• unlimited access with print and download
    $ 37 00
  • read full document, no print or download, expires after 72 hours
    $ 4 99
More info
Unlimited access including download and printing, plus availability for reading and annotating in your in your Udini library.
  • Access to this article in your Udini library for 72 hours from purchase.
  • The article will not be available for download or print.
  • Upgrade to the full version of this document at a reduced price.
  • Your trial access payment is credited when purchasing the full version.
Buy
Continue searching

Studies on the mechanism of action of quinone-based antitumor agents

ProQuest Dissertations and Theses, 2011
Dissertation
Author: Chao Yan
Abstract:
The mechanism of action of aziridinylbenzoquinones and indolequinones (IQs) were examined in this thesis. The role of multiple reductases in the bioactivation and cytotoxicity of the aziridinylbenzoquinone RH1, a novel bioreductive alkylating agent designed to be activated by the two-electron reductase NAD(P)H:quinone oxidoreductase 1 (NQO1), were defined. The potential contributions of other reductases including cytochrome b5 reductase (b5R), cytochrome P450 reductase (P450R), NRH:quinone oxidoreductase 2 (NQO2), and xanthine oxidase/xanthine dehydrogenase (XO/XDH) to RH1 bioactivation was examined in MDA468 human breast cancer cell lines over-expressing various levels of the above reductases. Data demonstrated that NQO1 and NQO2 were the principal enzymatic determinants of RH1 bioactivation and that b5R, P450R and XDH/XO were unlikely to play major roles. The data also suggested that NQO2 may be particularly relevant as a bioactivation system for RH1 in NQO1-deficient tumors such as leukemias and lymphomas. The mechanism of action of novel IQs in human pancreatic cancer was also examined. IQs were found to be potent growth inhibitors and efficient inducers of caspase-dependent apoptosis in human pancreatic cancer cell lines. Thioredoxin reductase 1 (TR1) was identified as a molecular target of the IQs. The C-terminal selenocysteine of TR1 was found to be the primary adduction site by the IQ-derived iminium electrophile. Inhibition of TR1 by IQs in pancreatic cancer cells resulted in the shift of thioredoxin redox state and activation of the ASK1-p38/JNK apoptosis signaling cascade, providing a possible mechanism of action of the IQs in human pancreatic cancer. Finally, compounds in the IQ series were characterized as mechanism-based inhibitors of NQO2 in both cell-free systems and in K562 human leukemia cells. Using biochemical, computational modeling and mass spectrometric approaches, structural requirements for effective and selective inhibition of NQO2 were identified and a novel mechanism of inhibition involving electrophilic adduction of FAD was characterized. These IQs represent the first mechanism-based inhibitors of NQO2 to be characterized in cellular systems, providing valuable tools for studying the functions of NQO2 both under physiological conditions and in disease.

TABLE OF CONTENTS CHAPTER I. REVIEW OF LITERATURE AND STATEMENT OF PURPOSE 1 Introduction 1 Mechanism of action of antitumor quinones 4 Bioreductive alkylating agents 7 NAD(P)H:Quinone Oxidoreductase 1 8 NQOl-directed bioreductive alkylating agents 12 Indolequinones 14 Aziridinylbenzoquinones 16 Statement of purpose 19 II. DISSECTING THE ROLE OF MULTIPLE REDUCTASES IN BIOACTIVATION AND CYTOTOXICIY OF THE ANTITUMOR AGENT RH1 21 Introduction 21 Materials and methods 22 Results 30 Role of NQOl in RH1 bioactivation and cytotoxicity 30 Role of P450R in RH1 bioactivation and cytotoxicity 31 Role of b5R in RH1 bioactivation and cytotoxicity 40 Role of XO/XDH in RH1 bioactivation and cytotoxicity 43 Role of NQ02 in RH1 bioactivation and cytotoxicity 46 Discussion 51 VI

MECHANISM OF ACTION OF NOVEL INDOLEQUINONES AS POTENT ANTITUMOR AGENTS AGAINST HUMAN PANCREATIC CANCER 58 Introduction 58 Materials and methods 67 Results 80 Structure of IQs used in this study 80 Growth inhibitory activity of the IQs in human pancreatic cancer cell lines 81 Cytotoxic activity of the IQs in human pancreatic cancer cell lines 84 Antitumor activity of the IQs in human pancreatic tumor xenografts 84 Induction of caspase-dependent apoptosis by the IQs in human pancreatic cancer cell lines 87 Effects of IQ treatment on general oxidative stress and DNA damage in human pancreatic cancer cell lines 87 Antitumor activity profile of the IQs in the NCI-60 tumor cell line panel 93 Characterization of TRl inhibition by IQs in cell-free systems 95 Identification of the TRl C-terminal selenocysteine active site as the target for IQ modification using BIAM 101 Conformation of the TRl C-terminal selenocysteine active site as the target for IQ modification using mass spectrometry 103 Inhibition of TRl by IQs in human pancreatic cancer cell lines 103 Effect of IQ treatment on the redox state of thioredoxin-1 in human pancreatic cancer cell lines 107 Effect of IQ treatment on p38 and JNK activation in human pancreatic cancer cell lines 107 VI1

Effect of IQ treatment on the activation of ASK1 in human pancreatic cancer cell lines 111 Discussion 111 IV. DEVELOPMENT OF INDOLEQUINONE INHIBITORS OF NRH:QUINONE OXIDOREDUCTASE 2 (NQ02). CHARACTERIZATION OF MECHANISM OF INHIBITION IN BOTH CELL-FREE AND CELLULAR SYSTEMS 121 Introduction 121 Materials and methods 133 Results 140 Structure of IQs 140 NRH-dependent NQ02 inhibition by IQs 141 Kinetic analysis of NQ02 inhibition by IQs 145 Partition ratio determination 146 Potency of IQ inhibitors relative to previously characterized competitive inhibitors of NQ02 149 Inhibition of NQ02 activity by IQs in K562 cells 149 Growth inhibitory effect of IQs in K562 cells 152 Molecular modeling of IQs into NQ02 152 MS analysis 157 Discussion 159 V. SUMMARY AND FUTURE DIRECTIONS 171 REFERENCES 180 APPENDIX 201 A. Table of IQs studied in Chapter III 201 B. Table of IQs studied in Chapter IV 224 viii

TABLES NQOl, NQ02, P450R, and b5R activity and RHl IC50 values (MTT/SRB) in all transfected MDA468 clones 33 Percent of DNA in comet tail for all the transfected clones following RHl treatment 36 IC50 values for IQs in human pancreatic cancer cell lines 83 Biochemical and physical characterization of the IQ inhibitors of NQ02 144

FIGURES Figure 1.1 Some naturally occurring quinones that participate in physiological processes 2 1.2 Some naturally occurring quinones with anticancer properties 3 1.3 Metabolism reactions of quinones 5 1.4 Crystal structure of the human NQOl dimer 9 1.5 Bioreductive activation of mitomycin C by NQOl 13 1.6 Structure of mitomycin C analogs 15 1.7 Structure of aziridinylbenzoquinones 17 1.8 Mechanism of RHl bioactivation by NQOl 18 2.1 Immunoblotting of NQO1, P450R, b5R, and NQ02 in MDA468 cells 32 2.2 Clonogenic survival curves of MDA468 and NQ16 cells following RHl treatment 34 2.3 RHl-induced DNA cross-linking in MDA468 and NQ16 cells 35 2.4 Characterization of MDA468 cells stably transfected with human P450R 38 2.5 RHl-induced DNA single-strand breaks and cross-linking in parental and P450R-transfected MDA468 cells 39 2.6 Characterization of MDA468 cells stably transfected with human b5R 41 2.7 RHl-induced DNA single-strand breaks and cross-linking in parental and b5R-transfected MDA468 cells 42 2.8 Scheme of xanthine oxidase/xanthine dehydrogenase 44 2.9 Role of XO/XDH in RHl activation and toxicity in cell-free and cellular systems 45 2.10 Reduction of RHl by recombinant human NQ02 47 2.11 Characterization of MDA468 cells stably transfected with human NQ02 48

2.12 Clonogenic survival curves of MDA468 and NQ2C4 cells following RH1 treatment 49 2.13 RH1-induced DNA cross-linking in parental and NQ02-transfected MDA468 cells 50 2.14 Levels of NQ02 expression in a panel of human leukemia cell lines 57 3.1 Mechanism of NQOl-mediated reductive activation of IQs 59 3.2 Mechanism of NQOl inhibition by the IQ ES936 61 3.3 Catalytic properties of human TR1 64 3.4 Structure of some known inhibitors of human TR1 66 3.5 Structure of the IQs used in this study 82 3.6 Effect of IQ treatments on colony formation in human pancreatic cancer cells 85 3.7 Inhibition of pancreatic xenograft tumor growth in nude mice following treatment with IQs 86 3.8 Induction of caspase-dependent apoptosis by IQ 5 in pancreatic cancer cells 88 3.9 Time course of IQ-induced apoptotic events in MIA PaCa-2 cells 89 3.10 Effect of IQ treatment on general oxidative stress in MIA PaCa-2 cells 91 3.11 Effect of IQ treatment on DNA strand breaks and DNA cross-linking in MIA PaCa-2 and BxPC-3 cells 92 3.12 Antitumor activity of IQs in the NCI-60 tumor cell line panel 94 3.13 Metabolism of IQ 5 by recombinant human NQ02 96 3.14 Inhibition of TR1 by IQ 5 in cell-free systems 98 3.15 Inhibition of TR1 by IQ 5 in cell-free systems, continued 100 3.16 Identification of the TR1 selenocysteine as a potential target of IQs using biotinylated iodoacetamide (BIAM) 102 3.17 Mass spectrometric analysis of TR1-IQ5 adducts 104 3.18 Inhibition of TR1 activity by IQs in MIA PaCa-2 cells 106 XI

3.19 Effects of IQ treatment on thioredoxin-1 redox state in MIA PaCa-2 cells 108 3.20 Effects of IQ treatment on the activation of the MAPK apoptotic signaling pathway 109 3.21 Effects of p38 and JNK inhibitors on IQ-induced apoptosis in MIA PaCa-2 cells 110 3.22 Effects of IQ treatment on the activation of ASK1 in MIA PaCa-2 cells 112 3.23 A proposed mechanism of TR1 inhibition by the IQs 116 3.24 A proposed apoptotic signaling cascade induced by IQ treatment in human pancreatic cancer cells 119 4.1 Crystal structure of the human NQ02 dimer 123 4.2 Comparison between the active site of human NQ02 and NQOl 124 4.3 CofactorsofNQ02 127 4.4 Substrates of NQ02 129 4.5 Inhibitors of NQ02 132 4.6 Structure of IQs used in this study 142 4.7 Inhibition of NQ02 (A) and NQOl (B) by IQs in cell-free systems 143 4.8 Kinetic analysis of NQ02 inhibition by IQs in cell-free systems 147 4.9 Partition ratio determinations for IQs in cell-free systems 148 4.10 Comparison of IQs with known inhibitors of NQ02 150 4.11 Selective inhibition of NQ02 by IQs in K562 cells 151 4.12 Molecular modeling of IQs 1-6 in the active site of NQ02 154 4.13 Molecular modeling of IQs 9-12 in the active site of NQ02 155 4.14 Molecular modeling of IQ 1 in the active site of NQOl (A) and NQ02 (B) 156 4.15 Molecular modeling of FAD in the active site of NQ02 158 4.16 HPLC analysis of the adduction of the FAD cofactor in NQ02 by IQs 160 xii

4.17 ESI-LC/MS analysis of the IQ 1-FAD adduct 161 4.18 ESI-LC/MS analysis of the IQ 2-FAD adduct 162 4.19 ESI-LC/MS analysis of the IQ 4-FAD adduct 163 4.20 ESI-LC/MS analysis of the IQ 5-FAD adduct 164 4.21 Proposed mechanism of FAD adduction by the IQ inhibitors of NQ02 165 5.1 Selective inhibition of NQOl, TRl, and NQ02 by IQs 178 xin

ABBREVIATIONS ASK1, apoptosis signal-regulating kinase 1 AZQ, 2,5-bis(l-aziridinyl)-3,6-bis(carboethoxyamino)-l,4-benzoquinone b5R, NADH: cytochrome b5 reductase BNAH, N-benzyldihydronicotinamide BCNU, l,3-bis-(2-chloro-ethyl)-l-nitrosourea BIAM, biotin-conjugated iodoacetamide CB1954, 5-[aziridin-l-yl]-2,4-dinitrobenzamide CDNB, l-chloro-2, 4-dinitrobenzene DCFH-DA, 2',7'-dichlorfluorescein-diacetate DCPIP, 2,6-dichlorophenol-indophenol DHE, dihydroethidium DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid) DZQ, 2,5-diaziridinyl-l,4-benzoquinone E09, 5-aziridinyl-3-hyroxymethyl-l-methyl-2[lH-indole-4,7-dione]prop-|3-en-a-ol FAD, flavin adenine dinucleotide GSH, glutathione (reduced form) GSSG, glutathione (oxidized form) H2O2, hydrogen peroxide HPLC, high performance liquid chromatography HQ, hydroquinone HSP90, heat shock protein 90 xiv

IC50, drug concentration at 50% growth inhibition i.p., intraperitoneal IQs, indolequinones JNK, c-Jun N-terminal kinase MAPK, mitogen-activated protein kinase MeDZQ, 2,5-diaziridinyl-3,6-dimethyl-l,4-benzoquinone MMC, mitomycin C MS, mass spectrometry MTT, 3-(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium NADH, nicotinamide adenine dinucleotide (reduced) NADPH, nicotinamide adenine dinucleotide phosphate (reduced) NF-KB, nuclear factor kappa-light-chain-enhancer of activated B cells NMeH, N-methyldihydronicotinamide NMNH, dihydronicotinamide mononucleotide NQOl, NAD(P)H:quinone oxidoreductase 1 NQ02, NRH:quinone oxidoreductase 2 NRH, dihydronicotinamide riboside P450R, NADPH: cytochrome P450 reductase PI, propidium iodide PQQ> pyrroloquinoline quinone RHl, 2,5-Diaziridinyl-3-(hydroxymethyl)-6-methyl-l,4-benzoquinone ROS, reactive oxygen species s.c., sub-cutaneous xv

SD, standard deviation of the mean SRB, sulforhodamine B TR1, thioredoxin reductase 1 TR2, thioredoxin reductase 2 XDH, xanthine dehydrogenase XO, xanthine oxidase xvi

CHAPTER I REVIEW OF LITERATURE AND STATEMENT OF PURPOSE Quinones are an important class of natural compounds that exhibit a wide range of biological activities. A major work on naturally occurring quinones has compiled over 700 botanical sources of quinones and more than 200 sources in the animal kingdom (1). In humans, endogenous quinones participate in many important physiological processes. For example, ubiquinone (coenzyme Q10, Figure 1.1) is a component of the mitochondrial electron transport chain and participates in aerobic cellular respiration (2). The phylloquinone (vitamin Kl, Figure 1.1) is involved in the post-translational modification of proteins by carboxylation of glutamate residues (3). The more recently discovered pyrroloquinoline quinone (coenzyme PQQ, Figure 1.1) functions as a cofactor for various dehydrogenases such as alcohol dehydrogenase and glucose dehydrogenase and has been found to have antioxidant effects (4). Some naturally occurring quinones also have antibiotic and anticancer properties. These include, but are not limited to the following classes: anthracyclines, streptonigrin, naphthoquinones, mitomycins, and benzoquinones (Figure 1.2). Anthracyclines, including daunorubicin and doxorubicin, are widely used as frontline chemotherapeutic agents (5). Streptonigrin (6) and the naphthoquinone (3-lapachone (7) are extremely cytotoxic agents and although they are not yet approved for clinical use both compounds have been widely used as experimental drugs because of their ability to induce redox cycling and oxidative stress. Mitomycin C (MMC), another natural quinone compound in clinical use, is the archetypal bioreductive drug, a family of anticancer compounds that 1

coenzyme Q10 H02C &x O^Y^N^C02H O PQQ vitamm Kl Figure 1.1. Some naturally occurring quinones that participate in physiological processes. 2

O OH Me Me daunorubicin doxorubicin (3-lapachone mitomycin C MeO HoN Mev OMe OCONH2 streptonigrin geldanamycin Figure 1.2. Some naturally occurring quinones with anticancer properties. 3

require metabolic activation to be transformed into their active forms (8). The benzoquinone ansamycins, represented by geldanamycin and analogs, are potent inhibitors of heat shock protein 90 (HSP90) and are promising agents against a variety of human cancers (9). Mechanism of action of antitumor quinones Reductive metabolism of antitumor quinones is frequently a prerequisite for their cytotoxicity. Quinones readily undergo facile reduction by multiple reductases in cells (10). One-electron reduction of a quinone generates a semiquinone radical, while two- electron reduction generates a hydroquinone (Figure 1.3). A semiquinone radical can also be formed by a comproportionation reaction between a quinone and a hydroquinone (11). Conversely two molecules of semiquinone radical can disproportionate to a quinone and a hydroquinone (11). Under aerobic conditions, a semiquinone radical depending on its redox potential can be rapidly re-oxidized by molecular oxygen forming superoxide anion radical and regenerating the parental quinone (Figure 1.3). The repetitive reduction and re-oxidation of a quinone under aerobic conditions with the generation of superoxide anion radicals is known as redox cycling (12). Dismutation of the superoxide anion radical either chemically or enzymatically generates hydrogen peroxide (H2O2) which can then undergo Fenton chemistry to generate hydroxyl radicals via metal-catalyzed reactions (13). Many quinones are very efficient redox cycling agents and readily induce reactive oxygen species (ROS) generation and oxidative stress (11). Reactive oxygen species are toxic to cells and can induce DNA degradation, lipid peroxidation, thiol depletion, 4

o o 1e" 1H+ O OH 1e" reduction o OH 2e 2H+ 2e" reduction O OH o OH o comproportionation/disproportionation equilibrium o OH OH o o + o, H+ OH 7 semiquinone radical redox cycling 2 to generate superoxide T + ± SOD 2 02 + 2 H H202 + 02 superoxide dismutation H202 + Fe(ll) OH +OH + Fe(lll) metal-catalyzed hydroxy radical generation (Fenton/Haber Weiss reaction) Figure 1.3. Metabolism reactions of quinones. 5

protein damage and may also play a role in cellular signaling (14). The most common mechanism of quinone-induced cytotoxicity is oxidative DNA damage (11). Reactive radicals can readily bind to DNA and induce deoxyribose breakdown and DNA strand breakage. Both streptonigrin and the naphthoquinone (3-lapachone are potent redox cycling agents and induce DNA single strand breaks in cancer cells (6, 7). Generation of free radicals during anthracycline (daunorubicin and doxorubicin) metabolism has been proposed as a determinant of both their antitumor activity and cardiotoxicity (5). Another good example is the naphthoquinone menadione, a vitamin K analog whose cytotoxicity relies on redox cycling of the semiquinone and is prevented by two-electron reduction to the hydroquinone form (15, 16). MMC can also undergo one-electron reduction generating semiquinone radicals and ROS leading to DNA strand breaks which contribute to its overall antitumor activity (8). DNA damage by antitumor quinones can also occur through topoisomerase inhibition. Anthracyclines and streptonigrin, for example, are potent inhibitors of topoisomerase II at therapeutic concentrations (6, 17) and function by stabilizing the topoisomerase II-DNA cleavable complex in an open (cleaved) conformation of DNA. Topoisomerase II inhibition can lead to both DNA single and double strand breaks, which subsequently lead to apoptosis and cell death (18). In addition to redox cycling and free radical formation, reductive metabolism of antitumor quinones can also generate alkylating species that can bind covalently to proteins, lipids and DNA (19). Quinones are electrophihc species and, depending on their substitution pattern can alkylate biological nucleophiles directly (11, 20). In many cases however and particularly with fully substituted quinones the metabolic formation of an 6

electrophilic reactive intermediate is required for antitumor activity and is achieved through two-electron reduction of quinones and subsequent rearrangement reactions (21). Anthracyclines can undergo two-electron reduction to form alkylating quinonemethides which can alkylate both protein and DNA (5). After two-electron reduction, MMC, the prototypical bioreductive alkylating agent, can act as a monofunctional agent and bind to DNA, RNA, and protein or it can act as a bifunctional agent and cross-link double strands of DNA or cross-link DNA with protein (8). Since the vast majority of quinones undergo reductive metabolism in cells, most research in the quinone antitumor drug area has been focused on developing better quinone-based bioreductive alkylating agents. Bioreductive alkylating agents The conception of bioreductive alkylation was developed by Sartorelli and colleagues in 1972 (22). These drugs are non-toxic to tumor cells on their own but once reduced in cells are transformed to cytotoxic alkylating species. Numerous cellular reductases are capable of reducing quinones (10). These include the one-electron reductases NADPH: cytochrome P450 reductase (EC 1.6.2.3, P450R), NADH: cytochrome b5 reductase (EC 1.6.2.2, b5R), xanthine oxidase (EC 1.2.3.2, XO), NADH: ubiquinone oxidoreductase (EC 1.6.5.3), ferredoxin-NADP+ reductase and the two- electron reductases NAD(P)H:quinone oxidoreductase 1 (EC 1.6.22.1, NQOl), NRH:quinone oxidoreductase 2 (EC 1.10.99.2, NQ02), and xanthine dehydrogenase (EC 1.1.1.204, XDH). Alkylating species can be generated following either one- or two- electron reduction. However, one-electron reduction of quinones also leads to redox cycling and ROS-mediated oxidative stress, a mechanism which contributes to tumor cell 7

killing but also undesired toxicities (23). Two-electron reduction by NQOl, on the other hand, prevents the formation of semiquinone radicals and inhibits redox cycling. Moreover, since NQOl levels were found to be elevated in a variety of solid tumors when compared to normal tissues (24, 25), it was hypothesized that agents that were specifically metabolized by NQOl would exhibit selective toxicity to human cancer cells. Below is a brief introduction to NQOl enzymology and NOOl-directed quinone antitumor agents that have been developed to date. NAD(P)H: Quinone Oxidoreductase 1 NQOl is a two-electron quinone reductase that can use both NADH and NADPH as cofactor (26). NQOl is a homodimer of two interlocked monomers of 274 amino acid (30.5 kDa), each containing a non-covalently bound molecule of flavin adenine dinucleotide (FAD). The crystal structural of human NQOl was resolved in 2000 (Figure 1.4) (27). Two equivalent catalytic sites are formed by residues from both monomers and located at the dimer interface. Each catalytic site has a FAD binding site and a cofactor/substrate binding site and the FAD cofactor is tightly bound to the protein non- covalently under physiological conditions. The cofactor and substrate share the same binding site which is the basis for the ping-pong mechanism of catalysis (28). In a catalytic cycle, reduced pyridine nucleotide binds to the active site first and reduces FAD to FADH2 through a postulated hydride transfer mechanism; the oxidized pyridine nucleotide is then ejected allowing binding of the substrate which is reduced by FADH2, again via a hydride transfer and the reduced substrate is then released from the active site 8

Figure 1.4. Crystal structure of the human NQOl dimer. One monomer is colored in purple; the other one in light blue. FAD in one of the two active sites at the dimer interface was also shown in stick mode with carbon atoms colored in yellow, nitrogen in blue, and oxygen in red. (The crystallographic coordinates for the structure of human NQOl co-crystallized with ES936, PDB code IKBQ, were from Protein Data Bank). 9

(28). The substrate binding site is highly flexible and can accommodate a wide range of quinone substrates (27). The major proposed physiological role of NQOl in the body is the protective detoxification of endogenous and exogenous quinones (29). Two-electron reduction of quinones by NQOl generates the hydroquinone form, therefore bypassing the formation of potentially toxic semiquinone radicals and protecting cells from oxidative stress. In addition, the hydroquinone products of NQOl reduction can be more easily conjugated and excreted compared to their quinone forms; therefore NQOl is usually classified as a phase II detoxifing enzyme (30). Two-electron reduction by NQOl has also been implicated in the metabolism of vitamin K and the generation of antioxidant forms of ubiquinone (coenzyme Q10) and vitamin E (30). NQOl mediated reduction of ubiquinone results in the formation of ubiquinol, an excellent antioxidant against lipid peroxidation (31). NQOl can reduce a-tocopherolquinone, a product of vitamin E oxidation, to a-tocopherolhydroquinone, a compound with antioxidant properties (32). Notably, NQOl has been reported to scavenge superoxide directly (33), further demonstrating the antioxidant activity of this enzyme. More recent studies have suggested that NQOl can stabilize the tumor suppressor protein p53 by inhibiting its proteasomal degradation (34). Importantly, the ability of NQOl to function as a chaperone is not related to its enzymatic activity but is a result of direct protein-protein interactions (35). In addition to p53, NQOl has also been shown to stabilize p73 (36), p33 (37), p63 (38) and ornithine decarboxylase (ODC) (39) and was proposed to function as a gatekeeper for protein degradation through the 20S proteasome (39). NQOl does not appear to interact directly with the 20S proteasome but co-purifies with the proteasome 10

(38, 40). The precise mechanism underlying the effect of NQOl on the 20S proteasome is still under investigation. Recently, NQOl has been shown to modulate expression of adhesion molecules in endothelial cells, a process regulated primarily by NF-KB transcription (41). Interestingly in melanoma cells NQOl has been shown to be an important modulator of cell growth mediated by an effect on NF-KB transcription (42). The mechanisms of NQOl modulation of NF-KB transcription are unclear but Garate et al. demonstrated that effects in melanoma cells were mediated by the NF-KB family member BCL3 (42). There is another structurally similar quinone reductase in the quinone reductase family, NRH:quinone oxidoreductase 2 (NQ02), which is also a two-electron reductase but differs from NQOl in terms of cofactor and substrate preference (43). NQ02 is 43 amino acids shorter than NQOl at the C-terminal, resulting in differences in the conformation of the active site (44). Instead of NAD(P)H, NQ02 uniquely uses dihydronicotinamide riboside (NRH) as a cofactor. In the presence of NRH, NQ02 is able to catalyze the two-electron reduction of a variety of quinone substrates in vitro (43, 45); however, the physiological substrate of NQ02 in the body remains unclear. The possibility of limited NRH availability in cells has also been recognized (46). NQ02 is of particular interest with respect to the bioactivation of the anticancer drug CB1954 [5- (aziridin-l-yl)-2,4-dinitobenzamide] into a bifunctional alkylating species (46), suggesting a potential role of NQ02 in the metabolic activation of bioreductive alkylating agents. NQ02 enzymology and biochemistry will be described in detail in Chapter IV. 11

NQOl-directed bioreductive alkylating agents A variety of structurally diverse quinone compounds have been screened as NQOl-directed antitumor agents, including cyclopropyl-mitosenes, mitosenes, indolequinones, aziridinylbenzoquinones (47). Structure-based rational design of quinone compounds as specific NQOl substrates has also been extensively explored (48). MMC, the prototypic bioreductive alkylating agent in clinical use, is effective against a variety of human tumors, including breast, stomach, head and neck, lung, prostate, and bladder (8). The mechanism of action and metabolism of MMC has been extensively studied after both one- and two-electron reduction (49-54) and it is generally agreed that bioreduction results in the formation of reactive metabolites that cross-link adjacent guanines in DNA (55). The proposed mechanism of two-electron mediated reductive activation of MMC is shown in Figure 1.5. Two electron reduction generates the hydroquinone resulting in the loss of the methoxy group and the opening of the aziridine ring to form an electrophile at C-l capable of alkylating DNA. The loss of the carbamate generates a second electrophile at the C-10 position and DNA cross-linking can then occur via another alkylation reaction (56). However, MMC is a relatively poor substrate for NQOl, and the metabolism of MMC by NQOl is known to be pH- dependent (49, 57). MMC is also bioactivated by one-electron reductases resulting in redox cycling and free radical generation (58). The design and synthesis of MMC analogs that are specifically activated by NQOl has generated lots of interest. Development of NQOl-directed MMC analogs resulted in the identification of a novel family of antitumor compounds based on the indolequinone backbone. 12

O 10r - OCONH2 H2Nk JK J .OMe H,N H,N 2e N 1 :NH 2H+ OCONH2 HoN NH2-DNA-NH2 H,N OCONH2 OMe NH V MeOH OCONH2 HoN OCONH2 OH OH DNA O, HoN DNA Figure 1.5. Bioreductive activation of mitomycin C by NQOl (Adjusted from Colucci et al, 2008 (47)). 13

Indolequinones. The indolequinone E09 (5-aziridinyl-3-hyroxymethyl-l-methyl-2[lH-indole-4,7- dione]prop-(3-en-oc-ol) was the first synthetic indolequinone developed in the late 80' s (59). E09 is a synthetic analog of MMC (Figure 1.6) but a much better NQOl substrate (60). E09 is a potential tri-functional alkylating agent after loss of the hydroxyl leaving group from both the indole 2- and 3-positions, and aziridine ring opening (60). E09 has been shown to be selectively toxic to cancer cell lines with elevated NQOl levels (61). However, E09 hydroquinone auto-oxidizes and redox cycles more readily than MMC after reduction by NQ01(62). E09 underwent phase I and II clinical trials but data from the phase II studies was disappointing due to rapid clearance of E09 in humans, limited activity of the drug metabolites in vivo and proteinuria as the dose-limiting toxicity (63). E09 has recently re-entered phase I/II clinical trials for treatment of bladder cancer (64). Collaborative work between the Ross and Moody (University of Nottingham) laboratories has resulted in the design and synthesis of many potent bioreductive quinones in the mitosene, cyclopropyl-mitosene, and indolequinone (IQ) families (Figure 1.6) (47). The initial goal of these studies was to simplify the structure of MMC and develop more potent compounds that are specifically activated by NQOl. The IQ compounds have the simplest structure but retain potent antitumor activities (47). In the IQ class an extensive series of compounds with various substituent combinations were synthesized and the structural requirements for efficient reduction by NQOl as well as for toxicity in cancer cells were examined previously (65-68). These studies were continued as a part of this thesis (Chapter III). 14

E09 O 10r - OCONH2 H2N^ J-L J ,OMe O 10^-OCONH2 mitomycin C cyclopropyl-mitosenes indolequinones MeO ES936 ure 1.6. Structure of mitomycin C analogs.

An unexpected finding during the early screening of the IQ compounds as NQOl substrates was that some of the compounds tested inactivated NQOl after the initial metabolism by the enzyme. This led to the development of ES936 (Figure 1.6) as the first mechanism-based inhibitor of NQOl (69). The mechanism of action of ES936 and analogs is a focus of this thesis and will be discussed in detail in Chapter III. Aziridinylbenzoquinones. A variety of aziridinylbenzoquinones have been developed as potential NQOl- directed bioreductive alkylating agents. AZQ (2,5-bis(l-aziridinyl)-3,6- bis(carboethoxyamino)-l,4-benzoquinone; diaziquone) was one of the first aziridinylbenzoquinones developed (Figure 1.7). Originally synthesized as an antitumor agent against brain tumors (70), AZQ was extensively studied for its bioreductive activation along with its analogs DZQ (2,5-diaziridinyl-l,4-benzoquinone), and MeDZQ (2,5-diaziridinyl-3,6-dimethyl-l,4-benzoquinone) (Figure 1.7) (71, 72). The alkylating activity of aziridinylbenzoquinones comes from the aziridine groups, which can be activated by ring opening following one- or two-electron reduction, resulting in alkylating intermediates (71). AZQ is a bi-functional alkylating agent capable of inducing DNA cross-linking and its alkylation properties are significantly enhanced by NQOl reduction to the hydroquinone (73); however, AZQ is a relatively poor substrate for NQOl. MeDZQ is an excellent substrate for NQOl and has demonstrated selective cytotoxicity in vitro to cells with high NQOl levels (74). However, similar to AZQ it has very limited solubility and therefore, clinical formulation was problematic. RH1 (2,5- 16

Full document contains 248 pages
Abstract: The mechanism of action of aziridinylbenzoquinones and indolequinones (IQs) were examined in this thesis. The role of multiple reductases in the bioactivation and cytotoxicity of the aziridinylbenzoquinone RH1, a novel bioreductive alkylating agent designed to be activated by the two-electron reductase NAD(P)H:quinone oxidoreductase 1 (NQO1), were defined. The potential contributions of other reductases including cytochrome b5 reductase (b5R), cytochrome P450 reductase (P450R), NRH:quinone oxidoreductase 2 (NQO2), and xanthine oxidase/xanthine dehydrogenase (XO/XDH) to RH1 bioactivation was examined in MDA468 human breast cancer cell lines over-expressing various levels of the above reductases. Data demonstrated that NQO1 and NQO2 were the principal enzymatic determinants of RH1 bioactivation and that b5R, P450R and XDH/XO were unlikely to play major roles. The data also suggested that NQO2 may be particularly relevant as a bioactivation system for RH1 in NQO1-deficient tumors such as leukemias and lymphomas. The mechanism of action of novel IQs in human pancreatic cancer was also examined. IQs were found to be potent growth inhibitors and efficient inducers of caspase-dependent apoptosis in human pancreatic cancer cell lines. Thioredoxin reductase 1 (TR1) was identified as a molecular target of the IQs. The C-terminal selenocysteine of TR1 was found to be the primary adduction site by the IQ-derived iminium electrophile. Inhibition of TR1 by IQs in pancreatic cancer cells resulted in the shift of thioredoxin redox state and activation of the ASK1-p38/JNK apoptosis signaling cascade, providing a possible mechanism of action of the IQs in human pancreatic cancer. Finally, compounds in the IQ series were characterized as mechanism-based inhibitors of NQO2 in both cell-free systems and in K562 human leukemia cells. Using biochemical, computational modeling and mass spectrometric approaches, structural requirements for effective and selective inhibition of NQO2 were identified and a novel mechanism of inhibition involving electrophilic adduction of FAD was characterized. These IQs represent the first mechanism-based inhibitors of NQO2 to be characterized in cellular systems, providing valuable tools for studying the functions of NQO2 both under physiological conditions and in disease.