strand breaks: homologous recombination repair (HRR) and non-homologous end joining (NHEJ). HRR is a high fidelity, potentially error-free repair pathway that uses a DNA strand with long regions of homology to the damaged region, normally a sister chromatid, as a template for repair. This involves recognition of the DNA damage by sensor proteins, followed by endo/exonuclease processing of the damaged strands which allows for invasion of the damaged strand into the template strand and subsequent replication of the damaged strand using the intact template (7). Although HRR can be a high fidelity DSB repair pathway, the requirement for a homologous strand to be in close proximity to the DSB restricts its activity to the S and G2 phases of the cell cycle (8). It can be argued that since HRR is limited to cells within S and G2 phase, targeting HRR could provide a therapeutic index by damaging the actively proliferating cancer cells while having less effect on most normal quiescent tissue.   3 Problems could arise however when targeting HRR in chronically hypoxic cells because a key protein of the HRR pathway, Rad51, has been found to be down regulated in chronically hypoxic cells and this deficiency in Rad51 has been found to limit the use of the HRR pathway in hypoxic cells (9-11). Additionally, loss-of-function mutations in proteins involved in HRR increase sensitivity to X-rays, but more severe radiosensitivity occurs in response to X-rays when components of the NHEJ pathway are deficient, implicating NHEJ as the predominant pathway involved in repair of ionizing radiation induced damage (12). Non-homologous end joining NHEJ is a low fidelity DSB repair pathway in which the ends of a DSB are detected by DNA damage sensor proteins in a similar manner to HRR, however in NHEJ the DNA ends are processed by endo/exonucleases and DNA polymerases into a suitable substrate for ligation. In contrast to HRR, this process occurs without the use of a homologous DNA template strand and inevitably leaves insertion and/or deletion mutations flanking the repaired region. In NHEJ the sensor proteins are two units of a ring-shaped heterodimeric protein complex called Ku70 and Ku80. The Ku proteins bind with high affinity to the ends of dsDNA, with very minimal to no selectivity for DNA sequence or structure (13). The large 470kDa catalytic subunit of the DNA dependent protein kinase (DNA-PKcs) is then recruited to each of the Ku 70/80-DNA complexes (7). The entire holoenzyme complex of DNA, Ku 70/80, and DNA-PKcs is referred to as DNA-PK. Once formed, DNA-PK becomes activated by multiple autophosphorylation events, the specific details of which are yet to be well characterized. It is clear that this activation initiates movement of the DNA-PK complex away from the broken ends, allowing for recruitment of processing enzymes, such as the endonuclease artemis, and a ligation complex composed of XLF, XRCC4, and ligase IV (12). Mutagenesis of DNA-   4 PKcs at amino acid residues that are known to be autophosphorylated diminishes NHEJ activity and can either inhibit or enhance the HRR pathway depending on the region mutated, demonstrating the crucial role of DNA-PK activation in DBS repair (14). DNA-PKcs is a member of the PI3-Kinase-like Kinase (PIKK) family of proteins, which also includes other DNA damage responsive proteins, ATM, ATR, and mTOR (15). Members of the PIKK family have a characteristic catalytic domain bearing sequence homology to that of the lipid phosphotidylinositol 3-kinases (15). Structural information is limited for DNA-PKcs due to the lack of a high-resolution X-ray crystal structure or solution NMR structure. Difficulty obtaining such structures is likely because of the proteins large 470 kDa size. Despite this lack of structural information, selective inhibitors of DNA-PKcs have been developed based on homology modelling with the crystal structure of PI3-K and pharmacophore mapping of the non-selective inhibitor LY294002 (16, 17). 1.3 Hypoxic cell radiation sensitizers and hypoxia activated prodrugs A number of treatment approaches and anti-cancer agents have and continue to be designed to target hypoxic cells in solid cancers, including many trials of hyperbaric oxygen, hypoxic cell radiosensitizers, bioreductive hypoxic cytotoxins, carbogen, and accelerated radiotherapy with carbogen and nicotinamide (ARCON) (18). Aside from select hypoxic cytotoxins currently undergoing clinical evaluations, most of these agents or modalities have had disappointing results due to high toxicity profiles or impracticality, and have been stymied by an inability to select appropriate patient populations that would most benefit from treatment. Despite the disappointing results of individual trials, meta-analysis of multiple clinical trials of nitroimidazole based agents and the hyperbaric oxygen trials suggests that targeting the hypoxic cell population is a valid strategy and   5 can enhance both local tumour control and patient survival, particularly in patients with head and neck, bladder, and uterine cervix cancers (19-22). The nitroimidazole based hypoxic cell radiosensitizers are sometimes referred to as oxygen mimetic radiosensitizers because there are some parallels with oxygen in their reactivity towards DNA base radicals (23). The exact mechanism of action of these agents is somewhat unclear due to the short half-life of reactive intermediates, but it is thought to involve the generation of DNA strand breaks through a fixation of DNA damage, as well as reaction with protein thiols and DNA bases to form detrimental macromolecule-drug adducts (23-25). As radiosensitizers, nitroimidazole drugs require very high peak serum concentrations in order to be effective. For example, in humans a 0.5-4 g dose of misonidazole (Ro 07-0582) is required such that peak plasma concentrations reach to the order of 0.1-1 mM (26). Similarly, in order to sensitize hypoxic cells in culture such that they become similar in radiosensitivity to oxygenated cells, >15 mM misonidazole in cell culture media is required (27). In this work, some of the prodrugs utilize a nitroimidazole as the basis for a hypoxia activated trigger, however the concentration of prodrugs administered is up to 150x lower than that of misonidazole in culture because the radiosensitizing activity of these prodrugs is derived from a more potent and molecularly targeted DNA-PK inhibitor. The components of a prototype prodrug and mechanism of prodrug activation are displayed in figure 1.1. We have taken a modular approach to the design of hypoxia activated DNA repair inhibitors (HADRI) prodrugs, as first described by Denny et al. (28). In this approach, prodrug design is considered with regard to three modules, those being 1) a trigger, which is required to undergo selective activation in tumour cells, 2) a linker, which is required to deactivate the effector and transmit the trigger activation signal, 3) an effector component, which exerts a potent cytotoxic effect. With this design, the   6 nitroimidazole acts as a means for hypoxia selective bioreduction, defining it as the trigger component of the prodrug. The prodrugs developed herein are designed to undergo intramolecular through-bond fragmentation upon bioreduction in hypoxic tissue. This mechanism has been previously described for prodrugs containing nitroaromatic triggers, and involves a four electron reduction of the nitroaromatic into a hydroxylamine (29). For the prodrugs described herein, this reduction is designed to result in fragmentation of a strategically placed ether linker, releasing the effector, a potent DNA- PK inhibitor. By including a DNA-PK inhibitor as the effector module of these prodrugs, we hope to prevent DNA repair in the hypoxic regions of tumours following radiotherapy. It is hoped that this design will dramatically increase the potency of these prodrugs over that of previous hypoxic cell radiosensitizers, thereby requiring lower doses for effective treatment and lessening the systemic toxicity. 1.4 Current status of DNA-PK inhibitors for use in cancer treatment In contrast to nitroimidazole based hypoxic cell radiosensitizers, which have undergone many clinical trials, DNA repair inhibitors have been developed more recently and entered clinical trials in 2005 with inhibitors of a DNA single strand break repair protein called poly (ADP-ribose) polymerase or PARP. PARP inhibitors are showing early success, particularly in treating patients with hereditary breast ovarian cancer syndrome where tumours carry mutations in both copies of the DNA repair associated BRCA1 and BRCA2 genes (30). Inhibitors of the DSB repair proteins ATM, a critical protein in the HRR pathway, and DNA-PK are also being developed for therapeutic use in cancer and are currently in various stages of pre-clinical development (7, 31-36). We have chosen a DNA-PK inhibitor called IC86621 as the focus of our initial proof of principle and drug development studies; see Appendix C for chemical structure.   7 Although IC86621 is designed to target NHEJ through the inhibition of DNA-PK, it has been shown that treatment with IC86621 also decreases the amount of HRR repair (37, 38). It is suspected that inhibition of DNA-PK using the ATP competitive inhibitor IC86621 or the non-competitive inhibitor wortmannin stalls the DNA-PK complex at the site of a DSB by disallowing DNA-PK autophosphorylation (37). With DNA-PK stalled at the broken DNA ends, the access of other repair proteins to the site of the break is restricted.    8  Figure 1.1  Mechanism and components of hypoxia activated DNA repair inhibitor prodrugs    9 Chapter 2:  Materials and methods 2.1 Hypoxia and radiation treatments For hypoxia treatments, cells plated on glass were placed into a custom-built aluminium chamber, described in detail in chapter 3, which was then sealed and equilibrated to the desired gas mixture through cyclic evacuation and pressurization of the chamber with constant bilateral agitation of the culture media. This protocol is similar to that described previously (39). Briefly, the process consisted of eight 1 min cycles of 1) evacuation to 46.5 centimetres of mercury (cm Hg) vacuum over 5 s, 2) holding this vacuum for 20 s, 3) release of the vacuum over 5 s, 4) pressurization to 25.8 cm Hg over 5 s, 5) holding the pressure for 20 s, 6) release of the pressure over 5 s, which completes one cycle. Pressure is described in reference to standard atmospheric pressure being zero (gauge pressure). After eight cycles, a final cycle with the same evacuation procedure followed by pressurization to 5.2 cm Hg was completed and the chamber was left to agitate for 1 min. The total time used to reach radiobiological hypoxia was 10.3 min. This procedure was shown to be non-toxic to cells, however for experimental control, all cells in both oxygenated and hypoxic conditions were treated with this gassing procedure, using mixtures of 0-20% O2, 5% CO2, 95-75% N2 (Praxair). X-ray irradiation was delivered to the aluminium chambers with a 300 keV Pantak Siefert XRAD320 irradiation system at 3.3 Gy/min with no filtration other than that produced by the chamber (5 mm aluminium).   10 2.2 Inhibitors, drugs and prodrugs 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)ethanone (IC86621) was synthesised in the laboratory of Dr. Gregory Dake, University of British Columbia Department of Chemistry according to previously published methods (40). A stock solution of 15 mg/mL IC86621 in DMSO was prepared and small volume aliquots were prepared to avoid freeze thaw cycles. Aliquots were stored in the dark at -20 °C. The laboratory of Dr. Gregory Dake at the University of British Columbia Department of Chemistry synthesized HADRI1, HADRI2, HAPI2, and IC86621, the structures and IUPAC names for which are listed in appendix C. HAPI3 was synthesized by Geoff Winters at the Centre for Drug Research and Development (Vancouver, BC). 2-(Morpholin-4-yl)-benzo[h]chomen-4-one (NU7026) was purchased from Sigma-Aldrich Canada Ltd(catalogue no. N1537) A stock solution of 3 mM NU7026 in DMSO was prepared and similarly stored in small aliquots away from light at -20 °C. Hydroxy-3H-phenoxazin-3-one-10-oxide sodium salt (Resazurin, Sigma-Aldrich Canada Ltd, R7017) was prepared as a 2.2 mM stock solution in 0.9% NaCl. Resazurin was prepared in a biosafety cabinet and sterile filtered through a sterile 0.2 µM filter (Sarstedt Inc, 83.1826.001). The sterile resazurin stock solution was stored away from light at 4 °C. 2.3 Cell culture CB.17 and SCID/st mouse embryonic fibroblast cell lines, M059K and M059J human glioma cells, and HeLa human cervical carcinoma cells were a gift from Dr. Peggy Olive. All cells were maintained in vented cap cell culture flasks (Sarstedt Inc, 83.1810.002, 83.1812.002, 83.1813.002) with Minimum Essential Medium with Earle’s buffered salt solution (MEM/EBSS, Hyclone, Thermo Scientific, SH30013.03) supplemented with 10% fetal bovine serum (FBS, Hyclone, Thermo Scientific or Gibco,   11 Invitrogen). In some experiments antibiotics were included in the media, 50 mg/mL penicillin and 50 U/mL streptomycin (Gibco, Invitrogen). For the purpose of this thesis, standard incubation conditions include a humidified incubator at 37 °C in a 5% CO2/ 5% O2/ 90% N2 atmosphere and any referral to cell culture media or medium, unless otherwise stated, refers to MEM/EBSS containing 10% FBS. 2.4 Resazurin reduction assay This assay has been previously described for use in assessing cell viability following exposure to ionizing radiation(41). Exponentially growing cells were harvested using trypsin (Sigma-Aldrich Canada Ltd, T4049) and counted by haemocytometer. Cells were then seeded into 1.5 cm diameter custom made glass tissue culture inserts fitted for standard non-treated 24 well plates (BD Biosciences, 351147) at a density of 5 x 103 cells/well (CB.17) or 7.5 x 103 cells/well (HeLa). After seeding, cells were incubated in standard conditions for 18-24 h with 500 µL of medium/well. After 18 h the medium was removed and replaced 1 h pre-irradiation, by 200 µL of medium containing DMSO, DNA- PK, or prodrugs as specified. 24 well plates were placed into aluminium chambers and gassed and irradiated as described in section 2.1. As specified, between 0-6 h following irradiation plates were removed from the chambers and placed back under standard incubation conditions. Drug treatments remained on the cells for 4-24 h following irradiation as specified, at which point the medium was replaced with 500 µL of fresh medium. In all experiments, 72 h following irradiation a stock solution of 2.2 mM resazurin was added to the cell culture medium to a final concentration of 220 µM. Approximately 4-8 h following the addition of resazurin, when the medium within the untreated control wells turned a dark pink colour, the amount of reduced resazurin (resorufin) was assayed by fluorescence using a TECAN GENios plate reader, excitation   12 "535 nm, emission "590 nm. Percent viability was determined by normalizing fluorescent values such that the mean resorufin fluorescence of non-irradiated controls represents 100% viability and the background fluorescence from 220 µM resazurin in cell culture medium without cells represents 0% viability. Graphs were generated with Prism 4.0 (GraphPad Software Inc.). 2.5 Clonogenic survival assay For clonogenic survival assays 1 x 105 HeLa cells were plated into 1.5 cm diameter custom glass inserts in non-treated 24 well plates (BD Biosciences, 351147), or 2 x 106 CB.17 or SCID/st cells were seeded in sterilized 60 mm diameter glass petri dishes (VWR International, 89000-300). Cells were incubated under standard incubation conditions for 18-24 h before use in experiments. For drug treatments in section 4.4, HeLa cells were pre-incubated for 1 h before irradiation with 100 µM IC86621, 100 µM HADRI1, or 0.1% DMSO as a vehicle control. Drug treatments remained on the cells for 4 h following irradiation, making for a 5 h total drug exposure time. Immediately following irradiation (CB.17 and SCID/st cells) or drug removal (HeLa cells), cells were harvested by trypsinization and kept on ice before being counted with a Z1 Coulter particle counter (Beckman Coulter). Dilutions were made in media such that approximately 100 surviving colonies would result, and cells were plated on standard 60 mm and 100 mm diameter tissue culture dishes (BD Falcon) with 10 mL media. Cells were incubated in standard conditions for 10-14 days before the media was removed and surviving colonies were stained with 1% crystal violet / 30% ethanol solution. Colonies of >50 cells were counted and the surviving fraction was determined by the following formula: Surviving fraction = number of cells plated x plating efficiency number of colonies    13 2.6 Flow cytometry of !H2AX and cell cycle analysis 1.5-2 x 106 HeLa cells were plated in 60 mm glass dishes and incubated overnight in a 5% O2/ 5% CO2 incubator. The following day plates were pre-treated for one hour with 100 µM IC86621 or 0.1% DMSO in media. Cells were then gassed and irradiated as described in section 2.1.  Following irradiation cells remained in the chambers at a given oxygen tension at 37 oC for one hour and were then removed to normal incubation in a 5% O2/ 5% CO2 incubator for a further 23 h. Cells were harvested 24 h following irradiation, fixed in methanol and stained for flow cytometry with a monoclonal antibody to !H2AX (Upstate mouse mAb anti-phospho H2AX Ser 139 clone JBW301), either directly conjugated to fluorescein isothiocyanate (FITC) or by using an AlexaFluor 488 anti-mouse secondary antibody (Invitrogen). The same cells were also treated with 5 mg/mL RNase A and stained with 50 mg/mL propidium iodide (Sigma, P4170) for cell cycle analysis. Results were analyzed with FlowJo software (Tree Star Inc.) using the Dean/Jett/Fox cell cycle algorithm. 2.7 Mouse liver microsome preparation  This protocol is a modified version of that described by Wu and McKown (42). Mouse liver mircosomes were prepared using livers from 10 week old male C57/Bl6 mice.  Following euthanasia by CO2 asphyxiation, livers were excised and rinsed briefly in sterile 0.9% NaCl. Tissue was minced with a scalpel and homogenized in a dounce homogenizer with the addition of 2-4 mL/liver ice cold Tris-HCl buffer (1.15% KCl, 0.05 M Tris-HCl pH 7.4). The liver homogenate was divided into equal volumes in ultracentrifuge tubes (Ultra-clear open top, Beckman Coulter) and the volume of liquid within the tubes was brought to 38 mL with Tris-HCl buffer. The liver homogenate was then centrifuged at 9,000 xg in an SW28 rotor (Beckman Coulter) for 30 min at 4 °C. The   14 resulting supernatant (S9) was then transferred to fresh ultracentrifuge tubes and brought to 38 mL volume with Tris-HCl buffer. The S9 was then centrifuged for 1 h at 100,000 xg at 4 °C. The supernatant was then discarded and the resulting microsomal pellet was re-suspended in 1 mL per liver cold 0.1 M HEPES/ 1.15% KCl buffer. Aliquots of microsomes were stored at -80 °C until use. Protein concentration of the microsome solutions was determined to be 20 mg/mL by the Biorad DC Assay. 2.8 Microsomal stability assays  This protocol is modified from that of Lan et al. (43) Microsomal incubations occurred in a total volume of 0.5 mL containing 5 µL of 0.5 M MgCl (5 mM MgCl final), 50 µL of 20 mM nicotinamide adenine dinucleotide phosphate (NADPH, Sigma, N6505) (2 mM NADPH final), 10 µL of microsomes (200 µg protein), and either 420 µL of 178 µM HAPI3  (150 µM HAPI3 final) or 1.19% DMSO (1% DMSO final) in Dulbecco’s phosphate-buffered saline (D-PBS, Hyclone). Microsomes were added last to start the reaction and experimental controls in which NADPH and/or microsomes were replaced with distilled H2O were performed in parallel. Reactions were incubated at 37 °C in either room air or in a hypoxic glove box at 0.2 ± 0.2% O2 for 2 h. 50 µL samples were removed from reactions every 20 min. Reactions were stopped by the addition of 200 µL ice cold methanol. Samples were stored at -20 °C for 1-2 days before HPLC analysis was performed. 2.9 High pressure liquid chromatography (HPLC) Chromatographic analysis was performed on Waters HPLC equipment (Waters Limited, Mississauga, ON, CA), including a model 510 pump, model 712 WISP injector, and model 996 photodiode array detector. A Symmetry C18 column (3.9 x 150 mm) was   15 used for sample separation with a mobile phase consisting of a mixture of 36% acetonitrile flowing at 1.5 ml/min where by HAPI3 eluted after 3.6 min and IC86621 after 4.6 min. Samples of 30 µL were injected and detection was carried out at "320 nm for HAPI3 and "340 nm for IC86621. Concentrations were determined from absorbance values using standard curves of external standards. 2.10 Cell lysis and western blotting  1-5 x 106 HeLa cells were plated in 10cm diameter standard tissue culture dishes (Sarstedt) and were incubated in standard conditions for 24-72 h before being placed in a humidified 37 °C incubator within a glove box containing 1% O2/ 5% CO2/ balance N2 for 4-72 h. Cell lysis occurred within the glove box by removing the culture media, rinsing the plates once with D-PBS, and adding 1.5 mL of Mammalian Protein Extraction Reagent (M-PER, Pierce, Thermo Fisher Scientific) containing 1 mM Sodium Fluoride, 1 mM Sodium Orthovanadate, 2 mM phenylmethanesulphonylfluoride (PMSF) and 10 µL/mL Protease Inhibitor Cocktail (BioShop Canada Inc, SFL001, SOV664, PMS123, PIC004).  Cells were scraped into the 1.5 mL of lysis buffer and the volume from each plate was collected into a 1.5 mL tube (Sarstedt). Cell solutions were shaken in the hypoxic glove box following and protein concentration was determined using the Biorad DC Assay (BioRad Laboratories Canada Ltd.).  For western blots, 100 µg lysate samples in Laemmelli buffer were separated by discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblot transferred to 0.45 mM nitrocellulose membrane using the mini-PROTEAN 3 system (Bio-Rad Laboratories Canada Ltd). Membranes were blocked for 1 h with 2% bovine serum albumin (BSA) in PBST buffer (D-PBS containing 0.01% Tween-20) before being incubated with primary antibodies against Ku70 (mouse anti-Ku70, Santa Cruz Biotechnology, Ab-9), Ku80 (mouse anti-Ku80, Neomarkers Thermo Scientific, Ab-2), DNA-PKcs (rabbit anti-DNA-   16 PKcs, Calbiochem, Ab-1), and $-tubulin (mouse anti-$-tubulin, Invitrogen). Following washing 5x in PBST over 1h, secondary antibodies solutions of horseradish peroxidase (HRP) conjugated goat anti-mouse (Invitrogen, 65-6420) or goat anti-rabbit (AbCam, ab6721-1) were applied to the membranes.  Membranes were again washed 5x in PBST over 0.5 h before the addition of enhanced chemiluminescence (ECL) detection reagents (Thermo Scientific).   17 Chapter 3:  Development of prodrug screening assays Preliminary drug discovery screening assays can be broadly broken down in to two categories, cell-based screening assays that measure the activity of test compounds on cell cultures, and cell-free in vitro assays that measure the ability of test compound to directly affect the pathway or protein target of interest in a biochemical assay. The benefits of these assays are complementary and when both are used, they allow for assessment of drug activity and cellular toxicity in the complex milieu of the cell and a more mechanistic understanding of effects on the specific protein target.  This chapter describes the development of both cell based and cell-free assays that have been designed to screen candidate hypoxia activated DNA repair inhibitor prodrugs. A focus has been deliberately placed on the cell-based assays, as these have had to be developed in house to be used as the primary screening tool, while commercial assays are readily available for in vitro assessment of DNA-PK kinase activity. 3.1 Hypoxia chamber development: the effect of cyclic evacuation and pressurization on cell viability Introduction In order to develop hypoxia activated radiosensitizing agents in an efficient and economical manner, it was necessary to develop a series of screening assays that employ systems for rendering cells hypoxic whilst allowing them to be irradiated. Many groups studying hypoxia perform cell culture based experiments within sealed plexi- glass glove boxes, wherein cells can be manipulated within the glove box, but cannot be easily transported out of the glove box while remaining hypoxic. Furthermore, performing   18 experiments in which multiple oxygen tensions are tested can take many days in a glove box due to the lengthy time required for gases to exchange throughout the large volume of the box. To circumvent these problems, custom hypoxia chambers were designed and constructed by Dr. Andrew Minchinton and Dr. Alastair Kyle, in which cells can be rapidly rendered hypoxic due to the small volume of the chamber. This smaller chamber is also easily transportable, making possible transfer to an X-ray machine for irradiation. A diagram and photograph of the chamber can be seen in figure 3.1. The design of this chamber is based upon that of a similar aluminium hypoxia chamber previously described by Dr. Cameron Koch (39, 44). Earlier efforts using aluminium hypoxia chambers involved a relatively slow gas exchange process in which the gaseous contents of the chamber were exchanged between 5-8 times over the course of 1-48 h (44-46). In aluminium hypoxia chambers, evacuation at reduced pressure causes rapid out-gassing of residual cellular oxygen, while using a thin medium layer allows rapid equilibration with the replacement gas mixture. Seeing as mild positive and negative pressure cycles had no effect on cell viability in these systems, we used a wider range of gauge pressure within the gas exchange cycles, including pressurization to 25.8 cm Hg and vacuum to 46.5 cm Hg. This gas exchange protocol was tested by examining cell viability 72 h following a 10.3 min gas exchange procedure with 21% O2/ 5% CO2 / balance N2 gas. Approach HeLa human cervical carcinoma cells were plated on glass inserts within 24 well plates as per section 2.4. In this experiment control cells were not treated to the gas exchange procedure, but were left in laboratory atmosphere conditions for 10 min. The viability of control cells was compared to cells that had been gassed using our standard gassing protocol of eight 30 s evacuation/ 30 s pressurization gas exchange cycles in   19 the aluminium chamber with 21% O2/ 5% CO2/ balance N2. Non-irradiated cells were placed immediately back in to standard incubation conditions following the 10 min wait or the 10 min gas exchange procedure, while irradiated cells were placed back in standard incubation conditions immediately following irradiation. Irradiated cells received a dose of 5 Gy immediately following either the 10 min wait period in air or following the 10 min gassing protocol. A minimum of n = 24 wells for each treatment group and an unpaired two-tailed t-test analysis was performed on the groups using Prism GraphPad V5.0b for Macintosh software. A P-value <0.01 was considered to be significant. Cell viability was assayed with the resazurin reduction assay 72 h following treatment as per section 2.4. Results Figure 3.2 displays the results of a cell viability assessment following the gas exchange procedure.  Contrary to our expectation of some toxicity imposed by the gassing, the gas exchange protocol confers a slight but significant increase in viability over cells left outside of the incubator in normal air for 10 min (P<0.05, two tailed unpaired t-test). Although the cause of this increase was not further investigated, it may be due to appropriate buffering of the media with 5% CO2 during the gassing protocol, while the cells left in atmospheric air may become somewhat alkaline due to the low pCO2 in atmospheric air. This effect on cell viability becomes indiscernible in cells treated with 5 Gy IR, as there is no significant difference found in viability between cells that were irradiated following gas exchange or following 10 min in laboratory air. Despite only minor effects of cell viability, in attempt to rule out any effects on survival caused by the gassing protocol, all cells were treated with the gas exchange protocol in both control and treatment groups for all of the experiments in this work unless otherwise indicated.    20  Figure 3.1 Photograph and diagram of an aluminium hypoxia chamber   21   Figure 3.2 Cell viability following gassing protocol with and without 5 Gy irradiation    22 3.2  Hypoxia chamber development: the number of gas exchange cycles necessary to achieve maximum radiobiological hypoxia Introduction  Hypoxia can be defined as having a pO2 lower than normal, so a standard working definition of radiobiological hypoxia for the purpose of this thesis, is such that cells are rendered radiobiologically hypoxic if cell viability is greater in hypoxic cells following a given dose of radiation, compared to the viability of cells irradiated under conditions of &3% O2. To further test the hypoxia chambers, the number of 30 s evacuation/ 30s pressurization gas exchange cycles in the hypoxia chamber necessary to achieve maximal radiobiological hypoxia was determined. Approach The chamber was tested for its ability to render cells hypoxic by examining cell viability with the resazurin reduction assay, see section 2.4 for details. Briefly, cell viability was assessed 72 h following a gas exchange procedure consisting of 0-10 gas exchange cycles. Following gassing, cells were irradiated with 5 Gy. A control treatment representing 100% cell viability was included in which cells were neither gassed nor irradiated. As per the definition of radiobiological hypoxia, it is expected that if cells are being rendered hypoxic, their survival will increase following ionizing radiation treatment until a maximum level of radioresistance imparted by the hypoxia is reached, at which point cells are effectively anoxic. Prism GraphPad software for Macintosh V5.0b was used to fit these data to a variable slope sigmoidal dose-response curve. Results   The results of this experiment are displayed in figure 3.3. Following increased numbers of gassing cycles, cells became increasingly resistant to the effects of ionizing   23 radiation as displayed by the corresponding increase in cell viability. This increase in cell viability occurred up to ten gas exchange cycles. A protocol using eight gas exchange cycles was used throughout this work because it achieves a level of hypoxia that is close to, albeit slightly decreased from complete anoxia, which has been shown to induce cellular apoptosis (47). Although this level of hypoxia is sufficient for the purposes of this prodrug screening program, which requires only that cells display practical radiobiological hypoxia, future work could address more specifically the oxygen tensions achievable using this system. In order to specifically determine the level of dissolved oxygen within the cell cultures, an aluminium chamber will need to be modified to include an oxygen sensor that is in contact with the cell culture media.  This likely could be achieved using a fibre optic oxygen sensor enclosed within the chamber through a sealed port.   24   Figure 3.3 HeLa cell viability following 2-10 cycles of gas exchange in hypoxia chambers and 5 Gy ionizing radiation.    25 3.3  Hypoxia chamber development: differentiation between pO2 tensions achieved using the hypoxia chambers Introduction To further characterize the hypoxia chambers and assess the ability of the resazurin reduction assay to differentiate between levels of cell viability following radiation, we employed the resazurin reduction assay following gas exchange with various gases containing between 0-5% O2 to induce different levels of hypoxia in combination with radiation dose response. Approach To test for the ability to differentiate between differences in oxygen tension, CB.17 MEF cells plated on glass inserts within 24 well plates were placed within the hypoxia chamber on the multi-attenuator insert, described in section 3.4. The chambers were sealed and gassed with 5% CO2/ balance N2 gas mixtures containing 0%, 0.2%, 0.5%, and 5% oxygen before being irradiated. Following irradiation cells were removed from the chambers and returned to normal incubation conditions where they were allowed to recover for 72 h before being assayed for viability with the resazurin reduction assay; see section 2.4 for assay details. Results The results presented in figure 3.4 demonstrate that cell viability was found to decrease along with oxygen tension at each of the radiation dose administered. Although statistical significance was not achieved between the various oxygen tensions at a given radiation dose, the trend of increasing radioresistance along with decreasing oxygen tension is consistent with the known radiation response to hypoxia, indicating that the chambers are functioning consistently in the gas exchange protocol and that the   26 resazurin reduction assay is sensitive enough to detect relatively small changes in cell viability.                 27  Figure 3.4 Percent viability of CB.17 cells equilibrated to various levels of hypoxia in response to IR.   28 3.4 Testing of a multi-attenuator insert to achieve multiple radiation doses with a single administration Introduction In order to increase the throughput of the candidate compound screening assays, a multi-attenuator insert was designed and built by Dr. Alastair Kyle. This insert was tested for radiation dosimetry and compatibility with the resazurin reduction assay by the author in concert with Dr. Kyle. The multi-attenuator insert fits within the hypoxia chamber, housing two 24-well plates, functioning to attenuate the intensity of the radiation beam across the plates. It is designed such that each of six columns in the 24 well plates receives different radiation doses when a single radiation administration is delivered from below, see figure 3.5, left. The ability to achieve multiple radiation doses with a single administration drastically shortens the time required to perform experiments assessing the effect of candidate prodrugs across various doses of radiation. Approach The amount of attenuation achieved across each column was determined using thermoluminescent dosimetry (TLD). TLD chips were placed within the wells of a 24-well plate containing glass inserts, corresponding to where cells would be in a typical experimental set up for either the clonogenic or resazurin reduction assays. A control chip that remained outside of the chamber was included to determine a background radiation reading. The background TLD value was subtracted from all readings and values were normalized to column 1 which contains no attenuating material and was designated as 0% attenuation. To visualize the attenuation, a piece of X-ray film was placed over top of the multi-attenuator and sealed within the chamber in a dark room. The chamber was then   29 irradiated with the equivalent of 3 Gy before the film was removed in the darkroom and developed. Results The results of the TLD measurments are displayed in Table 3-1. The attenuation was also visualized using X-ray film placed within the chamber prior to radiation delivery, see figure 3.5, right. Table 3-1 A list of the attenuating agents used to make the multi-attenuator and corresponding percent attenuation 24 well plate column Attenuating agent Background subtracted TLD reading (arbitrary units) Percent attenuation of administered dose 1 None 8073 0% 2 0.25 mm brass 6089 25% 3 0.76 mm brass 4513 44% 4 1.52 mm brass 3614 55% 5 1.52 mm brass, 0.38 mm lead 1867 77% 6 1.52 mm brass, 11.38mm lead 436 95%   30   Figure 3.5 Photograph and X-ray film of the multi-attenuator insert.   31 Chapter 4: Establishment of proof of principle A number of studies have demonstrated the radiosensitizing ability of broadly selective PI3K and PIKK inhibitors such as wortmannin and LY294002 as well as inhibitors that are more selective to DNA-PK such as PI-103, NU7441, IC87361 in oxic cells, see Appendix B. However, very few studies have been published exploring the inhibition of DNA-PK in hypoxic cells, and thereby it was necessary to establish that inhibition of DNA-PK in hypoxic cancer cells would sensitize hypoxic cells to ionizing radiation. This chapter includes experimental evidence that all three subunits of DNA-PK are expressed in hypoxic cells, and that genetic inactivation and chemical inhibition of DNA-PK can radiosensitize hypoxic mouse embryonic fibroblast (MEF) and human cervical carcinoma cells. Also included is a proof of principle experiment surrounding the design of hypoxia activated prodrugs of the DNA-PK inhibitor IC86621, referred to as HAPI2 and HAPI3. 4.1 DNA-PKcs, Ku70, and Ku80 protein expression is stable in HeLa human cervical cancer cells under hypoxic conditions Introduction  The expression of key proteins within the HRR pathway, including Rad51, Rad54, and XRCC3 has been shown by Chan et al. to decrease under conditions of chronic hypoxia, affecting the function of HRR in hypoxic cells (9). It is well established that a number of cellular processes are altered in response to hypoxic stress, and arguably the most characterized mediator of these processes is a transcription factor protein called hypoxia inducible factor 1 alpha (HIF1-#). In the presence of molecular   32 oxygen HIF1-# is hydroxylated at two critical proline residues by prolyl hydroxylase (PHD) enzymes (48). This hydroxylation targets HIF1-# for proteasomal degradation. However, in hypoxic conditions HIF1-# expression is stabilized, resulting in HIF1-# mediated gene expression changes which affect various cellular processes such as energy metabolism, proliferation, apoptosis, and angiogenesis (48). In order for the proposed prodrugs to be effective it is crucially important for the target protein, DNA-PK, to be expressed in hypoxic cells. There are only a small number of reports investigating the expression of either protein or mRNA expression for DNA-PK under hypoxic conditions (49, 50). Therefore, in order to confirm the presence of the drug target we have assessed the protein expression level of the three DNA-PK subunits, DNA-PKcs, ku70, and ku80, in acute and chronically hypoxic HeLa cells. Approach  Details of this protocol are presented in section 2.9. Briefly, HeLa cells were grown in a humidified 37 °C incubator within a 1% O2/ 5% CO2 glove box. At time points between 4-72 h cells were lysed. Whole cell lysate was separated by SDS-PAGE, transferred to 0.45 µm pore size nitrocellulose membrane and probed with antibodies against Ku70, Ku80, DNA-PKcs, HIF1-#, and $-tubulin. Secondary antibodies were HRP conjugated and enhanced chemiluminescence was carried out with film detection. Densitometry was performed on digital images of scanned films using NIH ImageJ V1.42 for Macintosh. Results Figure 4.1 presents the protein expression of each of the DNA-PK components from HeLa cell whole cell lysate collected following exposure to 1% oxygen for 4, 24, 48, and 72 h; $-tubulin is included as a protein loading control. Also displayed is the HIF1-#   33 expression, which was found to increase with time under hypoxia up to 72 h, at which point HIF1-# expression decreased. This decrease in HIF1-# expression is consistent with the up-regulation of PHD enzymes, which de-stabilize HIF1-# following prolonged exposure to hypoxia (51). Expression of DNA-PKcs was found to be relatively stable throughout hypoxia, however there is an increase in both Ku70 and Ku80 expression visible after 4 h and peaking after 48 h, and with levels remaining higher than in oxic cells after 72h. This result confirms a strong presence of DNA-PK in hypoxic cells, supporting its suitability as a target for hypoxia activated DNA repair inhibitors.      34  Figure 4.1 HeLa cell protein expression of DNA dependent protein kinase subunits following 4-72 hours of hypoxia at 1% oxygen   35 4.2 Genetic DNA-PK deficiency causes sensitivity to ionizing radiation independent of hypoxia Introduction Previous work with DNA-PK inhibitors has shown their ability to sensitize cancer cells in culture and human xenograft tumours in mice to the effects of DNA damaging agents under normoxic conditions, however, the effects of DNA-PK inhibitors on the sensitivity of hypoxic cells to ionizing radiation has not yet been thoroughly explored (52- 54). In one study by He et al., adenoviral mediated expression of a dominant negative Ku70 variant was shown to radiosensitize hypoxic human glioma and colorectal carcinoma cells that were equilibrated to 0.5% O2 for 24 h prior to irradiation (55). A second study by Murray et al. has examined the effects of oxygen on the radiosensitivity of two human glioma cell lines derived from the same tumour, one of which expresses functional DNA-PKcs, called M059K, and the other which lacks functional DNA-PKcs, M059J. This study demonstrated that DNA-PKcs deficient M059J cells are hypersensitive to ionizing radiation compared to DNA-PKcs wild-type M059K cells, independent of hypoxia (6). Both of these results support the proposal that inhibition of DNA-PK activity would sensitize mammalian cells to the cell killing effects of ionizing radiation independent of the presence of molecular oxygen. To further assess the effect of DNA-PK inhibition on cellular radiosensitivity in hypoxic conditions, we took advantage of two mouse embryonic fibroblast (MEF) cell lines that were originally derived from the CB.17 mouse strain, however one of which is from a mouse harbouring the naturally occurring SCID mutation (56). The SCID mutation codes for a nonsense mutation at tyrosine 4046 in both copies of the prkdc gene, resulting in low level transcription of a c-terminal truncated form of the DNA-PKcs protein, which lacks the kinase domain required for DNA-PK dependent NHEJ activity   36 (57). The degree to which cell survival is affected following IR is relative to the extent of hypoxia within the cultures, and is commonly expressed by the oxygen enhancement ratio (OER) as mentioned previously. Approach Details of the clonogenic survival assay are described in section 2.5. Briefly, DNA-PK deficient cells, SCID/st, and DNA-PK wild-type cells, CB.17, were plated into glass petri dishes and allowed to adhere overnight. The next day cells were gassed and irradiated under oxic (21% O2/ 5% CO2/ balance N2) and hypoxic conditions (0% O2/ 5% CO2/ balance N2) using the aluminium chambers described in section 3.2. Immediately following irradiation, cells were removed from the chambers, harvested by trypsinization, and re-plated on round tissue culture dishes at dilutions predicted to yield 100 colonies. Plates were left undisturbed under standard incubation conditions for two weeks before the medium was removed and colonies of cells were stained purple with crystal violet and counted. Results MEF cells derived from a wild type CB.17 mouse were irradiated following equilibration to atmospheric oxygen conditions (21% O2) or hypoxic conditions (0% O2). As anticipated from previous studies, the surviving fraction of CB.17 cells was increased when radiation was administered under hypoxic conditions, producing an OER of ~2.6, see figure 4.2 red circles. MEF cells derived from a CB.17 mouse harbouring the SCID defect, SCID/st, were similarly irradiated and assessed for clonogenic survival. As with the DNA-PKcs proficient CB.17 cells, SCID/st cells lacking functional DNA-PK activity display enhanced clonogenic survival following irradiation under hypoxic conditions, resulting in an OER of ~2.4, see figure 4.2 black triangles. Notably, the hypoxic DNA-   37 PKcs deficient SCID/st cells are similar in radiosensitivity to oxygenated DNA-PKcs proficient CB.17 cells.   38  Figure 4.2  The survival of oxic and hypoxic CB.17 and SCID/st cells following exposure to ionizing radiation    39 4.3 Chemical inhibition of DNA-PK can sensitize both oxic and hypoxic cells to the effects of ionizing radiation Introduction As we are interested in chemically inhibiting DNA-PK selectively in hypoxic cells, it is necessary that chemical inhibitors of DNA-PK can act to sensitize hypoxic cells to radiation in a manner similar to that displayed by genetic deficiency. To assess the affects of chemical inhibition on the radiosensitivity of hypoxic cells we treated CB.17 cells with the DNA-PK inhibitors IC86621 and NU7026 either alone or in combination with radiation under oxic and hypoxic conditions. These two compounds were chosen for their selectivity against DNA-PK as well as the availability of published preclinical data regarding their efficiency at radiosensitizing oxic cells (31, 58). We were particularly interested in IC86621 due to its chemical structure, which has a 2-hydroxyl group that is particularly amenable for conjugation to a hypoxia activated trigger.   For the chemical structures and published IC50 values of the individual inhibitors see Appendices A and B. As the graph of radiation induced DSB repair versus time displays a biphasic exponential curve, it is widely accepted that DSB repair consists of both a fast and slow repair component (59). The reported half lives of DSB repair for each of these components is on the order of 5-15 min for fast repair, which is thought to occur for simple or clean breaks that are easily repaired by the cell, and 1-15 h for the slower component of repair of more complex breaks (59-61). Because the fast component of DSB repair can occur very quickly, an anti-cancer agent that inhibits DSB repair must be administered prior to the delivery of the DNA damaging agent in order for it to be maximally effective. In the case of hypoxia activated prodrugs, administration of the prodrug to patients would need to occur prior to administration of radiation therapy.   40 Thus, in experiments involving DNA-PK inhibitors and radiation we have pre-treated cells with the DNA-PK inhibitor prior to the administration of IR. Approach In these experiments, CB.17 cells were pre-treated for 1h with DNA-PK inhibitors or an equivalent amount of DMSO as a vehicle control; 1% DMSO for 30 µM NU7026 and 0.1% DMSO for 100 µM IC86621. Following the pre-treatment period, cells were equilibrated to hypoxic conditions (0% O2/ 5% CO2/ balance N2) or oxic conditions (21% O2/ 5% CO2/ balance N2) and subsequently irradiated using our aluminum chambers containing the multi-attenuator insert described in section 3.4. The DNA-PK inhibitor or DMSO (vehicle control) remained on the cells for 24 h following administration of IR. Cells were then allowed to recover for an additional 48 h in the absence of DNA-PK inhibitor or DMSO before cell viability was assessed by the addition of resazurin dye (refer to section 2.4 for details of this assay). The values reported are the mean ± standard error of the mean for at least three intra-experiment replicates. Percent viability was determined by normalizing fluorescent values such that the non-irradiated control in each treatment group represents 100% viability and background fluorescence represents 0% viability. Results Figure 4.3 displays the results of a cell viability assay performed after treating both oxic and hypoxic CB.17 cells with the DNA-PK inhibitor IC86621 in combination with various radiation doses. Oxic and hypoxic CB.17 cells were sensitized to the effects of IR when treated with the DNA-PK inhibitor. Both 50 and 100 µM IC86621 were able to sensitize hypoxic CB.17 cells to the effects of ionizing radiation, and did so in a dose dependent manner.   41 Similarly, the more selective DNA-PK inhibitor NU7026 also radiosensitized both oxic and hypoxic CB.17 cells, as displayed in figure 4.4. Here, hypoxic CB.17 cells treated with 30 µM NU7026 were sensitized to the effects of IR. It is important to note that the radiosensitization effect observed with the use of DNA-PK inhibitors occurs at clinically relevant doses of administered radiation, which is normally delivered in fractions of 2-3 Gy.     42  Figure 4.3  Radiosensitization of oxic and hypoxic CB.17 cells by IC86621   43  Figure 4.4  Radiosensitization of oxic and hypoxic CB.17 cells by NU7026    44 4.4 Residual DNA damage is increased in cells when treated with the DNA-PK inhibitor IC86621 in combination with ionizing radiation Introduction Among the numerous post-translational modifications known to occur on histone proteins is the phosphorylation of serine residue 139 on histone H2A.X. When phosphorylated at serine 139, H2A.X is termed !H2AX. H2A.X is a variant of the minor histone protein, H2A. Phosphorylation of H2A.X generally occurs rapidly in large kilobase regions of chromatin surrounding newly generated DNA DSB, and is thought to act as an amplification signal to recruit proteins involved in the DNA damage response(62). Foci of !H2AX can be identified in the nuclei of cells containing DSB by using antibodies generated against serine 139 phosphorylated H2A.X. Analysis of cellular !H2AX levels by immunofluorescence, using either microscopy or flow cytometry, is a well-described method used to monitor the generation and repair of DSB, through the formation and resolution of !H2AX foci respectively (63, 64). The primary kinase responsible for phosphorylating H2A.X at serine 139 in response to DNA damage is ATM. However DNA-PK has been shown to contribute to !H2AX formation and acts redundantly in cells where ATM is deficient (62, 65, 66). As cell cycle checkpoint activation tends to occur in cells with extensive DNA damage, we have also analyzed the distribution of cells throughout the cell cycle by assessing DNA content (67). Approach Following treatment with hypoxia, DNA-PK inhibitor IC86621, and IR, we have used flow cytometry to analyze cellular levels of !H2AX as a marker of DSB and have assessed cell cycle distribution using the DNA binding dye, 4',6-diamidino-2- phenylindole (DAPI). Flow cytometry and cell cycle analysis was performed by Dr.   45 Robyn Seipp as outlined in section 2.6. Briefly, HeLa cells plated on 60 mm diameter glass petri dishes were pre-treated for 1 h with either the DNA-PK inhibitor IC86621, or DMSO as a vehicle control and were gassed immediately following drug administration. Cell cultures were gassed and irradiated according to the method outlined in section 2.1. The gassing occurred 1 h prior to irradiation and cells were released from the hypoxia chamber to normal incubation conditions 1 h following irradiation. IC86621 or DMSO treatment continued following the administration of radiation until cells were collected and fixed with cold methanol 1 h or 24 h following irradiation. In these experiments HeLa human cervical carcinoma cells were used because they provide a more relevant model for human cancer than the CB.17 MEF cell line. The hypoxic conditions used in these experiments represent cells treated with 0.2% O2/ 5% CO2/ balance N2 while the oxic conditions represent cells treated with the equivalent of atmospheric air, 21% O2/ 5% CO2/ balance N2. Results DNA-PK inhibition resulted in higher !H2AX levels 24 h after irradiation, see figure 4.5 A. The increase in !H2AX levels 24 h after irradiation is indicative of increased residual DNA damage in the DNA-PK inhibitor treated cells, relative to vehicle treated controls. This effect was found to be independent of the dose of radiation to which cells were subjected, as at both 2.5 Gy and 5 Gy irradiation !H2AX levels are increased in the DNA-PK inhibitor treated cells relative to vehicle treated controls irradiated at the same dose. Similarly, this trend of increased !H2AX in IC86621 versus DMSO controls was found to be independent of the amount of oxygen present at the time of irradiation, as seen in figure 4.5 B. At all oxygen tensions measured, treatment with the DNA-PK inhibitor lead to increased !H2AX levels 24 h following irradiation relative to vehicle   46 treated controls. !H2AX levels were found to increase with increasing oxygen tension, indicating that increasing the pO2 of cells results in increasing levels of DNA damage from IR. This result is consistent with the oxygen effect and the oxygen fixation hypothesis described in section 1.1. IC86621 did not affect the initial amount of !H2AX induced one hour following irradiation, or in non-irradiated control cells, see figure 4.5 C. The increase in !H2AX levels with DNA-PK inhibitor treatment controls was found to be dependent on the dose of IC86621 treatment in the 50-150 µM range, see figure 4.6 A. 24 h following 5 Gy irradiation under both oxic and hypoxic conditions, !H2AX increases along with the concentrations of IC86621 relative to the vehicle control. IC86621 treatment displays no obvious effects on !H2AX levels of non-irradiated control cells, outside of a minor increase in !H2AX in both oxic and hypoxic cells at the 150 µM treatment level. In our analysis of cell cycle distribution we found that !H2AX levels correlated closely with increased numbers of cells in the G2 phase of the cell cycle following irradiation, see figure 4.6 B. Treatment with 100µM IC86621 increased the proportion of cells in the G2 phase of cell cycle relative to vehicle treated controls, while non-irradiated cells treated with IC86621 did not demonstrate large differences in cell cycle relative to controls, outside of a small increase in the G1 population of cells in IC86621-treated non-irradiated cells which was often observed. This increase in G1 cells may be due to off target effects of the IC86621 inhibitor, which is likely to also be inhibiting the PI3K signalling pathway through inhibition of P110$ at the 100 µM treatment level, see appendix B for IC50 values. Inhibition of PI3K signalling results in decreased phosphorylation of the signal transduction protein Akt, whose downstream targets are mediators of cell cycle entry (68).   47 Figure 4.5  DNA-PK inhibitor IC86621 increases residual DNA damage in HeLa cells 24h following treatment  combination with ionizing radiation    48  Figure 4.6 DNA-PK inhibitor IC86621 in combination with ionizing radiation causes an IC86621 does dependent increase in residual DNA damage and elicits a G2 block   49 4.5 IC86621 loses DNA-PK inhibitory activity when modified at the 2-hydroxyl site Introduction The inhibitor IC86621 was chosen as a lead compound for the generation of hypoxia activated DNA-PK inhibitors as it was suspected that the 2’-hydroxyl moiety on IC86621 would provide a suitable site for an ether linkage to the hypoxia activated trigger. This would theoretically allow for reduction of the prodrug as per the proposed scheme in figure 4.7 A. The binding pocket of DNA-PKcs is predominantly composed of hydrophobic amino acid residues, and correspondingly, many of the more selective inhibitors of DNA- PK contain lipophilic scaffolds. Unfortunately there is no high-resolution structure available for DNA-PKcs, likely due to its very large 470 kDa size, and it is unknown where exactly on the folded protein the ATP binding domain resides. However, DNA- PKcs has a high degree of homology with the ATP binding domain of the structurally well-characterized protein PI-3K!. An X-ray diffraction co-crystal structure of PI-3K! with the inhibitor LY294002 bound in the ATP binding domain was published by Walker et al. in 2000 (69). Several drug development groups have used LY294002 as a scaffold structure, from which chemical libraries have been designed and screened for DNA-PK inhibitory activity (16, 31). From the PI-3K! -LY294002 co-cystal structure, it has been discerned that a hydrogen bond is formed between the morpholino oxygen of LY294002 and the backbone amide of Val-882(69). As this valine residue is conserved between PI3K!  and DNA-PKcs, and the morpholino group is a common scaffold between LY294002 and IC86621, it is suspected that the binding of IC86621 to DNA-PKcs is structurally similar to that of PI3K! -LY294002, see figure 4.7 B. It was also detected from the Walker at al.   50 study that there is a putative hydrogen bond between the ketone moiety of LY294002 and the 'N of Lys-833 of PI3K! (69). This lysine is also conserved between the ATP binding pockets of PI3K!  and DNA-PKcs. Interestingly, Walker et al. observed that the space surrounding the ketone moiety of LY294002 is restricted and this ketone moiety is analogous in structure to the 2’-hydroxyl moiety in IC86621, see figure 4.7 B (69). If the binding of IC86621 to DNA-PK is as expected based on structural similarities, this model suggests there to be limited space surrounding the 2’-hydroxyl region on IC86621. This limited space would signify the 2’-hydroxyl site to be ideal for trigger attachment because conjugation of a trigger at this site could provide the steric hindrance necessary to deactivate the effector component of the prodrug, IC86621, until hypoxia mediated fragmentation of the prodrug occurs. Approach To further investigate whether the 2’ hydroxyl site of IC86621 is a good candidate for trigger attachment two analogs of IC86621 were synthesized, each of which is blocked at the 2’hydroxyl site by an O-linkage to a small methyl (HADRI1) and allyl (HADRI2) functional group, see Appendix C. Only HADRI1 was tested for its ability to inhibit DNA-PK by assessing its radiosensitization ability, as HADRI2 had poor aqueous solubility. HADRI1 was assessed for radiosensitizing ability using the clonogenic survival assay described in section 2.5.  As we had previously determined that DNA-PK inhibition sensitizes hypoxic cells and it is expected that the HADRI compound would act similarly under both oxic and hypoxic conditions, for simplicity this assay was performed under normal atmospheric conditions (21% O2). The multi-attenuator insert was used within the aluminium chambers to achieve various does of radiation. However, the cells were not treated with the gas exchange procedure. Triplicate dishes were plated for each   51 treatment group and the combined results of two experiments are presented with error bars representing the mean ± standard deviation.   52  Figure 4.7  Reduction scheme of HAPI 3 in hypoxia and structural comparison of IC86621 to LY294002   53 Results The results displayed in figure 4.8 are the average ± standard deviation from two experiments. Treatment with HADRI 1 displayed no radiosensitization over the vehicle control cells treated with 0.14% DMSO, indicating that no inhibition of DNA repair occurs with HADRI1 treatment. Also of note is that no apparent toxicity was observed in treatment with HADRI1 over that of the vehicle control. This result is in accordance with the structural model prediction suggesting the modification at the 2’hydroxyl will impair the DNA-PK inhibitory activity of IC86621. As expected, however, treatment with the unmodified IC86621 in combination with ionizing radiation decreased the surviving fraction of cells at all radiation doses, indicating an inhibition of DNA repair attributable to DNA-PK inhibition.   54  Figure 4.8  Modification of the 2'-hydroxyl site on IC86621 to an O-methyl eliminates radiosensitization activity   55 Chapter 5: Screening and development Throughout the development process the cell viability and clonogenic survival screening assays have been refined and have served as useful tools to assess radiation sensitivity under both oxic and hypoxic conditions. We have thereby continued to use these assays for the screening of two of three candidate prodrug compounds. The first compound synthesized, HAPI1, contained a nitrobenzene trigger moiety, see Appendix C. Unfortunately, HAPI1 had poor aqueous solubility and thereby was not thoroughly screened as a part of this work. There is a possibility however that HAPI1 could display promising hypoxia selective radiosensitizing activity and future work could address the opportunity to modify HAPI1 with polar functional groups in order to improve its aqueous solubility. Results from the preliminary cell-based screening of two other prodrug candidates, HAPI2 and HAPI3, along with in vitro screening of HAPI3 with a microsomal stability assay, are presented in this chapter. A majority of this work was done with HAPI3 as the chemical synthesis of HAPI2 was problematic and only a small amount of HAPI2 was available for screening. 5.1 Prodrug HAPI2 displays hypoxia selective radiosensitization and dose dependent toxicity under both oxic and hypoxic conditions Introduction Only 3 mg of the 2-nitroimidazole-IC86621 prodrug HAPI2 was synthesised by Dr. Bhaskar Rheddy in Dr. Gregory Dake’s laboratory in the UBC Department of   56 Chemistry. However, results with HAPI2 have been included because this prodrug showed interesting and desirable activity in the resazurin reduction assay, which at a minimum displays further validation of the screening approach and beyond that, the data indicates the potential for candidate prodrug if a higher yielding synthetic route were to be developed in the future. Approach HAPI2 was screened on CB.17 MEF cells using the resazurin reduction assay as described in section 2.4. It is important to note that this compound was approximately 80% pure by nuclear magnetic resonance analysis and contained unknown impurities that may have affected the experimental results presented below. CB.17 cells were used in this assay because our decision to screen primarily on HeLa cells, which provide a more relevant model of human cancer, had not yet been made. It has been established that human and non-human primate cells have significantly higher expression levels of DNA-PK than mice and other mammals(70). Additionally, as no humans have been known to carry a DNA-PK deficiency, it is suspected that non-functional DNA-PK mutations are embryonic lethal in humans, contrary to other mammals including mice, dogs, and horses(70). Despite these differences, comparable results were obtained between HeLa and CB.17 cells when testing the DNA-PK inhibitors IC86621. In this experiment HAPI2 was added to the cells at 0, 25, 50, or 100 µM immediately before gassing with 0.2% O2 or 21% O2 gas each containing 5% CO2/ balance N2. The 0 µM treatment contained 0.4% DMSO as a vehicle control such that the volume of DMSO added equalled that of the 100 µM HAPI2 condition. Following gassing, the chambers were incubated for 1 h at 37 °C before irradiation with 5 Gy or mock irradiation. Sealed chambers were then placed in a 37 °C oven for 6 h while cells were allowed to recover from the IR in the presence or absence of HAPI2 and hypoxia.   57 After 6h the chambers were opened to the air and prodrug-containing media was removed and replaced with fresh media and the 24 well plates were placed under normal incubation conditions. 72 h from the time of IR administration resazurin was added to assess cell viability according to the protocol described in section 2.4. Results The results of this experiment are presented in figure 5.1. In examining the non- irradiated (0 Gy) treatment groups, the decreasing resorufin signal with increasing doses of HAPI2 clearly displays a dose dependent toxicity for HAPI2 over the concentration range tested in both oxic and hypoxic conditions. However, in cells where DNA damage has been induced by the administration of 5 Gy ionizing radiation, HAPI2 does appear to have hypoxia selective radiosensitizing ability indicated by the dose dependent decrease in resorufin fluorescence only under hypoxic conditions. We were surprised to find slightly lower viability in the hypoxic cells compared to oxic cells in both the non- irradiated and irradiated cells. However, upon investigation of the experimental protocol it became clear that during incubation the hypoxic chambers were heated up to a temperature above 37 °C, which likely accounts for the overall decreased viability of the hypoxic cells and cannot be ruled out as a confounding factor. The results presented with HAPI2 are representative of three independent experiments. Unfortunately, due to the small amount of HAPI2 available we have been unable to repeat this experiment with appropriate incubation conditions.    58  Figure 5.1  HAPI2 displays dose dependent hypoxia selective radiosensitization ability     59  5.2  Prodrug HAPI3 decreases cell viability selectively in hypoxic cells treated with ionizing radiation Introduction The third candidate prodrug to be synthesized, HAPI3, is structurally very similar to HAPI2, with the exception of an additional methyl group attached at the 1-position on the imidazole ring. A synthetic route for this compound was developed by members of Dr. Gregory Dake’s laboratory at the UBC Department of Chemistry and was further optimized by Geoff Winters at the Centre for Drug Research and Development (Vancouver, Canada). Approach In this experiment HAPI3 was added to HeLa cells at 0, 25, 50, 100 or 150 µM immediately before gassing with 0% O2, hypoxic conditions, or 21% O2, oxic conditions, with each gas containing 5% CO2/ balance N2. The 0 µM treatment group contained 1% DMSO as a vehicle control such that the volume of DMSO equalled that of the 150 µM HAPI3 treatment. Additionally included are cells treated with 100 µM IC86621 for comparison as a positive control of radiosensitization. Following gassing, the chambers were incubated for 1 h at 37 °C before the chambers receiving radiation were treated with either 5 Gy (oxic cells) or 12.5 Gy (hypoxic cells). In this experiment the hypoxic cells were treated with a dose of radiation 2.5x higher than that of the oxic cells in order to achieve approximately equal cell kill between the two conditions. Chambers were then returned to the 37 °C oven for 4 h while cells were allowed to recover in the presence or absence of HAPI2 and hypoxia. After 4 h the chambers were opened to the air and the prodrug containing media was removed and replaced with 500 µL fresh media. The cells   60 were then allowed to recover for a further 72 h under normal incubation conditions before resazurin was added to assess cell viability as per section 2.4. Results Figure 5.2 displays the results of both the irradiated and non-irradiated cells in the screen. As expected, IC86621 displays no obvious toxicity in the absence of DNA damage. However both oxic and hypoxic cells are sensitized to ionizing radiation by the DNA-PK inhibitor. As per the experimental design, hypoxic DMSO control cells irradiated at 12.5 Gy were found to have similar, albeit slightly increased viability 72 h following irradiation compared to oxic DMSO control cells irradiated with 5 Gy. Equalizing the cell viability between the oxic and hypoxic cells eases comparison between these treatment groups, while also showing that the hypoxic cells are resistant to the effects of radiation. In contrast to HAPI2, no obvious off target toxicity was observed in the non-irradiated cells treated with 25-100 µM HAPI3. However, with 150 µM HAPI3 treatment cell viability did decrease somewhat in both oxic and hypoxic cells. As displayed in figure 5.3, increasing concentrations of HAPI3 beyond 25 µM were found to cause dose dependent radiosensitization selectively in hypoxic cells. Figure 5.3 displays results from the same screening assay, however in this figure only the irradiated treatment groups are displayed and individual replicates are plotted. As these preliminary screening results display the desired hypoxia selective radiosensitizing activity, and because sufficiently more of the HAPI3 compound has been made available for screening, future work with HAPI3 will include clonogenic survival assays and potentially anti-cancer activity and pharmacological evaluation in mice.   61  Figure 5.2  HAPI3 displays no off-target toxicity in the 25-100µM range         62  Figure 5.3  HAPI3 has activity as a hypoxia selective radiosensitizer   63 5.3 Prodrug HAPI3 is reduced by an NADPH dependent enzyme to the DNA-PK inhibitor IC86621 selectively under hypoxic conditions Introduction To assess activity of candidate prodrugs more mechanistically we have employed a cell-free microsomal stability assay. Microsomes are vesicular artifacts from the endoplasmic reticulum of lysed eukaryotic cells that are rich in metabolic enzymes (42). Liver microsomal stability is often used under normoxic conditions in drug development efforts to estimate the extent of hepatic metabolism a compound will undergo in vivo (42). However, for our purposes microsomal stability is assessed under both oxic and hypoxic circumstances to measure differences in the bioreduction products formed from hypoxia activated prodrug candidates. Approach Details of mouse liver microsome preparations and prodrug incubations are described in section 2.7. High pressure liquid chromatography was used to separate the parent prodrug HAPI3 from the reduction product IC86621 (see section 2.8 for details). Incubations were performed in triplicate and error bars displayed represent the mean ± standard deviation. Results The concentration of prodrug HAPI3 and the DNA-PK inhibitor IC86621 are plotted against microsomal incubation time in figure 5.4 A, B. During incubation in the presence of NADPH, seen in figure 5.4A, under atmospheric oxygen the concentration of HAPI3 decreases from 34 to 16 µM over 2 h, indicating microsomal enzyme mediated metabolism of the prodrug. However, only a small amount of this metabolism resulted in   64 the production of IC86621, the concentration of which increased only slightly from 0 to 1 µM over the same 2 h time period. This result is in contrast to that obtained from performing the same assay under hypoxic conditions. In hypoxia, HAPI3 was more rapidly metabolized, falling from 30 to 1.5 µM over 2 h. Also, in hypoxia the reduction product IC86621 was favoured, with up to 18 µM IC86621 being formed over 2 h. This result suggests that the reduction scheme proposed in figure 4.7A is likely to be occurring. However, because an incomplete yield of HAPI3 to IC86621 resulted, additional metabolic processes which do not result in IC86621 are also apparent. 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Chem. Lett. (2004) vol. 14 pp. 6083–6087 3  Ismail et al. Oncogene (2004) vol. 23 pp. 873–882 4  Knight et al. Bioorg. Med. Chem  (2004) vol. 12 pp. 4749–4759 5  Block et al. Nucleic Acids Res (2004) vol. 32 (6) pp. 1967-72 6  Foukas et al. J Biol Chem (2002) vol. 277 (40) pp. 37124-30 7  Durant et al. Nucleic Acids Res (2003) vol. 31 (19) pp. 5501-12 8  Sarkaria et al. Cancer Res (1998) vol. 58 (19) pp. 4375-82 9  Izzard et al. Cancer Res (1999) vol. 59 (11) pp. 2581-6  IC50 (µM) Inhibitor common name DNA- PKcs ATM ATR mTOR P110a P110b P110d P110g PI-1031 0.002 0.92 0.85 ND 8 88 48 150 NU74412 0.014 >100 >100 1.7 5 ND ND ND SU117523 0.13 ND ND ND ND ND ND 1.1 IC866214 0.17 >100 ND >100 16 0.99 3.8 10 NU70262 0.23 >100 >100 6.4 13 ND ND ND AMA374 0.27 >100 >100 >100 32 3.7 22 ~100 Caffeine5,6 ~0.4 0.2 1.1 ND 400 400 75 1000 LY2940024 0.66 >100 >100 8.9 9.3 2.9 6 38 DMNB7 15 ND ND ND ND ND ND ND Vanillin6 ~1500 ND ND ND ND ND ND ND Wortmannin8,9 0.016/ 0.25* 0.15 1.8 ND 0.003 0.003 0.003 0.003   75 Appendix C: Chemical structures and table of common and International Union of Pure and Applied Chemistry names for compounds synthesised as part of the HADRI project H 3 C     O N O OH H 3 C     O N O O CH 3 CH 3 O O N O CH 2 CH 3 O O HN N N + O – O N O H 3 C N O CH 3 O N O N N +       O – O H 3 C     O N O O N + O O – IC86621 HADRI1 HADRI2 HAPI1 HAPI2 HAPI3 Common name International union of pure and applied chemistry (IUPAC) name IC86621 1-(2-Hydroxy-4-morpholin-4-yl-phenyl)ethanone HADRI 1 1-[2-methoxy-4-(morpholin-4-yl)phenyl)ethan-1-one HADRI 2 1-[4-(morpholin-4-yl)-2-(prop-2-en-1-yloxy)phenyl]ethan-1-one HAPI 1 1-[4-(morpholin-4-yl)-2-[(4-nitrophenyl)methoxy]phenyl)ethan-1-one HAPI 2 1-[4-(morpholin-4-yl)-2-[(2-nitro-1H-imidazol-5-yl)methoxy]phenyl]ethan- 1-one HAPI 3 1-{2-[(1-methyl-2-nitro-1H-imidazole-5-yl)methoxy]-4-(morpholin-4- yl)phenyl}ethan-1-one

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